Nutrition and skeletal muscle 9780128104224, 0128104228

Table of contents :
Content: Part I. General aspects: skeletal muscle physiology and nutrition. Skeletal muscle mass indices in healthy adults / Heliodoro Alemán-Mateo and Roxana E. Ruiz Valenzuela
Reduced skeletal muscle mass and lifestyle / David Scott
Molecular mechanisms of postmeal regulation of muscle anabolism / Olivier Le Bacquer
Adaptation of skeletal muscle mass and metabolism to physical exercise / Xavier Bigard --
Part II. Pathophysiology of skeletal muscle: the important role of diet and nutrients. The role of specific nutriments in sarcopenia associated with chronic diseases: a focus on cancer / Sami Antoun
Sarcopenic obesity / Andrea P. Rossi, Sofia Rubele and Mauro Zamboni
Effects of sarcopenic obesity on cardiovascular disease and all-cause mortality / Janice L. Atkins
Skeletal muscle in obesity and chronic overfeeding / Christelle Guillet
Skeletal muscle mitochondrial, obesity, and high-fat feeding / Christelle Guillet
Muscle immune cells, obesity, and high-fat feeding / Carla Domingues-Faria, Nicolas Goncalves-Mendes and Marie-Chantal Farges
Physiological regulation of skeletal muscle mass: resistance exercise-mediated muscle hypertrophy / Joaquín Pérez-Schindler and Christoph Handschin
Training, changes in nutritional requirements and dietary support of physical exercise / Irène Margaritis
Proteins and amino acids and physical exercise / Stephen D. Patterson, Mark Waldron and Owen Jeffries
Obesity and respiratory skeletal muscles / Richard Severin, Samantha Bond, Adriana Mazzuco, Audrey Borghi Silva, Ross Arena and Shane A. Phillips
Physical exercise in chronic diseases / Bente K. Pedersen --
Part III. Nutrition as a therapeutical tool for skeletal muscle. Whey protein and muscle protection / Yves Boirie
Branched-chain amino acids (leucine, isoleucine, and valine) and skeletal muscle / Stefan H.M. Gorissen and Stuart M. Phillips
Glutamine and skeletal muscle / Vinicius F. Cruzat
Arginine and skeletal muscle / Jean-Pascal De Bandt
Citrulline and skeletal muscle / Charlotte Breuillard, Arthur Goron and Christophe Moinard
Sulfur amino acids and skeletal muscle / Isabelle Papet, Didier Rémond, Dominique Dardevet, Laurent Mosoni, Sergio Polakof, Marie-Agnès Peyron and Isabelle Savary-Auzeloux
Regulation of skeletal muscle metabolism by saturated and monounsaturated fatty acids / Cedric Moro and Frederic Capel
Polyunsaturated omega-3 fatty acids and skeletal muscle / Gordon I. Smith
Vitamin D signaling and skeletal muscle cells / Carla Domingues-Faria and Stéphane Walrand
Vitamin D deficiency and myopathy / Christian M. Girgis
Phytochemicals, their intestinal metaboolites, and skeletal muscle function / Kazumi Yagasaki
Antioxidants and polyphenols mediate mitochondrial mediated muscle death signaling in sarcopenia / Stephen E. Alway
Beta-conglycinin and skeletal muscle / Satoshi Wanezaki and Nobuhiko Tachibana
Effects of quercetin on mitochondriogenesis in skeletal muscle: consequences for physical endurance and glycemic control / Alfredo Fernández-Quintela, Iñaki Milton-Laskibar, Leixuri Aguirre, Saioa Gómez-Zorita, Marcela González and Maria P. Portillo --
Part IV. Adverse effects due to drugs and alcohol. Statins and muscle damage / Matthew J. Sorrentino
Alcoholic myopathy / Emilio González-Reimers, Geraldine Quintero-Platt, Emilio Ganzález-Arnay, Candelaria Martín-González, Lucía Romero-Acevedo and Francisco Santolaria-Fernández.

Citation preview

NUTRITION AND SKELETAL MUSCLE

NUTRITION AND SKELETAL MUSCLE Edited by

STE´PHANE WALRAND

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright r 2019 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-810422-4 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: John Fedor Acquisition Editor: Stacy Masucci Editorial Project Manager: Sam W. Young Production Project Manager: Swapna Srinivasan Cover Designer: Matthew Limbert Typeset by MPS Limited, Chennai, India

List of Contributors Leixuri Aguirre CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Spain; Nutrition and Food Sciences Department, Faculty of Pharmacy, University of the Basque Country, Paseo de la Universidad 7, 01006, VitoriaGasteiz, Spain Heliodoro Alema´n-Mateo Departamento de Nutricio´n y Metabolismo, Coordinacio´n de Nutricio´n, Centro de Investigacio´n en Alimentacio´n y Desarrollo (CIAD), A.C. Hermosillo, Sonora, Me´xico Stephen E. Alway Department of Physical Therapy, College of Health Professions, Memphis, TN, United States; Department of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, United States Sami Antoun Emergency Unit, Ambulatory Care, Gustave Roussy Cancer Campus, Villejuif, France Janice L. Atkins University of Exeter Medical School, Exeter, Devon, United Kingdom Xavier Bigard French Antidoping Agency, Paris, France Yves Boirie Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CHU Clermont-Ferrand, Service de Nutrition Clinique, CRNH Auvergne, ClermontFerrand, France Charlotte Breuillard Laboratoire de bioenerge´tique fondamentale et applique´e, Universite´ Grenoble Alpes, Grenoble, France Frederic Capel Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, Clermont-Ferrand, France Vinicius F. Cruzat School of Pharmacy and Biomedical Sciences, Curtin University, Perth, Western Australia, Australia; Faculty of Health, Torrens University, Melbourne, Victoria, Australia Dominique Dardevet Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Jean-Pascal De Bandt EA4466 PRETRAM Centre de recherche pharmaceutique de Paris Faculte´ de Pharmacie de Paris, Universite´ Paris Descartes 4, avenue de l’observatoire 75270 Paris cedex 06, APHP, Paris, France Carla Domingues-Faria Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Marie-Chantal Farges Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Alfredo Ferna´ndez-Quintela CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Spain; Nutrition and Food Sciences Department, Faculty of Pharmacy, University of the Basque Country, Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain

xi

xii

LIST OF CONTRIBUTORS

Christian M. Girgis Department of Endocrinology, Royal North Shore Hospital, Sydney, NSW, Australia; Department of Endocrinology, Westmead Hospital, Sydney, NSW, Australia; University of Sydney, Sydney, NSW, Australia Saioa Go´mez-Zorita CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Spain; Nutrition and Food Sciences Department, Faculty of Pharmacy, University of the Basque Country, Paseo de la Universidad 7, 01006, VitoriaGasteiz, Spain Nicolas Goncalves-Mendes Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Marcela Gonza´lez Nutrition and Food Science Department, National University of Litoral, National Council for Scientific and Technical Research (CONICET), Santa Fe, Argentina Emilio Gonza´lez-Arnay Department of Anatomy, Pathology and Histology, University of La Laguna, Tenerife, Canary Islands, Spain Emilio Gonza´lez-Reimers Internal Medicine Service, University Hospital of the Canary Islands, University of La Laguna, Tenerife, Canary Islands, Spain Stefan H.M. Gorissen Department of Kinesiology, McMaster University, Hamilton, ON, Canada Arthur Goron Laboratoire de bioenerge´tique fondamentale et applique´e, Universite´ Grenoble Alpes, Grenoble, France Christelle Guillet Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Christoph Handschin Biozentrum, University of Basel, Basel, Switzerland Owen Jeffries St Mary’s University, Twickenham, London, United Kingdom Olivier Le Bacquer Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, Clermont-Ferrand Cedex 1, France Ire`ne Margaritis Human Nutrition Risk Assessment French Agency for Food, Environmental and Occupational Health Safety (ANSES), Maisons-Alfort, France Candelaria Martı´n-Gonza´lez Internal Medicine Service, University Hospital of the Canary Islands, University of La Laguna, Tenerife, Canary Islands, Spain In˜aki Milton-Laskibar Nutrition and Food Sciences Department, Faculty of Pharmacy, University of the Basque Country, Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain Christophe Moinard Laboratoire de bioenerge´tique fondamentale et applique´e, Universite´ Grenoble Alpes, Grenoble, France Cedric Moro INSERM, UMR1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse, France; University of Toulouse, UMR1048, Paul Sabatier University, Toulouse, France Laurent Mosoni Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Isabelle Papet Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Stephen D. Patterson St Mary’s University, Twickenham, London, United Kingdom

LIST OF CONTRIBUTORS

xiii

Bente K. Pedersen The Centre of Inflammation and Metabolism and The Centre for Physical Activity Research, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Joaquı´n Pe´rez-Schindler Biozentrum, University of Basel, Basel, Switzerland Marie-Agne`s Peyron Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, Clermont-Ferrand, France Stuart M. Phillips Department of Kinesiology, McMaster University, Hamilton, ON, Canada Sergio Polakof Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Maria P. Portillo CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Spain; Nutrition and Food Sciences Department, Faculty of Pharmacy, University of the Basque Country, Paseo de la Universidad 7, 01006, VitoriaGasteiz, Spain Geraldine Quintero-Platt Internal Medicine Service, University Hospital of the Canary Islands, University of La Laguna, Tenerife, Canary Islands, Spain Didier Re´mond Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France Lucı´a Romero-Acevedo Internal Medicine Service, University Hospital of the Canary Islands, University of La Laguna, Tenerife, Canary Islands, Spain Andrea P. Rossi Department of Medicine, Geriatric Division, University of Verona, Verona, Italy Sofia Rubele Department of Medicine, Geriatric Division, University of Verona, Verona, Italy Roxana E. Ruiz Valenzuela Departamento de Salud, Universidad Iberoamericana, Ciudad de Me´xico-Tijuana, Tijuana, Baja California, Me´xico Francisco Santolaria-Ferna´ndez Internal Medicine Service, University Hospital of the Canary Islands, University of La Laguna, Tenerife, Canary Islands, Spain Isabelle Savary-Auzeloux Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France David Scott Department of Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia; Department of Medicine—Western Health, Australian Institute for Musculoskeletal Science, The University of Melbourne, St Albans, VIC, Australia Gordon I. Smith Center for Human Nutrition, Division of Geriatrics and Nutritional Science, Washington University, St. Louis, MO, United States Matthew J. Sorrentino Section of Cardiology, University of Chicago Medicine, Chicago, IL, United States Nobuhiko Tachibana Research Institute for Creating the Future, Fuji Oil Holdings Inc., Osaka, Japan Mark Waldron St Mary’s University, Twickenham, London, United Kingdom; University of New England, Armidale, NSW, Australia Ste´phane Walrand Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France

xiv

LIST OF CONTRIBUTORS

Satoshi Wanezaki Research Institute for Creating the Future, Fuji Oil Holdings Inc., Osaka, Japan Kazumi Yagasaki Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya, Tochigi, Japan; Department of Applied Biological Chemistry, Tokyo University of Agriculture and Technology, Tokyo, Japan Mauro Zamboni Department of Medicine, Geriatric Division, University of Verona, Verona, Italy

C H A P T E R

1

Skeletal Muscle Mass Indices in Healthy Adults Heliodoro Alema´n-Mateo1 and Roxana E. Ruiz Valenzuela2 1

Departamento de Nutricio´n y Metabolismo, Coordinacio´n de Nutricio´n, Centro de Investigacio´n en Alimentacio´n y Desarrollo (CIAD), A.C. Hermosillo, Sonora, Me´xico 2 Departamento de Salud, Universidad Iberoamericana, Ciudad de Me´xico-Tijuana, Tijuana, Baja California, Me´xico

INTRODUCTION Skeletal muscle (SM) increases during postnatal development through a process of hypertrophy of the individual muscle fibers; a similar process may be induced in adult SM in response to contractile activity like strength exercises, and to androgens and β-adrenergic agonists [1]. SM remains relatively constant during the third and fourth decades of life but begins to decline at B45 years of age in both genders [2]. In other words, age exerts a strong influence on SM, but gender does as well [3 8]. Several studies have demonstrated the effects of age and gender on SM, and some have examined the effects on its distribution [2,5 6]. Perhaps the most relevant study on this issue was published by Janssen et al. [2], who used an in vivo method to study body composition using, particularly, magnetic resonance imaging (MRI) to provide precise and reliable measurements of SM and its distribution in a broad sample of Caucasian men and women. The results of this study reported new findings on the behavior of SM mass during the lifecycle in both men and women subjects. Due to the growing awareness of the importance of SM on functionality and other clinical entities in older age groups [9 16], there is a need to establish reference values for relative SM in young adult populations using the most precise and accurate methods available, and considering the factors of age, gender, and ethnicity. It is important to stress that few countries have been able to conduct national-level studies of this kind [10,17 19]. Some published works based on small samples of healthy young adult subjects reported data on SM [2,6,20 21] and proposed cutoff points for diagnosing sarcopenia—i.e., low SM mass—and sarcopenia syndrome [9,10,17,22 42]. To our knowledge, 21 cutoff points

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00001-4

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© 2019 Elsevier Inc. All rights reserved.

4

1. SKELETAL MUSCLE MASS INDICES IN HEALTHY ADULTS

based on SM exist; specifically, 17 dual-energy X-ray absorptiometry (DXA)-derived appendicular skeletal muscle mass (ASM) indices, 3 DXA-derived total SM (TSM) indices, and one DXA-derived total lean body mass (LBM) index for younger adult populations around the world [9,17,22 36], together with seven indices derived from bioelectrical impedance analysis (BIA) [10,37 42], one that used an ultrasound technique [43], and one MRI-derived TSM index (TSMI) [19]. Before presenting the main results of the published ASM, TSM, and LBM indices, we will first review the biological bases that underlie them.

THE BIOLOGICAL BASES THAT UNDERLIE THE INDICES Indices are association measurements that can be very useful in classifying nutritional status or, as in the present case, the stadia of SM, evaluating the chronicity of the skeletal muscle loss and assessing the efficacy of nutrition on loss of SM during the intervention therapy. They may also serve to quantify various components of body mass. Indices of this kind are usually divided into (1) those relative to weight and height and (2) those relative to body composition. SM indices are the ones most closely related to body composition that are used to diagnose presarcopenia and sarcopenia syndrome in geriatric populations. As mentioned above, SM tissue is dependent on age, gender, body weight, height, and ethnicity [2,5 6]. It is important to note that much of our current understanding of SM is based on studies that used DXA and MRI, two in vivo methods for analyzing body composition [2,5 6] that can accurately measure total or regional SM. DXA- derived lean tissue in arms and legs or ASM account for .75% of total SM [44], which constitutes the primary portion of SM involved in ambulation, physical activities, and functionality across the lifespan. To clarify the origin of an index, particularly ASM, we examined the main findings reported by Gallagher et al. [6], who assessed SM components by DXA in 148 healthy adult women (80 African-Americans, 68 Caucasians) and 136 healthy adult men (72 African-Americans, 64 Caucasians) with an age range of 21.1 67.5 years. First, they noted a significant negative correlation between ASM and age in all four groups. After adjusting for age, they determined that ASM was significantly and positively correlated with body weight and height in both ethnic groups, and in both men and women. Using multiple regression models, they then explored the independent effects of height, weight, age, gender, and ethnicity on ASM. In their predicted ASM model, height and weight explained 64% and 67%, respectively, of ASM variance in the African-American and Caucasian women, and 63% and 39%, respectively, in the African-American and Caucasian men. Smaller contributions were found for age. Interestingly, after adjusting for height, body weight, and age, an effect of gender was also found, as the men were found to have greater ASM than the women in both groups of subjects across the entire age range. Finally, a significant effect of ethnicity on ASM was found, as the African-American men and women had greater adjusted ASM than the Caucasian subjects [6]. Three years later, Janssen et al. [2] examined the influence of age, gender, body weight, and height on TSM assessed by whole body MRI in a large, heterogeneous sample of 468 men and women aged 18 88 years. It is important to mention that this study included two additional ethnic groups—Asians and Hispanics—in assessing TSM and its distribution [2]. As in the case of Gallagher et al. [6], these researchers also reported a significant

I. GENERAL ASPECTS: SKELETAL MUSCLE PHYSIOLOGY AND NUTRITION

THE BIOLOGICAL BASES THAT UNDERLIE THE INDICES

5

effect of height, body weight, age, and gender on total and regional or appendicular SM. In another significant finding related to this chapter, Janssen et al. [2], demonstrated that the substantial increase in body weight observed between 18 and 40 years of age was not associated with a corresponding increase in TSM. Instead, the absolute quantity of TSM was maintained into the fifth decade of life, but with noticeable losses thereafter. This finding is important so as not to limit the cutoff points for SM mass indices for people aged 18 40 years, as was originally proposed in Rosseta’s study [6], and the body composition reference values from National Health and Nutrition Examination Survey [17] or Korean studies [29,33 34]. Numerous body composition indices have been generated by considering the biological and statistical associations among certain aspects of body composition with age, and specific anthropometric variables; especially, the strong correlation between absolute SM and height. Originally, in order to define low SM mass or sarcopenia, it was necessary to have a measure related to the amount of existing SM [9], and as previously mentioned, the absolute values of SM correlates strongly with height. Therefore, ASM (kg) divided by height squared (ht2) produces an index of relative appendicular SM mass that serves as an indicator of muscularity. In fact, Baumgartner et al. [9] established that placing ht2 in the denominator constituted the best common power for minimizing the correlation of the index with height across all gender, ethnic and age groups, and study populations, since adjusting TSM or ASM for size eliminates the differences in the amount of SM associated with the greater height of young adults, as well as those related to gender and ethnicity. The resulting index permits better comparisons than absolute values of SM or any other parameter of body composition. SM has also been adjusted by body weight following the same principle as adjustment by ht2. Following Janssen et al. [10], SM adjusted by body weight considered adjustments for both stature and the mass of non-SM tissues (i.e., fat, organs, and bones) under the assumption that most mobility tasks and daily living activities are strongly influenced by body size. In older people, in addition to the adjustment by height, SM has also been adjusted by fat mass. Originally, it was hypothesized that SM adjusted by height and fat mass would better identify subjects with sarcopenia or with low SM than an index adjusted only for ht2 and that individuals so identified as such might be at even greater risk for poor lower extremity functioning [45]. With respect to the adjustments of absolute SM values by ht2, body weight, fat mass, or body mass index, a debate is ongoing as to which of these different indices is the best SM parameter and, therefore, the one that should be included in the criteria to define sarcopenia. Based on their results, Han et al. [42] proposed using absolute SM adjusted by height, because it correlated more closely with grip strength and with better muscular function than SM adjusted by body weight. Therefore, they recommended utilizing SM adjusted by height to diagnose sarcopenia syndrome. To the best of our knowledge, no established cutoff points adequately identify the level at which relative SM becomes deficient or low SM. In the most recent international consensus on the operational definition of presarcopenia and sarcopenia syndrome by the European Working Group on Sarcopenia in Older People (EWGSOP) [46], the International Working Group on Sarcopenia (IWGS) [47], and the Asian Working Group for Sarcopenia (AWGS) [48], the cutoff values for each gender were redefined,

I. GENERAL ASPECTS: SKELETAL MUSCLE PHYSIOLOGY AND NUTRITION

6

1. SKELETAL MUSCLE MASS INDICES IN HEALTHY ADULTS

respectively, as values one or two standard deviations (SD) below the gender-specific mean values of the reference data of a healthy young adult population [9 10,46 48]. The usefulness of these criteria and their corresponding cutoff points is based on a normal distribution which assumes that 68% of cases would fall within one standard deviation from the mean, and that 95% of all cases would fall within two standard deviations. As a result, the ASM index (ASMI) not only adjusts ASM according to height but also takes into account the lower correlation between height and ASM; this means, a deficit, expressing low appendicular SM mass or presarcopenia, or an excess, suggestive of high appendicular lean tissue or muscularity, expressed as high ASM. Finally, this criterion (22 SD of the mean values of the gender ethnic-specific SM indices from a young adult population) is the one most often used and associated with impaired physical performance and physical disability in geriatric populations [9 10].

DIFFERENT SKELETAL MUSCLE INDICES GENERATED WORLDWIDE Currently, sarcopenia syndrome is characterized by low SM plus low muscle strength or low physical performance [46 48]. To establish low SM, some researchers and different international consensus on sarcopenia (EWGSOP, IWGS, AWGS) have considered some indices based on measurements of ASM or TSM—determined by several methods—in a sample of a healthy young adults in the age range within which muscle reaches a plateau and then remains relatively constant (18 40 years). Computed tomography (CT) and MRI are considered the gold standards and the most accurate imaging methods for assessing SM, muscle cross-sectional area, and muscle quality [49]. However, due to the high cost and operational complexity of these methods, other imaging techniques, such as DXA and peripheral quantitative CT, have been recommended as accurate tools for assessing lean tissue or SM in both clinical and research contexts [50]. More recently, BIA technique has been used to estimate SM in clinical, research, and epidemiologic settings [51 52]. Estimates can be obtained by using BIA-derived TSM or ASM; their accuracy could depends on the methodology used to generate the body composition estimates [51 52] and the precision and validity of the equations. In the case of DXA-derived appendicular lean tissue, it is assumed that the entity measured represents limb SM mass or ASM with the additional assumption that limb SM mass represents 75% of the TSM. But DXA can also be used to estimate TSM by applying a correction factor of 1.33 [44]. Several cutoff points derived from samples of young adult populations have been obtained using the ASM, LBM, and TSM measured by DXA or BIA techniques (Tables 1.1 and 1.2). Recently, other cutoff points based on ASM or TSM measurements using old, new, and standard reference methodologies—including anthropometry in younger and older people [21,53], ultrasound in adults, and CT [54] and MRI [19] in subjects over a broad age range (18 78 years)—have been reported. Using specific cutoff points for different population sectors will enable researchers to more accurately assess the prevalence of sarcopenia across countries [49]. It has also been argued that applying gender- and ethnicspecific body composition predictive bioelectrical impedance or anthropometric equations,

I. GENERAL ASPECTS: SKELETAL MUSCLE PHYSIOLOGY AND NUTRITION

TABLE 1.1 Indices and Several Cutoff Points Based on DXA-derived ASM, TSM, and LBM in Young Adults From Different Regions of the World for Diagnoses of Sarcopenia in Older Adult Populations Country

n

Age, Years

Skeletal Muscle Indices

Male

Female

DXA

United States

229

18 40 ASM/ht2

7.26 kg/m2

5.45 kg/m2

Lunar, DPX, Madison, WI

96

18 50 LBM/ht2

15.4 kg/m2

11.7 kg/m2

QDR 2000

TSM/ht

2

558

20 25 ASM/ht2

Denmark 754

2

9.0 kg/m

2

6.0 kg/m

6.19 kg/m2

2

Software

Authors Baumgartner et al. [9]

Version 5.67

Melton III et al. [22]

Version 12.1

Kelly et al. [17]

Version V8.10a:33.1 and 3.2

Tanko´ et al. [23]

Hologic, Waltham, MA

4.73 kg/m2

QDR-4500A Hologic, Waltham, MA

19 39 ASM/ht

5.4 kg/m

2

QDR-4500A Hologic, Waltham, MA, o´ Lunar, DPX, Madison, WI

China

111

20 39 ASM/ht2

7.4 kg/m2

6.4 kg/m2

QDR 2000

TSM/ht2

8.5 kg/m2

9.9 kg/m2

Hologic, Waltham, MA

5.85 kg/m2

4.23 kg/m2

QDR-4500A

179

18 39 ASM/ht2

865

2

Lau et al. [24]

Wen et al. [28]

Hologic, Waltham, MA

Korea

145 70

18 40 ASM/ht

20 40 ASM/ht2 ASM/ht2

2513 20 39 ASM/ht

2

6538 18 39 ASM/ht2

2

2

Class I: 7.01 kg/m , Class 2: 5.42 kg/m2

Class I: 6.08 kg/m , Class 2: 4.79 kg/m2

Hologic Delphi A

7.4 kg/m2

5.14 kg/m2

Hologic Discovery A, Hologic; Bedford, MA

Kim et al. [25]

7.09 kg/m2

5.27 kg/m2

Lunar Madison, WI

Lim et al. [27]

2

2

Cheng et al. [31]

Hologic Inc.

Class I: 7.50 kg/m , Class 2: 6.58 kg/m2

Class I: 5.38 kg/m , Class 2: 4.59 kg/m2

Hologic Discovery-W, Hologic

Kim et al. [29]

6.09 kg/m2

4.09 kg/m2

QDR-4500

Kim et al. [33]

AM, Hologic, Waltham, MA 3338 30 40 ASM/ht2

4.4 kg/m2

QDR-4500

Kwon et al. [34]

Hologic Bedford, MA

(Continued)

TABLE 1.1 (Continued) Country

n

Taiwan

100

Italy Mexico

216

Age, Years

Skeletal Muscle Indices

Male

Female

DXA

20 40 ASM/ht2 TSM/w%

7.27 kg/m2 # 37.4%

5.44 kg/m2 # 28.0%

Prodigy; Lunar, Madison, WI and DPX-NT, Madison, WI

20 39 ASM/ht2

7.59 kg/m2

5.47 kg/m2

Hologic QDR 4500 W

2

5.86 kg/m

2

4.72 kg/m

2

2

2

5.22 kg/m

2

18 39 ASM/ht TSM/ht

Taiwan

6.6 kg/m

506

20 40 ASM/ht2

6.39 kg/m2

4.84 kg/m2

529

2

2

2

Software

Authors Lee et al. [36]

8.2

Coin et al. [30]

Lunar, DPX, Madison, WI

Alema´n and Ruiz [32]

Lunar DPX

Meng et al. [35]

General Electric, Madison, WI Japan

18 40 ASM/ht

6.87 kg/m

5.46 kg/m

QDR-4500

version 11.2:3

Sanada et al. [26]

Hologic, Waltham, MA DXA, dual-energy X-ray absorptiometry; ASM, appendicular skeletal muscle mass; TSM, total skeletal muscle mass (1.33*ASM); LMB, lean body mass; ht2, height square; w%, weight, kg x 100; Class I, ,1 SD; Class II, ,2 SD of skeletal muscle indices (ASM, TSM, LBM).

TABLE 1.2 Indices and Several Cutoff Points Based on BIA-derived ASM and TSM in Young Adults From Different Regions of the World for Diagnoses of Sarcopenia in Older Adult Population Age, Years

Country

n

United States

6414 18 39

Taiwan

200

Skeletal Muscle Indices TSM/w%

18 40

TSM/ht2

18 39

2

Male

Female

BIA

Frequency

Authors

Class I: # 31% 37%

Class I: # 22% 28%

1990B

Single frequency

Janssen et al. [10]

Class II: # 30%

Class II: # 21%

8.87 kg/m2

6.42 kg/m2

Single frequency

Chien et al. [37]

Valhalla Medical, San Diego, CA

920 Maltron BioScan, RaYLEIGHT, UK

782

1000 20 40

145

20 40

2

2

TSM/ht

8.6 kg/m

6.2 kg/m

ASM/ht2

6.76 kg/m2

5.28 kg/m2

2

2

2

TSM/ht

7.75 kg/m

5.67 kg/m

TSM/w%

# 30.4%

# 25.8%

2

2

Impedimed Multifrequency analyzer, Brisbane, Australia

Multifrequency Tichet et al. [38]

BC-418

Single frequency

Tanita Corp., Tokyo, Japan

TSM/ht

7.4 kg/m

5.14 kg/m2

720BIA

TSM/w%

# 35.70%

# 30.70%

InBody

Chang et al. [39]

Multifrequency Han et al. [42]

Biospace, Seoul, Korea Spain

230

20 40

2

8.25 kg/m

6.68 kg/m

2

2

2

TSM/ht

2

2

101 RJL Systems

Turkey

301

18 39

TSM/ht

9.2 kg/m

7.4 kg/m

BC532 Tanita Corp., Tokyo, Japan

Single frequency

Masane´s et al. [40]

Single frequency

Bahat et al. [41]

BIA, bioelectrical impedance analysis; ASM, appendicular skeletal muscle mass; TSM, total skeletal muscle mass (1.33*ASM); ht2, height square; Class I, ,1 SD; Class II, ,2 SD of total skeletal muscle; w%, weight, kg x 100.

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1. SKELETAL MUSCLE MASS INDICES IN HEALTHY ADULTS

or gender- and ethnic-specific cutoff points, improves the accuracy of body composition estimates and diagnoses of sarcopenia [49]. On the topic of generating SM indices, Alema´n-Mateo and Ruiz Valenzuela [32] recently published significant differences between the cutoff points based on DXA-derived SM indices in a young adult Mexican population with the cutoff points for a population of young male and female adult Caucasians from the United States and other ethnic groups. It was suggested that the indiscriminate use of published indices could produce biased results regarding the prevalence of low SM mass (presarcopenia) and sarcopenia syndrome in older people. Similar differences had been reported previously; for example, cutoff points for ASM for American females differed significantly from those for Italian women [55]. As mentioned previously, there are some BIA-derived SM indices. The usefulness and validity of the BIA technique for estimating SM is based on the relation between the volume of a conductor and its electrical resistance; there is a strong correlation between BIA resistance and SM measurements in the arms [56 57] and legs [57 58], and validity depends upon the criterion method for measuring SM. There are seven BIA-derived SM indices (ASMI and TSMI) generated in young adult populations from around the world, but it is important to note that most were derived from TSM measurements of young adult populations. The absolute values for BIA-derived TSM have also been adjusted by both height and body weight, while BIA-derived ASM have been adjusted only by height (Table 1.2).

CLINICAL IMPLICATIONS OF THE INDISCRIMINATE USE OF DIFFERENT PUBLISHED INDICES Currently, despite advances in defining and establishing diagnostic criteria for sarcopenia by the EWGSOP [46], IWGS [47], and AWGS [48], no universal consensus has been reached as to which criteria and method can best be used to measure SM, and which cutoff points should be applied to identify sarcopenia syndrome. This situation generates serious problems in determining the prevalence, effectiveness of clinical treatments, and prevention. With respect to the various published indices, their indiscriminate use in older adult populations from other countries or ethnic groups could result in over- or underestimations in the prevalence of sarcopenia in older people. Recently, a systematic review of the literature by Pagotto and Silveira [59] concluded that the heterogeneity in the prevalence of sarcopenia was due to the diagnostic method and cutoff points chosen and the characteristics of both the study and the reference populations. On the specific question regarding cutoff points, these authors found that applying the ASMI classification criteria to other populations (n 5 18) resulted in sarcopenia prevalence ranged from 0.0% to 56.7% in men and from 0.0% to 33.9% in women. They sustain that these results can be attributed to racial characteristics and emphasize the effects of physical characteristics and cultural aspects that entail different levels of physical activity, distinct dietary regimes, and varying levels of quality of life among older adult populations in different countries. For example, the low prevalence encountered among a Chinese study group led the authors to conclude that ASMI is not an appropriate method for diagnosing sarcopenia in this specific population. The cutoff points for the Chinese population are lower than for Americans (,5.72 versus 7.26 kg/m2 in men; ,4.82 versus ,5.45 kg/m2 in women), with mean values derived from a young people of the same I. GENERAL ASPECTS: SKELETAL MUSCLE PHYSIOLOGY AND NUTRITION

CLINICAL IMPLICATIONS OF THE INDISCRIMINATE USE OF DIFFERENT PUBLISHED INDICES

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ethnic group were used as references. The mean ASMI of young Asians was approximately 15% lower than that of Caucasians, even after adjusting for height. Therefore, low muscle mass in young Asians will result in lower prevalence of sarcopenia in the elderly [59]. Around the time of the Pagotto and Silveira [59] publication, and as mentioned before we reported significant differences between the cutoff points determined by DXA-derived ASMI and TSMI from young adult subjects from different parts of the world and those derived from a group of young Mexican adult men and women [32]. More recently, Masanes et al. [60] assessed how changes in cutoff points for muscle mass, gait speed, and grip strength affected the prevalence of sarcopenia according to EWGSOP, 2010 criteria [46]. They found an increase from 5.45 to 6.68 kg/m2 in the SM index for female outpatients and nursing-home residents, together with an increase in the prevalence of sarcopenia from 4% to 23% and 9% to 47%, respectively; the corresponding increases for men were 7.25 8.87 kg/m2, and the prevalence of sarcopenia from 1% to 22% and 6% to 41%, respectively. Those authors further determined that changes in gait speed and grip strength had only a limited impact on the prevalence of sarcopenia. Their conclusion was that the cutoff points used for SM affect reported prevalence rates for sarcopenia, and as a result, interstudy comparability. Finally, according to their work, the main factors that influence the magnitude of change are the distribution of the SM index in the population and the absolute value of the cutoff points used, since the same difference between two references (e.g., 7.5 7.75 or 7.75 8 kg/m2) may produce variations in the prevalence [60]. In addition to gender, ethnicity, height, and body weight, other factors may also significantly affect SM mass or its indices and, therefore, the reported prevalence of sarcopenia. Tyrovolas et al. [61] have shown that lower levels of education and wealth, higher percentages of body fat, current consumption of alcoholic beverages, and having one chronic condition were all significantly associated with lower estimated SM indices. Importantly, higher percentages of body fat were consistently associated with lower estimated SM indices in all countries included in their study. This multicontinent study confirms the need for specific cutoff points for the ASM or TSM indices in the reference values derived from younger adult populations in order to ensure accurate diagnoses of low SM (presarcopenia) and sarcopenia syndrome [61]. Finally, these distinct indices of SM mass have been associated with other nutritionalrelated diseases or clinical entities, including vertebral hip fractures in women and hip and ankle fractures in older male Chinese subjects [13], while in elderly Korean people they have been related to bone mineral density and osteoporosis [62]. In Italy, meanwhile, low appendicular lean mass has been significantly associated with low bone mineral density in women with hip fractures [63]. Other studies have reported a significant association of low ASMI and obesity with insulin resistance, metabolic syndrome, and risk factors for cardiovascular disease [64]. There is also a recent report that weight-adjusted ASM was able to identify cardiometabolic risk factors such as triglycerides and high-density lipoprotein cholesterol, while height-adjusted ASM could identify factors for osteoporosis [65]. These findings highlight the relationship between SM indices and the risk of certain lifestyle-related diseases and clinical entities, though it is not yet clear what the best method or anthropometric variable for normalizing muscle mass might be. For this reason, cohort studies are required to clarify these associations by going beyond the classic association of SM indices with impaired physical performance and physical disabilities in geriatric populations. I. GENERAL ASPECTS: SKELETAL MUSCLE PHYSIOLOGY AND NUTRITION

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1. SKELETAL MUSCLE MASS INDICES IN HEALTHY ADULTS

CONCLUSIONS There are several published cutoff points for TSMI and ASMI based on SM measurements by DXA and BIA for diagnoses of sarcopenia. It seems that the use of specific indices for different ethnic populations improves the accuracy of diagnoses of sarcopenia and decreases estimates of high prevalence generated by applying cutoff points derived from other populations. Based on the evident bias in diagnoses of the prevalence of sarcopenia produced by using nonethnic-specific reference values, the main challenge is to ensure that SM is measured by accurate and precise methods or estimated by valid BIA or anthropometric equations in young adult populations. These more accurate SM indices should be apply to estimate the prevalence of sarcopenia and other new clinical entities in geriatric populations. Finally, in the continuing absence of a universal consensus as to the best criteria and cutoff points available, using distinct cutoff points for specific ethnic groups is advisable for diagnosing sarcopenia, treating this condition, and preventing it in the growing older adult population worldwide.

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[37] Chien M-Y, Huang T-Y, Wu Y-T. Prevalence of sarcopenia estimated using a bioelectrical impedance analysis prediction equation in community-dwelling elderly people in Taiwan. J Am Geriatr Soc. 2008;56(9):1710 15. [38] Tichet J, Vol S, Goxe D, Salle A, Berrut G, Ritz P. Prevalence of sarcopenia in the French senior population. J Nutr Health Aging 2008;12(3):202 6. [39] Chang C-I, Chen C-Y, Huang K-C, Wu C-H, Hsiung CA, Hsu C, et al. Comparison of three BIA muscle indices for sarcopenia screening in old adults. Eur Geriatr Med. 2013;4(3):145 9. [40] Masane´s F, Culla A, Navarro-Gonza´lez M, Navarro- Lopez M, Sacanella E, Torres B, et al. Prevalence of sarcopenia in healthy community-dwelling elderly in an urban area of Barcelona (Spain). J Nutr Health Aging. 2012;16(2):184 7. [41] Bahat G, Tufan A, Tufan F, Kilic C, Akpinar TS, Kose M, et al. Cut-off points to identify sarcopenia according to European Working Group on Sarcopenia in Older People (EWGSOP) definition. Clin Nutr 2016;35 (6):1557 63. [42] Han D, Chang K, Li C, Lin Y, Kao T. Skeletal muscle mass adjusted by height correlated better with muscular functions than that adjusted by body weight in defining sarcopenia. Sci Rep. 2016;6:1 8. [43] Abe T, Thiebaud RS, Loenneke JP, Loftin M, Fukunaga T. Prevalence of site-specific thigh sarcopenia in Japanese men and women. Age (Dordr). 2014;36(1):417 26. [44] Snyder WS, Cook CMJ, Nasset ES, Karhausen LR, Parry G, Tipton HIH, et al. Report of the task group on reference man. 1992. [45] Newman AB, Kupelian V, Visser M, Simonsick E, Goodpaster B, Nevitt M, et al. Sarcopenia: alternative definitions and associations with lower extremity function. J Am Geriatr Soc. 2003;51(11):1602 9. [46] Cruz-Jentoft AJ, Baeyens JP, Bauer JM, Boirie Y, Cederholm T, Landi F, et al. Sarcopenia: European consensus on definition and diagnosis: report of the European Working Group on Sarcopenia in Older People. Age Ageing. 2010;39(4):412 23. [47] Fielding RA, Vellas B, Evans WJ, Bhasin S, Morley JE, Newman AB, et al. Sarcopenia: an undiagnosed condition in older adults. Current consensus definition: prevalence, etiology, and consequences. International Working Group on Sarcopenia. J Am Med Dir Assoc. 2011;12(4):249 56. [48] Chen LK, Liu LK, Woo J, Assantachai P, Auyeung TW, Bahyah KS, et al. Sarcopenia in Asia: consensus report of the Asian Working Group for Sarcopenia. J Am Med Dir Assoc. 2014;15(2):95 101. [49] Visser M, Schaap L. Measurements of muscle mass, equations and cut-off points, in sarcopenia. In: CruzJentoft AJ, Morley JE, editors. Sarcopenia. Chichester, UK: John Wiley & Sons, Ltd; 2012. p. 205 25. [50] Erlandson MC, Lorbergs AL, Mathur S, Cheung AM. Muscle analysis using pQCT, DXA and MRI. Eur J Radiol. 2016;85(8):1505 51. [51] Janssen I, Heymsfield SB, Baumgartner RN, Ross R. Estimation of skeletal muscle mass by bioelectrical impedance analysis. J Appl Physiol. 2000;89(2):465 71. [52] Kyle UG, Genton L, Hans D, Pichard C. Validation of a bioelectrical impedance analysis equation to predict appendicular skeletal muscle mass (ASMM). Clin Nutr. 2003;22(6):537 43. ´ ngel B, Sa´nchez H, Picrin Y, Hormazabal MJ, Quiero A, et al. Validation of cut points of skeletal [53] Lera L, A muscle mass index for identifying sarcopenia in Chilean older people. Nutr Hosp. 2014;31(3):1187 97. [54] Hamaguchi Y, Kaido T, Okumura S, Kobayashi A, Hammad A, Tamai Y, et al. Proposal for new diagnostic criteria for low skeletal muscle mass based on computed tomography imaging in Asian adults. Nutrition. 2016;32(11 12):1200 5. [55] Zoico E, Di Francesco V, Guralnik JM, Mazzali G, Bortolani A, Guariento S, et al. Physical disability and muscular strength in relation to obesity and different body composition indexes in a sample of healthy elderly women. Int J Obes Relat Metab Disord. 2004;28(2):234 41. [56] Brown BH, Karatzas T, Nakielny R, Clarke RG. Determination of upper arm muscle and fat areas using electrical impedance measurements. Clin Phys Physiol Meas. 1988;9(1):47 55. [57] Nun˜ez C, Gallagher D, Grammes J, Baumgartner R, Ross R, Wang Z, et al. Bioimpedance analysis: potential for measuring lower limb skeletal muscle mass. J Parenter Enter Nutr. 1999;23(2):96 103. [58] Pietrobelli A, Morini P, Battistini N, Chiumello G, Nun˜ez C, Heymsfield SB. Appendicular skeletal muscle mass: prediction from multiple frequency segmental bioimpedance analysis. Eur J Clin Nutr. 1998;52 (7):507 11. [59] Pagotto V, Silveira EA. Methods, diagnostic criteria, cutoff points, and prevalence of sarcopenia among older people. ScientificWorldJournal. 2014;2014:231312.

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[60] Masane´s F, Rojano LX, Salva` IA, Serra-Rexach JA, Artaza I, Formiga F, et al. Cut-off points for muscle mass not grip strength or gait speed - determine variations in sarcopenia prevalence. J Nutr Health Aging 2017;21 (7):825 9. [61] Tyrovolas S, Koyanagi A, Olaya B, Ayuso-Mateos JL, Miret M, Chatterji S, et al. Factors associated with skeletal muscle mass, sarcopenia, and sarcopenic obesity in older adults: a multi-continent study. J Cachexia Sarcopenia Muscle. 2016;7(3):312 21. [62] Kim S, Won CW, Kim BS, Choi HR, Moon MY. The association between the low muscle mass and osteoporosis in elderly Korean people. J Korean Med Sci. 2014;29(7):995. [63] Di Monaco M, Castiglioni C, Di Monaco R, Tappero R. Association between low lean mass and low bone mineral density in 653 women with hip fracture: does the definition of low lean mass matter? Aging Clin Exp Res. 2017;3:1 6. [64] Chung J-Y, Kang H-T, Lee D-C, Lee H-R, Lee Y-J. Body composition and its association with cardiometabolic risk factors in the elderly: a focus on sarcopenic obesity. Arch Gerontol Geriatr. 2013;56(1):270 8. [65] Furushima T, Miyachi M, Iemitsu M, Murakami H, Kawano H, Gando Y, et al. Comparison between clinical significance of height-adjusted and weight-adjusted appendicular skeletal muscle mass. J Physiol Anthropol. 2017;36(1):15.

I. GENERAL ASPECTS: SKELETAL MUSCLE PHYSIOLOGY AND NUTRITION

C H A P T E R

2

Reduced Skeletal Muscle Mass and Lifestyle 1

David Scott1,2

Department of Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia 2Department of Medicine—Western Health, Australian Institute for Musculoskeletal Science, The University of Melbourne, St Albans, VIC, Australia

From around the fifth decade of life, adults lose approximately 1 2 kg of skeletal muscle mass per decade [1]. Age-related declines in muscle mass occur due to reduced number and size of muscle fibers through processes of denervation, apoptosis, and atrophy [2]. In 1988, Irwin Rosenberg proposed the descriptor “sarcopenia” from the Greek sarx for “flesh” and penia for “loss” [3], but since then definitions of sarcopenia have evolved to also include poor muscle function [4,5]. Nevertheless, research has demonstrated that low muscle mass alone is associated with a range of health disorders including insulin resistance [6], cardiovascular disease [7], osteoporosis and fractures [8,9], as well as increased mortality [10]. Thus, strategies which can contribute to the maintenance of skeletal muscle during aging are likely to improve quality of life and longevity in the older adult population. The plasticity of skeletal muscle to hypertrophic stimuli diminishes with age, but there are still opportunities to implement strategies to maintain muscle mass [11]. Lifestyle behaviors represent an attractive target because unlike other contributors to age-related muscle wasting, such as genetic and neuromuscular factors, they are inherently modifiable. Lifestyle modification strategies which can be implemented into public health recommendations can also potentially benefit a large proportion of the older adult population at relatively low costs compared with drug therapy interventions. This chapter summarizes current literature on associations of lifestyle behaviors and reductions in skeletal muscle mass during aging.

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00002-6

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© 2019 Elsevier Inc. All rights reserved.

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2. REDUCED SKELETAL MUSCLE MASS AND LIFESTYLE

PHYSICAL ACTIVITY Resistance training is the primary strategy recommended for older adults to maintain and improve both muscle mass and function [12]. A metaanalysis of 49 studies including over 1300 adults aged 50 years and older demonstrated that resistance training interventions result in mean improvements in lean body mass of around 1 kg, and this can be increased with higher volume [13]. Despite this, as few as 5% of adults over the age of 50 years meet guidelines for resistance training [14], suggesting that promotion is important in public health policies. However, aerobic exercise is also likely to be beneficial with master athletes (primarily runners, cyclists, and swimmers) who train 4 5 times per week demonstrating no significant declines in muscle with age [15]. Most observational studies explore associations between general physical activity (including both intentional and incidental movement) and muscle mass in older adults. Early studies utilized self-reported measurements of physical activity which are limited by recall bias but have demonstrated associations for self-reported physical activity, intensity of work activities, and performing exercise during leisure time, with higher muscle mass [16,17], and smaller declines in thigh girth over time [18]. Conversely, in a study of 337 US older adults, there was no association between self-reported physical activity and appendicular lean mass (the sum of lean mass in the arms and legs) [19], and odds for low appendicular lean mass normalized to height were not significantly decreased in participants of the New Mexico Elder Health Survey with high physical activity levels [20]. In Korean older men, likelihood for low appendicular lean mass normalized to body weight was 40% 70% lower in men with moderate and high levels of physical activity, but no such association was observed for women [21]. Furthermore, using the European Working Group on Sarcopenia in Older People (EWGSOP) definition of sarcopenia, which includes low gait speed and/or hand grip strength in addition to low relative appendicular lean mass [4], participants of the AGES-Reykjavik study who reported high levels of moderatevigorous physical activity (MVPA) had around 40% lower 5-year incidence of sarcopenia, although MVPA was not significantly associated with rate of muscle loss [22]. The different instruments used to assess self-reported physical activity are likely to explain conflicting associations with muscle mass in these studies. The advent of devices supporting objective assessments of physical activity has provided new opportunities to expand our understanding of its relationship with muscle mass in older adults. We previously reported that higher average steps per day counts, assessed by pedometers, are associated with higher leg lean mass over almost 3 years in older men and women of the Tasmanian Older Adult Cohort (TASOAC) study [23]. In the Nakanojo Study, a year-long assessment of physical activity using pedometers indicated positive effects of increasing steps/day on appendicular lean mass in older adults with a threshold at approximately 8000 and 6900 steps/day in men and women, respectively [24]. In TASOAC, accelerometers, which provide estimates of sedentary behavior and physical activity intensity by measuring accelerations (usually at the hip), demonstrated that sedentary time was associated with lower lean mass percentage. Furthermore, there was a dose response relationship for physical activity, with MVPA associated with higher lean mass percentage than light physical activity, although the magnitude of association was

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lowest in the oldest age group [25], consistent with the decreasing plasticity of skeletal muscle with aging. Similar results were observed in a UK study of almost 1300 older men which reported that each additional 30 minutes/day of accelerometer-determined MVPA was associated with almost half the risk of severe sarcopenia (defined as low mid-upper arm circumference, hand grip strength, and gait speed), but light physical activity and sedentary time were not significantly associated with sarcopenia [26]. In the Nakanojo Study, older adults who spent ,15 minutes/day at or above three accelerometer-determined metabolic equivalents, a level equivalent to moderate intensity physical activity, were more than twice as likely to have low appendicular lean mass relative to height compared with those who spent .23 minutes/day at this intensity [24]. Using ultrasound, Abe et al. also demonstrated that accelerometer-determined MVPA, but not light physical activity, was positively correlated with lower leg muscle thickness in older women [27]. Thus, objective assessments of physical activity generally demonstrate that MVPA is associated with greater muscle mass in older adults. While resistance training is likely to confer the greatest muscle mass improvements, minimizing sedentary behavior and increasing movement, particularly at higher intensities, should be a focus of guidelines for maintaining muscle mass during aging.

NUTRITION Dietary energy intake declines by up to 5000 kJ between the ages of 20 and 80 years [28]. This withdrawal of energy intake can contribute to weight loss which includes declines in skeletal muscle mass, and additional muscle mass and function deficits may occur due to inadequate intakes of specific macronutrients, micronutrients, and vitamins. Recent research has also focused on dietary patterns, which reflect how foods are consumed together, rather than as individual dietary components. Higher dietary variety is associated with greater mean arm muscle area and circumference in frail nursing home residents [29], and Chinese older adults with high scores on a diet quality index focusing on variety, adequacy, moderation, and overall balance of nutrition had half the likelihood of sarcopenia at baseline although not 4 years later [30]. In older Germans, adherence to a Mediterranean diet pattern was associated with greater appendicular lean mass relative to BMI and fat mass in women, but not men [31], and in the Helsinki Birth Cohort Study, adherence to the healthy Nordic diet was associated with better muscle strength, but not muscle mass [32]. To date, optimal dietary patterns for maintaining muscle mass during aging have not been thoroughly investigated, but future guidelines are likely to be informed by studies of specific nutrients. Adequate dietary protein intake is a key nutritional component for skeletal muscle because protein contains amino acids, including essential amino acids which cannot be synthesized in the body, required for muscle protein synthesis [33]. Due to blunted muscle protein synthesis during aging, older adults may need to exceed current recommendations of 0.8 g/kg/day of dietary protein [34] and leucine (a branched-chain essential amino acid) may be particularly important for muscle mass [35 37]. For example, in older adults randomized to receive a drink containing 2 3 g of β-hydroxy-β-methylbutyrate (HMB; a leucine metabolite), as well as L-arginine and L-lysine (also essential amino acids) for

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1 year, compared with those who received a nonessential amino acid drink, bioelectrical impedance analysis and dual-energy X-ray absorptiometry estimates of lean mass increased by 0.5 1 kg. Significant increases in whole-body protein turnover were also observed for the HMB/L-arginine/L-lysine group compared with controls at 12 months [38]. Nevertheless, in healthy older adults, there is limited evidence to suggest that protein or amino acid supplementation alone is beneficial for improving muscle mass [39]. Despite the mixed evidence for protein supplementation, longitudinal cohort studies support the beneficial role of higher dietary protein intakes in reducing muscle mass declines during aging. In the TASOAC study, older adults who reported failing to meet 0.8 g/kg/day of dietary protein had 0.8 kg lower appendicular lean mass than those who met or exceeded the recommendation, and baseline protein intakes were positively associated with change in lean mass over 3 years [40]. Older adults with dietary protein intakes in the lowest 20% in the US Health ABC Study also had around 40% greater decline in total and appendicular lean mass over 3 years [41], and in postmenopausal Australian women, baseline protein intake positively correlated with total and appendicular lean mass, up to 5 years later [42]. Protein sources may be an important consideration given meat contains higher amounts of essential amino acids; in the US Framingham Offspring Cohort, participants in the highest quartiles for total protein and animal protein had approximately 0.5 kg higher leg lean mass than those in the lowest quartiles, but leg lean mass did not differ across quartiles of plant protein intake [43] suggesting animal proteins are more beneficial for muscle mass in older adults. Distribution of protein consumption throughout the day may also influence muscle protein synthesis, with more even distributions across meals associated with higher lean mass in Canadian older adults [44]. Increased levels of inflammatory markers are associated with greater risk of sarcopenia [45]. Higher saturated and trans fatty acid intakes, associated with increased inflammation, were associated with lower fat-free mass, while the dietary ratio of polyunsaturated fatty acids (associated with decreased inflammation) to saturated fatty acids was positively associated with fat-free mass, in women in the TwinsUK study [46]. Omega-3 polyunsaturated fatty acids, primarily found in fish, plants, and nut oils, may enhance muscle mass by reducing inflammation and also by enhancing protein synthesis [2]. An 8-week trial in 16 older adults demonstrated that those randomized to receive omega-3 polyunsaturated fatty acids had increased muscle protein synthesis [47]. Similar effects have also been reported in a recent study of the effects of 16-weeks of omega-3 polyunsaturated fatty acids prior to and after a single bout of resistance exercise. Mixed muscle, mitochondrial, and sarcoplasmic protein synthesis rates increased prior to exercise, and mitochondrial and myofibrillar protein synthesis increased postexercise [48]. Six months of daily omega3 supplementation (equivalent to the omega-3 polyunsaturated fatty acid content of 200 400 g of freshwater fatty fish) has also resulted in a 4% increase in muscle thigh volume compared to control in older men and women [49]. Age-related increases in accumulation of reactive oxygen/nitrogen species (ROS/RNS), known as oxidative stress, are thought to contribute to muscle mass decline through proteolysis which shifts protein balance into deficit [50]. Higher intakes of fruits and vegetables have been postulated to protect against muscle mass losses because they are a major dietary source of antioxidants [51,52]. In the Korean National Health and Nutrition Examination Survey, older men in the highest quintile for fruit and vegetable intake had

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approximately 70% reduced likelihood of low appendicular lean mass relative to height and fat mass, and a similar effect was seen for women with high fruit intake [53]. Achieving the recommended intake of vegetables in women ($5 servings/day) was also associated with approximately half the likelihood for low muscle mass in the same cohort, although the effect of vegetable intake was not significant in men [54]. In Chinese older men, a high “vegetables fruits” dietary pattern was also associated with 40% lower likelihood of sarcopenia [30]. In terms of specific antioxidants, selenium and vitamins A, C, and E intakes appear to be lower in older adults with low muscle mass and physical function [55 57]. Other nutrients associated with higher fruit and vegetable intakes may also contribute to maintenance of muscle mass during aging. Potassium plays a key role in muscle contraction, and dietary and urinary potassium have been positively associated with lean mass and muscle strength in older adults [58,59], although not changes in lean mass over 3 years [40,58]. Dietary magnesium may influence muscle adenosine triphosphate, and also inflammation [60]. Baseline magnesium intakes were 6% lower in sarcopenic compared with nonsarcopenic older adults in the PROVIDE study despite similar total energy intakes [56]. In TASOAC, energy-adjusted magnesium intake was a positive predictor of maintenance of appendicular lean mass over 3 years [40], and in the TwinsUK study, women in the highest quintile of magnesium intake had 0.4 kg/m2 higher fat-free mass (normalized to height) than those in the lowest quintile; this effect size was seven times greater than that observed for higher protein intake [60]. Vitamin D, a secosteroid hormone produced in the epidermis following ultraviolet B light exposure and also obtained in smaller amounts from some foods, may have antiinflammatory properties [61]. Vitamin D deficiency is common in older adults due to lifestyle changes and age-dependent decreases in vitamin D metabolism [62]. In addition to indirect antiinflammatory effects, vitamin D may have direct effects on skeletal muscle as nuclear 1,25 vitamin D receptors (VDRs; which decrease with age) located in skeletal muscle may bind 1,25-dihydroxyvitamin D (1,25D; the active form of vitamin D) and promote protein synthesis [63]. Vitamin D may also augment mitochondrial oxidative phosphorylation in skeletal muscle [64]. Although VDR polymorphisms have been associated with low fat-free mass in older men [65], most research in this area focuses on 25-hydroxyvitamin D (25(OH)D) concentrations. 25(OH)D is produced in the liver and converted in the kidneys to 1,25D and levels less than 50 nmol/L are generally considered low and associated with increased falls and fracture risk through effects on muscle strength [66]. Effects on muscle mass are controversial however. Appendicular lean mass relative to body height was significantly lower in those with low 25(OH)D in the MINOS study of French middle-aged and older men [17], and in the Longitudinal Aging Study Amsterdam, odds for a loss of muscle mass greater than 3% over 3 years were around two times greater in older adults with low (,25 nmol/L) serum 25(OH)D levels, compared with participants with high ( . 50 nmol/L) 25(OH)D levels at baseline [67]. Conversely, in TASOAC, baseline 25(OH)D was a positive predictor of change in leg strength but not appendicular lean mass percentage over almost 3 years [68]. Dietary intakes of vitamin D have also been demonstrated to have no association with muscle mass in a cross-sectional older French women [69,70], although this may reflect the fact that relatively low proportions of total vitamin D are generally obtained from

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the diet. A recent analysis of the Concord Health and Aging in Men Project found that lower baseline levels of both 25(OH)D (,40 nmol/L) and the biologically active 1,25D (,62 pmol/L) are associated with over twofold increased odds of developing incident sarcopenia over the subsequent 5 years in men aged 70 years and older [71]. Given sarcopenia was defined in this study according to the Foundation for the National Institutes of Health definition (both low appendicular lean mass and low hand grip strength), it is not possible to determine whether the beneficial associations of 25(OH)D and 1,25D are attributable to effects on muscle mass and/or function. Consistent with the concept that vitamin D may benefit muscle function but not muscle mass, a metaanalysis of vitamin D supplementation studies reported a small but significant positive effect on muscle strength relative to placebo or control, but no effect on muscle mass [72]. Vitamin D may therefore exert indirect effects on muscle function that are not explained by muscle mass [73], through improvements in neuromuscular factors such as coordination [74]. However, a small 4-month randomized controlled trial (RCT) in mobility-limited older women reported a significant increase in type II muscle fiber crosssectional area in response to 4000 IU/day vitamin D3 compared with placebo [75], indicative of a benefit of vitamin D for muscle hypertrophy. A combination of nutritional supplements may be most effective in ensuring adequate dietary intakes and improving muscle mass in older adults. In PROVIDE, a 13-week double-blind, placebo-controlled randomized trial of 380 sarcopenic older adults, participants randomized to a twice-daily nutritional supplement containing 20 g whey protein, 3 g total leucine, 9 g carbohydrates, 3 g fat, and 800 IU vitamin D demonstrated a significant gain in appendicular lean mass of almost 0.2 kg compared with controls, and also improved more in a sit-to-stand physical function test [76]. Further clinical trials are required to confirm the optimal combinations of nutrients and dosing regiments, and it is likely that not all older adults, particularly those with adequate diets, demonstrate muscle hypertrophy due to supplementation alone. Indeed, a recent systematic review concluded that three out of four nutritional supplementation RCTs indicated a positive effect on physical function, but not muscle mass in older adults [77]. Nevertheless, combination supplements may be effective at minimizing multiple nutritional deficiencies which contribute to muscle mass declines in older adults over the long term.

COMBINING PHYSICAL ACTIVITY AND NUTRITION Given that adequate physical activity and nutrition are independently associated with improved muscle mass and function in older adults, combining these behaviors may be most beneficial. A recent metaanalysis concluded that, in healthy older adults, protein and amino acid supplementation may only improve muscle mass when combined with exercise [39]. Even moderate aerobic exercises may enhance muscle protein synthesis from protein supplementation [78], although in the US National Health and Nutrition Examination Survey (2003 06), significant positive associations for self-reported total beef and protein intakes with appendicular lean mass were observed only in middle-aged and older adults who reported participation in vigorous aerobic or muscle-strengthening exercise, rather than light intensity activity [79]. Adequate intakes of other nutrients in

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combination with physical activity may also be beneficial; women in the Korean National Health and Nutrition Examination Survey who achieved two, or three or more, healthy lifestyle behaviors (including adequate intake of meat, fish, eggs, and legumes; adequate intake of vegetables; adequate intake of fruits; sufficient moderate/vigorous intensity physical activity; and resistance exercise twice or more per week) had less than half the likelihood of low muscle mass compared with those who achieved no healthy behaviors [54]. Also, in over 600 community-dwelling older adults participating in TASOAC, participants who had high 25(OH)D and high physical activity levels at baseline lost significantly less appendicular lean mass (normalized to body weight) over 5 years, compared with those who had low vitamin D, low physical activity or low vitamin D, and low physical activity [80]. Factorial design RCTs are required to determine whether effects of exercise and nutrition interventions are synergistic (i.e., the combined benefits are greater than the sum of their individual benefits) [81], and few studies to date have been appropriately designed with adequate sample sizes. Based on the results of limited factorial RCTs, it is unclear whether benefits of combined protein supplementation and resistance training on muscle mass in older adults may be additive, rather than synergistic. In 155 Japanese women aged 75 years and older randomized to a 3-month exercise program and 3-g day of an amino acid supplement (containing 42.0% leucine, 14.0% lysine, 10.5% valine, 10.5% isoleucine, 10.5% threonine, 7.0% phenylalanine, and 5.5% other), exercise alone, amino acid supplement alone, or health education (control), a significant interaction was observed indicating greater leg muscle mass in the exercise plus supplement group compared with the health education group, although this improvement was not significantly greater than that observed for exercise or supplement alone groups [82]. Similarly, a 10-week factorial RCT in which obese older adults consuming a hypocaloric diet were randomized to a high vs low (1.3 g/kg/day vs 0.8 g/kg/day) with or without resistance training demonstrated no significant interaction effects, although fat-free mass increased significantly (0.6 kg) for the high protein plus training group only [83]. The majority of nonfactorial RCTs have investigated whether protein supplementation plus resistance training provides greater improvements in body composition and muscle function than resistance training alone. A recent metaanalysis of 49 trials in over 1800 adults not only reported significantly greater increases for fat-free mass and muscle cross-sectional area for resistance training when combined with protein intakes up to approximately 1.6 g/kg/day but also found that increasing age reduces these additive benefits [84]. Accordingly, some trials have not observed any effect of protein supplementation plus resistance training over resistance training alone for muscle mass in older adults [85,86]. Conversely, in female nursing home residents, 16 weeks of progressive resistance training combined with consumption 160 g of cooked lean red meat consumed 6 days per week resulted in 0.5 kg greater gains in total lean mass relative to progressive resistance training only [87]. In a 24-week study of frail older adults randomized to a twice-daily 250 mL protein drink (15 g protein) or placebo while completing resistance exercise, lean mass increased by 1.3 kg in the protein group but did not change in the placebo group [88]. Evidence is also lacking for a synergistic benefit of vitamin D (alone or in combination with protein) supplementation. A factorial RCT including 96 vitamin D deficient older adults randomized to 9 months of resistance training or no exercise, and to 400 IU/day

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per day of vitamin D plus calcium or calcium alone, observed no effects on muscle mass [89]. An 18-month factorial design RCT did demonstrate improved lean mass and muscle cross-sectional area in older men completing resistance training, but randomization to low-fat vitamin D fortified milk, providing an additional 1000 mg calcium, 800 IU vitamin D3, and 13.2 g protein per day, did not have additive or synergistic benefits [90]. However, in a two-arm RCT, early postmenopausal women who completed 24 weeks of resistance training while randomized to receive a supplement containing 10 g of whey protein, 31 g of carbohydrate, 1 g of fat, 200 IU of vitamin D, and 250 mg of calcium had an 0.8 kg increase in total lean mass, with no change observed for women in a placebo and resistance training group [91]. In a 12-week study, 130 sarcopenic older adults who participated in an exercise program including muscle-building, balance, and gait training, participants were randomized to receive an oral nutritional supplement containing whey protein (22 g), essential amino acids (10.9 g; 4 g leucine), and vitamin D (100 IU) or an isocaloric control. Participants in the supplement group gained an extra 1.7 kg of fat-free mass, and also had significant increases in appendicular lean mass relative to height, hand grip strength, and self-reported physical function, compared with placebo group [92]. The effects of antioxidant and omega-3 supplements on muscle mass have also been trialed in combination with exercise in older adults. A factorial RCT randomized 128 sarcopenic Japanese women to exercise plus tea catechin supplementation, exercise alone, tea catechin supplementation alone, or health education for 3 months [93]. Supplementation consisted of 350 mL of tea fortified with 540 mg of catechin (an antioxidant) each day for 12 weeks. Muscle mass changes did not differ between groups, but only the exercise plus tea catechin group demonstrated significant improvements in leg muscle mass from baseline to follow-up [93]. Fish oil supplements (rich in omega-3) have been shown to enhance improvements in muscle strength and function following a 12-week strength training program in older women [94], but it is unclear whether this relates to effects on muscle mass. Da Boit et al. randomized 50 older adults to receive 3 g of fish oil or safflower oil (placebo) each day during an 18-week lower-limb resistance exercise program but found no differences in the change in muscle cross-sectional area between groups [95]. This was consistent with a trial of 51 older adults who were randomized to 14 g/day of alpha-linoleic acid (an essential omega-3 polyunsaturated fatty acid) or placebo whilst completing resistance training for 12 weeks. A 62% decrease in interleukin-6 in the omega-3 group was supportive of the antiinflammatory benefits of omega-3 fatty acids, but little effect was seen for muscle thickness (assessed by ultrasound), total lean mass, and muscle strength [96]. It is possible that beneficial effects of omega-3 polyunsaturated fatty acids for muscle hypertrophy in response to exercise may be observed over longer duration interventions; a recent 24-week three-arm trial in which older women were randomized to control, resistance training, or resistance training plus a healthy diet rich in omega-3 polyunsaturated fatty acids reported that whole-body lean mass increased significantly only for the healthy diet group, and this group generally demonstrated greater increases in muscle force than the resistance training alone group [97]. While evidence is lacking for synergistic effects of nutritional supplements and exercise for improving muscle mass in older adults, supplementation may be beneficial for improving exercise benefits, particularly in patients with nutritional deficiencies. For the generally healthy older adult, ensuring adequate intake of important nutrients through the diet may

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FIGURE 2.1 Proposed role of adequate intake of key nutrients for optimising muscle hypertrophy responses to physical activity.

be sufficient for optimal muscle protein synthesis following exercise and everyday physical activity. As demonstrated in Fig. 2.1, muscle hypertrophy in response to physical activity will likely occur even in the presence of inadequate dietary nutrient intakes but may be maximized if the diet contains sufficient protein (with essential amino acids), omega-3 polyunsaturated fatty acids, vitamin D (through sun exposure and diet), and fruits and vegetables (particularly those high in antioxidants). By enhancing muscle gains, capacity for subsequent physical activity may also be increased, resulting in even greater exercise benefits in the long term.

SMOKING, ALCOHOL USE AND SOCIOECONOMIC STATUS Although physical activity and nutrition appear to be the most important modifiable factors contributing to age-related declines in skeletal muscle mass, other lifestyle behaviors have also been proposed to play a role. In the MINOS cohort of French older men, a significantly greater proportion of current and former smokers had low muscle mass relative to body height compared with men who had never smoked [17], and less than half as many older adults in the highest third for fat-free mass were current smokers (6%) compared with the lowest third for fat-free mass (B14%) in the Rancho Bernardo Study [98]. This was a similar finding to that of a recent Australian study which observed that 13% of older men with sarcopenia (defined by the EWGSOP consensus definition) were current smokers, compared with only 6% of healthy controls [99]. Research suggests that cigarette smoke components including ROS/RNS and aldehydes promote skeletal muscle damage through activation of muscle specific proteolytic pathways, and promotion of muscle catabolic processes and inhibition of anabolic processes [100].

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Excessive alcohol use may also be linked with decreased muscle mass because ethanol impairs muscle protein synthesis and increases autophagy, but a metaanalysis including 13 studies of over 13,000 participants actually reported that alcohol consumption was associated with a 23% reduced likelihood of sarcopenia [101]. This metaanalysis is limited by the varying definitions of sarcopenia and alcohol consumption assessments between studies. Furthermore, it is possible that minimal, or even beneficial, effects of moderate alcohol consumption influenced the overall results. Indeed, in postmenopausal women in the Korean National Health and Nutrition Examination Survey, those who reported high-risk alcohol consumption had a fourfold increased likelihood for low muscle mass relative to body weight compared with women who reported low-risk alcohol consumption [102]. Women who reported high-risk drinking in this study were also more likely to be current smokers, but the association of alcohol use and low muscle mass remained significant even after adjustment for potential confounders. Nevertheless, this highlights the fact that unhealthy lifestyle behaviors often aggregate and this is particularly true in individuals with low socioeconomic status. In the US Health and Retirement Study, the most disadvantaged participants had an almost threefold increased risk of 10-year mortality, and combined effects of physical inactivity, smoking, and alcohol use explained almost 70% of this increased risk [103]. In Australian women, smoking, larger food serving sizes, and low physical activity are most common in individuals from lower socioeconomic groups [104]. To date, there has been little direct research into the relationships between socioeconomic status and age-related muscle mass declines. The EWGSOP definition of sarcopenia is associated with lower incomes [105], and low calf circumference is also associated with lower education levels [106], in Brazilian older adults. In Australian middle-aged and older men, a significant trend for increasing lean mass percentage was observed with increasing socioeconomic status (assessed by the Index of Relative Socioeconomic Advantage/Disadvantage); the highest socioeconomic group had approximately 2.5% greater lean mass than the lower socioeconomic group [107]. Thus, it is likely that individuals in lower socioeconomic groups have increased risk for low relative muscle mass during aging and this may be explained to a large extent by poorer lifestyles. Declines in socioeconomic status may be particularly common during aging where many older adults transitioning to retirement are likely to experience reduced incomes preventing them from engaging in desirable health behaviors [108]; for example, food costs and travel are important predictors of nutritional choices in older adults [109], while access to facilities and neighborhood safety influence participation in physical activity [110]. Undoubtedly, it is a significant societal challenge to improve socioeconomic status of older adults on a population level. However, lifestyle interventions for older adults may be of particular benefit if they specifically target socioeconomic barriers to healthy behaviors.

CONCLUSIONS Older adults who demonstrate healthy lifestyle behaviors appear to maintain greater skeletal muscle mass as they age. It should, however, be acknowledged that interactions with genetic and environmental factors substantially influence this relationship (Fig. 2.2).

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FIGURE 2.2 Role of lifestyle in maximising peak muscle mass in early adulthood, extending the period of maintenance through mid-life, and minimising declines in older age.

A recent study of over 1500 twins in the United Kingdom reported a heritability estimate for appendicular lean mass of 80%, demonstrating that a large proportion of muscle mass variance is explained by our genetic profile [111]. Ethnicity is also important; Black populations generally demonstrate higher amounts of muscle mass than Caucasian and Asian populations but possibly experience more rapid declines with age [112]. While muscle mass insufficiency is generally considered a condition of aging, it is important to recognize the need for a life course approach to maintenance of muscle mass [112]. In the Hertfordshire Cohort Study including nearly 3000 UK older adults, birth weight and weight at 1 year of age positively predicted fat-free mass [113], and forearm and calf muscle cross-sectional area [114], at mean age 65 70 years. In a separate UK study, birth weight was found to explain approximately 50% of the variance in lean body mass at ages 70 75 years [115]. As demonstrated in Fig. 2.2, health behavior choices beginning in the prenatal and childhood environments can significantly influence muscle mass trajectory throughout the lifetime. By maximizing peak muscle mass achieved in early adulthood and also minimizing the rate of decline in older age through maintaining appropriate health behaviors, the likelihood that muscle mass will fall below a threshold where functional ability and metabolic health are compromised may be significantly reduced. Research into therapies for low muscle mass in older adults is increasing with promising pharmacological options including myostatin inhibitors [116,117]. Nevertheless, even if these or other pharmacotherapies are proven to be effective in future, it is likely they will be accessible only to small segments of the older adult population. As such, health policies and interventions which address desirable lifestyle behaviors must continue to be a primary focus of reducing the impact of sarcopenia in the wider older adult population.

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In summary, participation in higher intensity aerobic and resistance training activity from an early age and throughout the lifetime, while maintaining safe but adequate sun exposure and dietary patterns that promote increased intakes of key nutrients including vitamin D, protein high in essential amino acids, omega-3 fatty acids, and antioxidants, is likely to minimize the age-related decline in skeletal muscle mass. For most older adults, achieving these goals will be sufficient to avoid sarcopenia in later life, but even those who have low muscle mass can reverse this through participation in combined nutritional and exercise interventions.

References [1] Janssen I, Heymsfield SB, Wang Z, Ross R. Skeletal muscle mass and distribution in 468 men and women aged 18 88 yr. J Appl Physiol 2000;89(1):81 8. [2] Candow DG, Forbes SC, Little JP, Cornish SM, Pinkoski C, Chilibeck PD. Effect of nutritional interventions and resistance exercise on aging muscle mass and strength. Biogerontology 2012;1 14. [3] Rosenberg IH. Summary comments. Am J Clin Nutr 1989;50(5):1231 3. [4] Cruz-Jentoft AJ, Baeyens JP, Bauer JM, Boirie Y, Cederholm T, Landi F, et al. Sarcopenia: European consensus on definition and diagnosis: report of the European working group on sarcopenia in older people. Age Ageing 2010;39(4):412 23. [5] Studenski SA, Peters KW, Alley DE, Cawthon PM, McLean RR, Harris TB, et al. The FNIH sarcopenia project: rationale, study description, conference recommendations, and final estimates. J Gerontol A Biol Sci Med Sci. 2014;69(5):547 58. [6] Alema´n-Mateo H, Teros MTL, Astiazara´n-Garcı´a H. Association between insulin resistance and low relative appendicular skeletal muscle mass: evidence from a cohort study in community-dwelling older men and women participants. J Gerontol Ser A: Biol Sci Med Sci. 2014;69(7):871 7. [7] Chin SO, Rhee SY, Chon S, Hwang Y-C, Jeong I-K, Oh S, et al. Sarcopenia is independently associated with cardiovascular disease in older Korean adults: the Korea National Health and Nutrition Examination Survey (KNHANES) from 2009. PLoS ONE 2013;8(3):e60119. [8] Hars M, Biver E, Chevalley T, Herrmann F, Rizzoli R, Ferrari S, et al. Low lean mass predicts incident fractures independently from FRAX: a prospective cohort study of recent Retirees. J Bone Miner Res 2016;31(1):2048 56. [9] Scott D, Chandrasekara SD, Laslett LL, Cicuttini F, Ebeling PR, Jones G. Associations of sarcopenic obesity and dynapenic obesity with bone mineral density and incident fractures over 5 10 years in communitydwelling older adults. Calcif Tissue Int 2016;99(1):30 42. [10] Balogun S, Winzenberg T, Wills K, Scott D, Jones G, Aitken D, et al. Prospective associations of low muscle mass and function with 10-year falls risk, incident fracture and mortality in community-dwelling older adults. J Nutr, Health Aging. 2017;21(7):843 8. [11] Brook MS, Wilkinson DJ, Phillips BE, Perez-Schindler J, Philp A, Smith K, et al. Skeletal muscle homeostasis and plasticity in youth and ageing: impact of nutrition and exercise. Acta Physiol. 2016;216(1):15 41. [12] Cruz-Jentoft AJ, Landi F, Schneider SM, Zu´n˜iga C, Arai H, Boirie Y, et al. Prevalence of and interventions for sarcopenia in ageing adults: a systematic review. Report of the International Sarcopenia Initiative (EWGSOP and IWGS). Age Ageing 2014;43(6):748 59. [13] Peterson MD, Sen A, Gordon PM. Influence of resistance exercise on lean body mass in aging adults: a metaanalysis. Med Sci Sports Exerc 2011;43(2):249 58. [14] Bennie JA, Pedisic Z, van Uffelen JG, Charity MJ, Harvey JT, Banting LK, et al. Pumping iron in Australia: prevalence, trends and sociodemographic correlates of muscle strengthening activity participation from a national sample of 195,926 adults. PLoS ONE 2016;11(4):e0153225. [15] Wroblewski AP, Amati F, Smiley MA, Goodpaster B, Wright V. Chronic exercise preserves lean muscle mass in masters athletes. Phys Sportsmed. 2011;39(3):172 8. [16] Baumgartner RN, Waters DL, Gallagher D, Morley JE, Garry PJ. Predictors of skeletal muscle mass in elderly men and women. Mech Ageing Dev 1999;107(2):123 36.

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Molecular Mechanisms of Postmeal Regulation of Muscle Anabolism Olivier Le Bacquer Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, Clermont-Ferrand Cedex 1, France

Maintenance of muscle mass is a dynamic equilibrium regulated by the balance between muscle protein breakdown and synthesis rates. These processes are highly sensitive to physical activity and food intake. In particular, after consumption of a meal, the postprandial muscle protein synthesis rate is upregulated. Several factors such as ingested protein digestion, amino acid absorption, amino acid splanchnic sequestration, and skeletal muscle tissue perfusion are known to influence protein accretion in muscles [1]. Crucial components of this response are elevation of circulating hormones such as insulin and increased amino acid uptake by skeletal muscles. The postprandial elevation of insulin in response to carbohydrate ingestion can modulate muscle protein synthesis via an indirect mechanism by stimulating the endothelial nitric oxide synthase [2,3], which results in increased microvascular blood flow to skeletal muscle [4,5]. This increase perfusion of muscle is postulated to induce an increased exposure of muscle tissue to nutrients and growth factors which ultimately leads to increased protein synthesis. The postprandial stimulation of skeletal muscle protein synthesis also results from an increase amino acid uptake by amino acids transporters located on the cell membrane of muscle cells [6] and subsequent activation of intracellular molecular mechanism toward protein anabolism. The translation of messenger RNA is the key mechanism of protein synthesis regulation in numerous cells types including skeletal muscle cells [7,8]. Translation initiation is a tightly controlled process and is regulated by hormonal and nutrients cues through the mechanistic (a.k.a. mammalian) target of rapamycin (mTOR) kinase (for review see [8]). The mTOR is a highly conserved serine/threonine kinase protein and a central sensor linking nutrient availability to cell growth and proliferation as well as numerous cellular processes in skeletal muscle [9,10]. mTOR forms two distinct functional complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (for review, see [10]). mTORC1 is mainly defined by its three core components: mTOR, Raptor (regulatory protein associated

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00003-8

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with mTOR), and mLST8 (mammalian lethal with Sec13 protein 8, GβL), where mLST8 stabilizes the kinase activation loop and Raptor facilitates substrate recruitments to mTORC1 allowing their phosphorylation. Like mTORC1, mTORC2 contains mTOR and mLST8. However, instead of Raptor, it contains Rictor (rapamycin insensitive companion of mTOR). The mTORC1 pathway integrates inputs from a number of stimuli including nutrients (amino acids), hormones and growth factors (insulin, IGF1), energy status, and oxygen levels to regulated cell growth and proliferation. In order to maintain such processes, mTOR stimulates anabolic processes including protein synthesis, energy production in mitochondria, and control protein degradation processes.

CELLULAR PROCESSES UNDER mTORC1 Protein synthesis. Once activated by nutrients or growth factors, mTORC1 promotes protein synthesis largely through the phosphorylation of two key effectors, the p70S6 kinase 1 (S6K1) and the eukaryotic translation initiation factor 4E-binding proteins (4E-BPs) [11,12]. S6K1 and 4E-BPs associate with mRNAs and regulate translation initiation and progression thus controlling the rate of protein synthesis [13]. The 4E-BPs compete with eIF4G for a shared binding site on eIF4E as the binding of 4E-BPs and eIF4G to eIF4E are mutually exclusive [14]. This results in inhibition of mRNA translation initiation. Binding of 4E-BP to eIF4E is regulated through phosphorylation: whereas hypophosphorylated forms strongly interact with eIF4E, hyperphosphorylation of 4E-BPs dramatically weakens this interaction [15]. In response to growth factors, or nutrients, the mTORC1 induces the sequential phosphorylation of 4E-BPs [14,16,17], leads to their dissociation from eIF4E which allow the recruitment of eIF4G to the 50 end of most mRNA and results in increased cap-dependent translation initiation through assembly of the eIF4F complex [14]. When phosphorylated by mTORC1 on its Thr389 residue, S6K1 phosphorylates or binds a number of components of the translational machinery and related factors such as ribosomal protein S6 (rpS6), CBP80 (80 kDa nuclear cap binding protein), eEF2K (eukaryotic elongation factor 2 kinase), eIF4G, and eIF4B [1822], which collectively promote translation initiation and elongation. S6K1 also phosphorylates and promotes the degradation of PDCD4, an inhibitor of eIF4B [23] leading to increased interaction of SKAR (S6K1 Aly/ REF-like target, a component of the exon-junction complexes) [24] with eIF4B. This ultimately results in enhanced translation efficiency of spliced mRNA. Lastly, mTORC1 is able to phosphorylate MAF1 and to modulate the activity of TIF-IA which respectively control the synthesis of tRNA and rRNA [25,26]. It was originally believed that S6K1 activation and phosphorylation of its target rpS6 was able to control the translation of a subset of mRNAs including 50 terminal olygopyrimidine (50 TOP) mRNAs, which harbor a stretch of pyrimidines following the cap structure, referred to as 50 TOP motif [27,28]. 50 TOP mRNAs encode most ribosomal proteins and others components of the protein synthesis machinery [29,30]. Although mTOR is playing a central role in the control of their translation [27,31], subsequent studies demonstrated that this role is independent of S6K1 and rpS6 phosphorylation but rather involved the 4E-BPs [31,32]. Indeed, the acute treatment of mouse cells with mTOR inhibitor Torin1, a specific mTOR inhibitor, revealed that the subset of mRNAs that are specifically

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regulated by mTORC1 consists almost entirely of 50 TOP mRNAs. Remarkably, the loss of 4E-BPs is sufficient to render 50 TOP mRNA translation resistant to Torin1 [31]. However, in another study, Miloslavski et al. demonstrated that oxygen availability controls the translation of 50 TOP mRNA in an mTOR-dependent manner and that 4E-BPs are dispensable for this regulation [33]. Altogether, these data suggest that 50 TOP mRNA translation is context dependent. Energy production and mitochondrial biogenesis. Protein synthesis is one of the most energy consuming processes in the cell and must be tightly coordinated with cellular energy production [34,35]. Emerging studies establish that mTOR is able to modulate ATP production and mitochondrial biogenesis and function. This modulation can take place at different levels such as mitochondrial biogenesis, direct regulation of mitochondrial proteins and regulation of uptake, and utilization of carbohydrates. mTORC1 activation increases the number of mitochondrial DNA copies and the expression of genes encoding proteins which regulate mitochondrial metabolism such as PPARγ coactivator 1α (PGC1α) and hypoxia inducible factor 1α (HIF1α) [3639]. On the opposite, inhibition of mTORC1 activity by genetic deletion of Raptor or TSC2 in skeletal muscle reduces the expression of oxidative genes and the number of mitochondria [40,41]. mTORC1 mediates its effects on mitochondrial DNA content and oxygen consumption via the Ying-Yang 1 (YY1) transcription factor. The association with Raptor induces the phosphorylation of YY1 which enhances its interaction with PGC1α, a crucial transcription factor of the mitochondrial biogenesis process in skeletal muscle [36]. mTORC1 is also a positive regulator of HIF1α [39,4244] which promotes the expression of genes that regulate glucose transport and glycolysis and provides a mean to maintain energy production when cellular respiration is reduced [45]. Not only mTOR regulates mitochondrial activity and biogenesis at the transcriptional level but it also controls this process at the translational level. Indeed, short-term (i.e., 12 hours) mTOR inhibition does not induce major changes in the transcription of mitochondria-related genes [37]. Using genome-wide polysome profiling, Morita et al. revealed that mTORC1 stimulates the synthesis of a subset of nuclear-encoded mitochondrial regulators including TFAM (transcription factor A, mitochondrial), which promotes mitochondrial DNA replication and transcription [46], mitochondrial ribosomal proteins, and components of complex I and V by upregulating the translation of their corresponding mRNAs [37]. They also revealed that 4E-BPs mediate the stimulatory effect of mTORC1 on the mitochondrial respiration and biogenesis, ATP production, and the translation of mitochondria-related mRNAs [37]. Altogether, these findings demonstrate that mTORC1 is able to control mitochondrial biogenesis and function not only at the transcriptional level but also at the translational level. Last, mTORC1 is involved in balancing glycolytic flux with mitochondrial respiration via direct phosphorylation of mitochondrial proteins. It has been documented that mTORC1 is able to associate with mitochondria [47,48]. Particularly, Schieke et al. observed that overall mitochondrial activity was correlated with the level of complex formation between mTOR and Raptor and that disruption of this complex by 12-hour treatment with rapamycin was able to lowered mitochondrial potential, oxygen consumption, and ATP production [47]. The decreased ATP synthesis capacity was associated with a dramatic alteration in the mitochondrial phosphoproteome. Using a shorter treatment

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(i.e., 30 minutes), Ramanathan et al. demonstrate that rapamycin lowers mitochondrial respiration and induces a shift from oxidative phosphorylation (the mitochondrial process that uses energy release during the oxidation of nutrients to produce ATP) toward lactateproducing glycolysis [48]. They proposed that this process occurs through a mechanism involving an interaction between mTOR and the voltage-dependent anion selective channel (VDAC1) and Bcl-XL. VDAC proteins are situated in the outer mitochondrial membrane and are crucial for substrate transport into the mitochondria [49]. Bcl-XL is an antiapoptotic mitochondrial transmembrane protein and a mediator of mitochondrial function that is able to interact with VDAC to increase substrate permeability [50,51]. Once phosphorylated on serine62 residue, Bcl-XL may stimulate the permeability of TCA substrates and hence increased ATP production through oxidative phosphorylation. Protein degradation. In parallel to stimulating protein synthesis, mTORC1 is also able to promote anabolism by suppressing protein catabolism, mainly autophagy, a selfdegradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress [52]. ULK1 (unc-51-likekinase 1) is a kinase that forms a complex with ATG13 (mammalian autophagy-related gene 13) and FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) and drives autophagosome formation. Its activation is a crucial step in autophagy. Under postprandial conditions when amino acids, carbohydrates, and growth factors are abundant, mTORC1 inhibits autophagosome formation by phosphorylating two positive mediators of autophagy, ULK1 and ATG13, and a negative mediator, DAP1 (death-associated protein 1). The mTORC1-mediated phosphorylation of ULK1 prevents its activation by AMPK, a key regulator of autophagy [53], and the mTORC1-mediated phosphorylation of ATG13, a positive regulator of ULK1, inhibits its activity [54,55]. In such conditions, mTORC1 also phosphorylates DAP1 which suppresses autophagy [56]. Last, mTORC1 controls autophagy by blocking lysosome biogenesis via the phosphorylation and inhibition of the nuclear translocation of TFEB (transcription factor EB) [5759]. In mammalian cells under normal conditions, a large proportion of the protein breakdown occurs through the ubiquitin proteasome system (UPS) [6062]. In addition to autophagy, it was recently demonstrated that mTORC1 inhibition also activates protein degradation by the UPS [61]. The mechanisms by which mTORC1 suppresses UPS are still to be discovered but seem to be independent of 4E-BPs, S6K1, and ULK [61].

UPSTREAM mTOR SIGNALING A multitude of extracellular signals and intracellular cues have been implicated in the modulation of mTOR signaling including nutrients (i.e., amino acids), growth factors, energy, and stress [12]. These inputs are integrated to allow fine-tuning of the mTORC1 activity. The regulation of mTORC2 is only beginning to be discovered, and, to date, only growth factors have been documented to regulate this complex. Amino acids regulate mTORC1. In skeletal muscle and other cell types, amino acids (especially essential amino acids such as leucine) do not just act as building blocks or source of energy for protein synthesis but also as signaling molecules to the mRNA translational machinery. In human skeletal muscle, the amino acid-mediated increase in protein

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synthesis that occurs after a meal ingestion lasts for approximately 23 hours before returning to its basal level [63]. While the ability of amino acids to stimulate mTORC1 activity and protein synthesis is well described, the underlying mechanisms were unclear until recent discoveries (for review see [64]). Over the last decade, several candidates have been associated with amino acid sensing by mTORC1, including hVPS34 (mammalian vacuolar protein sorting 34 homologue), IPMK (inositol polyphosphate monokinase), and MAP4K3 (mitogen-activated protein kinase kinase kinase kinase) [6568]. The most exhaustive comprehension of the mechanisms by which amino acids activate mTORC1 came from the recent discovery by the Sabatini’s lab that mTORC1 activation requires its recruitment to the lysosomal surface [69,70]. This recruitment is mediated by heterodimers of Rag GTPases and requires a multisubunit complex called Ragulator [69,71,72]. The recruitment of mTORC1 to the lysosomal surface is controlled by two protein complexes, GATOR 1 (GTPase activating protein activity toward Rags) and GATOR 2 [73]. GATOR 1 has been shown to inactivate the Rag heterodimer complex which prevents the docking of mTORC1 to the lysosomal surface. In the presence of sufficient amount of amino acids, GATOR 2 removes the GATOR 1 inhibition toward the Rag GTPase heterodimer allowing the activation of mTORC1. An important breakthrough in the understanding of amino acid sensing came from the identification of CASTOR1 and Sestrin2 as arginine and leucine sensor, respectively [74,75]. Sestrin2 is a direct sensor of leucine that binds and inhibits GATOR2 function in the absence of leucine [74,76,77]. CASTOR1 (cellular arginine sensor for mTORC1) acts as an arginine sensor and unlike Sestrin2 inactivates GATOR2 in the absence of arginine [75,78]. Thus, after a meal ingestion, the supply of leucine and arginine stimulates mTORC1 activity by releasing CASTOR1 and Sestrin2 from GATOR2 which allows the subsequent inhibition of GATOR1 and localization of mTORC1 to the lysosomal surface. Several additional mechanisms through which amino acids regulate mTORC1 activity have recently been reported. In the presence of high intracellular leucine levels, the leucyltRNA synthetase seems to migrate to the lysosomal surface where it facilitates the assembly of the Rag GTPase heterodimer complex [79]. The vacuolar H1 ATPase (v-ATPase) is located at the lysosomal surface and is sensitive to amino acid concentration within the lysosome. v-ATPase appears to interact with the Ragulator to stimulate mTORC1 activation [70]. Last, glutamine, an abundant amino acid in skeletal muscle that is used as nitrogen and energy sources by proliferating cells, activates mTORC1 independently from the Rag GTPase complex through a mechanism that requires v-ATPase [80]. Regulation of mTOR by growth factors. The increased mTORC1 activity toward increased anabolism should only occur in the presence of sufficient amounts of nutrients, energy, and growth factors such as insulin. In response to growth factors, mTORC1 is activated via the PI3K/Akt pathway [9]. Binding of insulin to its receptor activates the PI3K/ Akt pathway, which leads to the phosphorylation and activation of Akt. In turn, Akt phosphorylates an heterodimer consisting of TSC1 (tuberous sclerosis 1, also known as hamartin) and TSC2 (also known as tuberin), which functions as a GTPase-activating protein for the Ras homolog enriched in brain (Rheb) GTPase [81,82]. Akt activation by insulin leads to an Akt-dependent phosphorylation of TSC2 [81,83,84] which inhibits the TSC complex activity and increases level of GTP-bound Rheb that directly interacts with mTORC1 and strongly activates its kinase activity [82,85]. Growth factors can also activate mTORC1 via

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alternative mechanisms. For example, receptor tyrosine kinase-dependent Ras signaling activates mTORC1, via ERK (extracellular signal-regulated kinase) which phosphorylates TSC2 to inhibit its activity [86]. The upstream mechanism leading to mTORC2 activation is still poorly understood. mTORC2 is insensitive to nutrients but responds to growth factors like insulin through a PI3K-dependent mechanism leading to the association of mTORC2 with ribosomes [87]. The most important role of mTORC2 is the phosphorylation of Akt, a key effector of the insulin signaling [88,89]. Once activated, Akt promotes several cellular process including proliferation, survival, growth through the phosphorylation of key substrates such as the mTORC1 inhibitor TSC2. Regulation of mTORC1 by energy. mTORC1 is sensitive to energy status and glucose availability. The AMP-activated protein kinase (AMPK) is considered the cellular energy sensor. Any cellular or metabolic state that reduces ATP levels causes AMPK activation [90]. Then, AMPK phosphorylates TSC2 and enhances its activity which leads to mTORC1 inhibition [91]. Glucose uptake induces localization of mTORC1 to the lysosome by regulating the binding of Ragulator to the v-ATPase [92]. Last, Hexokinase-II, which catalyzes the first step of glycolysis, is able to bind and inhibit mTORC1 in the absence of glucose [93].

CONCLUSION It is clear that the mTOR pathway is a crucial regulator of muscle anabolism in response to meal ingestion. Its activation by insulin, glucose, and amino acids not only stimulates protein synthesis but also inhibits proteolytic processes such as autophagy and the UPS. mTORC1 also modulate the production of energy to support the anabolic process by targeting mitochondrial biogenesis and substrate utilization. Despite the recent discovery of intracellular amino acid sensors (CASTOR1, Sestrin2), it is still unclear how diet, especially amino acid composition and amino acid-derivative metabolites, impacts the activity of mTORC1. While the mechanisms of leucine sensing have been largely characterized, as a branched-chain amino acid, it can be catabolized within muscle into α-ketoisocaproic acid (KIC) and β-hydroxy-β-methylbutyrate (HMB) [94]. Recent reports demonstrate unlike leucine, KIC and HMB are able to regulate protein homeostasis in skeletal muscle [9597]. In the liver, it has been proposed that intracellular sequestered calcium stores are responsible for maintenance of protein synthesis [98]. A report describes the possible involvement of endoplasmic reticulum calcium signaling in mediating the effect of leucine on protein synthesis in rat myotubes [99]. Further studies will be required to understand how signaling from amino acid sensing, which mediates the localization and activation of mTORC1 at the lysosomal surface, and calcium signaling at the endoplasmic reticulum are integrated to promote anabolism in skeletal muscle.

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[69] Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell. 2010;141 (2):290303 Epub 2010/04/13. [70] Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(1)-ATPase. Science 2011;334(6056):67883 Epub 2011/11/05. [71] Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 2008;320(5882):1496501 Epub 2008/05/24. [72] Kim J, Kim E. Rag GTPase in amino acid signaling. Amino Acids 2016;48(4):91528 Epub 2016/01/20. [73] Bar-Peled L, Chantranupong L, Cherniack AD, Chen WW, Ottina KA, Grabiner BC, et al. A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 2013;340(6136):11006 Epub 2013/06/01. [74] Wolfson RL, Chantranupong L, Saxton RA, Shen K, Scaria SM, Cantor JR, et al. Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 2016;351(6268):438 Epub 2015/10/10. [75] Chantranupong L, Scaria SM, Saxton RA, Gygi MP, Shen K, Wyant GA, et al. The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell. 2016;165(1):15364 Epub 2016/03/15. [76] Chantranupong L, Wolfson RL, Orozco JM, Saxton RA, Scaria SM, Bar-Peled L, et al. The Sestrins interact with GATOR2 to negatively regulate the amino-acid-sensing pathway upstream of mTORC1. Cell reports 2014;9(1):18 Epub 2014/09/30. [77] Saxton RA, Knockenhauer KE, Wolfson RL, Chantranupong L, Pacold ME, Wang T, et al. Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway. Science 2016;351(6268):538 Epub 2015/11/21. [78] Saxton RA, Chantranupong L, Knockenhauer KE, Schwartz TU, Sabatini DM. Mechanism of arginine sensing by CASTOR1 upstream of mTORC1. Nature 2016;536(7615):22933 Epub 2016/08/04. [79] Han JM, Jeong SJ, Park MC, Kim G, Kwon NH, Kim HK, et al. Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell. 2012;149(2):41024 Epub 2012/03/20. [80] Jewell JL, Kim YC, Russell RC, Yu FX, Park HW, Plouffe SW, et al. Metabolism. Differential regulation of mTORC1 by leucine and glutamine. Science 2015;347(6218):1948 Epub 2015/01/09. [81] Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 2003;17(15):182934 Epub 2003/07/19. [82] Tee AR, Manning BD, Roux PP, Cantley LC, Blenis J. Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Current biology : CB. 2003;13(15):125968 Epub 2003/08/09. [83] Cai SL, Tee AR, Short JD, Bergeron JM, Kim J, Shen J, et al. Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning. J Cell Biol 2006;173(2):27989 Epub 2006/04/26. [84] Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell 2002;10(1):15162 Epub 2002/08/02. [85] Parmar N, Tamanoi F. Rheb G-Proteins and the activation of mTORC1. Enzymes. 2010;27:3956 Epub 2010/ 01/01. [86] Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell. 2005;121(2):17993 Epub 2005/04/27. [87] Zinzalla V, Stracka D, Oppliger W, Hall MN. Activation of mTORC2 by association with the ribosome. Cell. 2011;144(5):75768 Epub 2011/03/08. [88] Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006;22(2):15968 Epub 2006/04/11. [89] Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictormTOR complex. Science 2005;307(5712):1098101 Epub 2005/02/19. [90] Hardie DG, Sakamoto K. AMPK: a key sensor of fuel and energy status in skeletal muscle. Physiology 2006;21:4860 Epub 2006/01/31. [91] Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell. 2003;115(5):57790 Epub 2003/12/04.

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[92] Efeyan A, Zoncu R, Chang S, Gumper I, Snitkin H, Wolfson RL, et al. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival. Nature 2013;493(7434):67983 Epub 2012/12/25. [93] Roberts DJ, Tan-Sah VP, Ding EY, Smith JM, Miyamoto S. Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol Cell 2014;53(4):52133 Epub 2014/01/28. [94] Hutson SM, Sweatt AJ, Lanoue KF. Branched-chain [corrected] amino acid metabolism: implications for establishing safe intakes. J Nutr 2005;135(6 Suppl):1557S1564SS Epub 2005/06/03. [95] Wilkinson DJ, Hossain T, Hill DS, Phillips BE, Crossland H, Williams J, et al. Effects of leucine and its metabolite beta-hydroxy-beta-methylbutyrate on human skeletal muscle protein metabolism. J Physiol 2013;591 (11):291123 Epub 2013/04/05. [96] Escobar J, Frank JW, Suryawan A, Nguyen HV, Van Horn CG, Hutson SM, et al. Leucine and alphaketoisocaproic acid, but not norleucine, stimulate skeletal muscle protein synthesis in neonatal pigs. J Nutr 2010;140(8):141824 Epub 2010/06/11. [97] Giron MD, Vilchez JD, Salto R, Manzano M, Sevillano N, Campos N, et al. Conversion of leucine to betahydroxy-beta-methylbutyrate by alpha-keto isocaproate dioxygenase is required for a potent stimulation of protein synthesis in L6 rat myotubes. J Cachexia Sarcopenia Muscle 2016;7(1):6878 Epub 2016/04/12. [98] Brostrom CO, Brostrom MA. Calcium-dependent regulation of protein synthesis in intact mammalian cells. Annu Rev Physiol 1990;52:57790 Epub 1990/01/01. [99] Miura Y, Nakazawa T, Yagasaki K. Possible involvement of calcium signaling pathways in L-leucinestimulated protein synthesis in L6 myotubes. Biosci Biotechnol Biochem 2006;70(6):15336 Epub 2006/06/24.

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C H A P T E R

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Adaptation of Skeletal Muscle Mass and Metabolism to Physical Exercise Xavier Bigard French Antidoping Agency, Paris, France

One of the key principles of physical training is the specificity of the tissue adaptations in response to the type of training performed [1,2]. Adaptations to resistance and endurance training (ET) are generally different, especially regarding the adaptive responses of skeletal muscle. Resistance training (RT) has only little effect on aerobic and cardiorespiratory capacities but leads to increased muscle mass, strength/power production, glycolytic enzyme activity, and intramuscular energy stores (i.e., ATP and phosphocreatine stores) [3]. In contrast, ET enhances the oxidative capacity of trained myofibers, their mitochondrial density, and muscle capillarity [4]. In addition, ET elicits a decrease in myofiber size, an adaptive alteration that could negatively impact muscle strength and power. In some ways, resistance and ET are placed in contrasting ends of the training adaptation continuum. A large number of sports such as rowing, rugby, swimming, decathlon, etc. require skeletal muscle properties combining both strength/power and endurance [5]. That is why many athletes train for strength and endurance simultaneously, combining both resistance and endurance exercises in a regular training plan named concurrent training (CT). Negative effects on the development of strength and muscle growth have been reported when CT is performed over long periods of time [6]. The specific adjustments of the skeletal muscle mass and phenotype to physical training result from homeostatic perturbations (i.e., metabolic stress, local hypoxia, mechanical stress) [7]. The exercise-induced stimuli lead to alterations in gene transcription, via specific intracellular signaling pathways, translation of transcripts into proteins, and assembly of these encoded proteins. These transient adjustments in gene expression are cumulated with repeated exercises and contribute to explain the structural/functional responses of skeletal muscles to physical training [7]. The aim of this chapter was to review the current knowledge on the muscle adaptive responses to physical training, their potential regulatory mechanisms, and relevance for protein requirement in athletes.

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00004-X

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THE MUSCULAR ADAPTATION TO ENDURANCE TRAINING ET entails both central and peripheral factors involved in performance, affects the motor-units recruitment, and elicits marked adaptive changes of myofiber phenotype. Briefly, the muscular adaptation to ET skeletal muscle encompasses increased fat oxidation and glycogen sparing at submaximal workloads, increased glycogen stores, and enhanced buffering capacity. Taken together, ET results in an increased ability of skeletal muscle to generate energy via oxidative metabolism, without improvements in muscle strength. The increased oxidative enzyme capacity and mitochondrial and capillary density elicit a muscle phenotype associated with improved resistance to fatigue during prolonged contractile activity.

The Oxidative Phenotype Early studies showed that muscles of highly trained athletes differed from those of sedentary subjects by many aspects [8], the most obvious being the greater resistance to fatigue. Whether the characteristics of skeletal muscle in athletes result from traininginduced changes has been examined. Several studies showed that ET per se elicits a variety of metabolic and structural changes that occur fairly rapidly in response to exercises, within the first 10 days after initiation of the training program [9,10]. These adaptive responses of the oxidative potential of human skeletal muscles are fiber-type dependent and consistent with the recruitment pattern of the motor units that follows the size principle of Henneman. The number and size of mitochondria increased with ET, as well as muscle capillarity, whereas the myofiber size was only scarcely affected. ET elicits a differentiation of muscle fibers toward a phenotype with a high mitochondrial volume density, through several highly coordinated adaptive processes. The increase in the number and size of mitochondria results from synthesis and incorporation of new proteins in the existing mitochondrial network, expanding from preexisting mitochondria, rather than de novo generation [11]. The mitochondrial function in skeletal muscles is improved with exercise training, especially ET. A highly significant correlation between maximal rates of oxygen consumption (state III respiration) measured in isolated mitochondria, and both the training status and the whole body maximal oxygen uptake were found in human skeletal muscle [12]. Moreover, using measures of respiration in mitochondria maintained in situ within muscle fibers permeabilized with saponin, it has been shown that physical activity leads also to qualitative alterations in mitochondrial function, as well in predominant glycolytic, as in predominant oxidative myofibers [13]. In addition to an enhancement of muscular oxidative capacity, ET is associated with an improvement of coupling and regulatory properties of mitochondrial respiration. Therefore, the phenotypic plasticity of mitochondria in response to repeated endurance exercises includes both structural and functional changes that contribute to increased muscle oxidative capacity [14]. In human, skeletal myofibers have been categorized not only according to their mitochondrial density (high or low oxidative fibers) but also according to their speed of contraction (fast-twitch or slow-twitch), based upon their myosin heavy chain (MHC)

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composition. While longitudinal studies clearly demonstrated that skeletal muscles of endurance athletes have a high proportion of slow-twitch fibers (type I fibers, mainly comprising the type I MHC) [8,15], most studies failed to demonstrate a conversion between fast-twitch and slow-twitch fibers with specific ET programs (for review, see [16]). In the majority of studies, only a slight shift toward a slower profile occurred in human skeletal muscles in response to ET, with only a conversion between fast-twitch fibers [17]. There is now a consensus to consider that endurance-type repeated exercises in humans induce fiber-type switching to a more oxidative phenotype but do not alter the type I to type II fiber ratio and do not increase switching to type I/slow myofibers [18]. But whether long-term (years) ET in humans can increase type I percentage in skeletal muscle remains a matter of debate [18]. The possibility exists that many years of aerobic ET could increase the type I myofiber percentage, but this remains to be documented; longitudinal human studies in the same subjects from childhood to adulthood require repeated muscle biopsies beginning in children, and such studies are not permitted for evident ethical reasons. Taken together, these results suggested that physical training only hardly affected the relative expression of myosin isoforms, emphasizing the dissociation between metabolic and contractile phenotype adaptations [13,19]. Higher values of capillary density have been demonstrated in skeletal muscles of endurance athletes in comparison with nonactive people [20]. Both the capillary-to-fiber ratio and the capillary density increased as a result of ET. The increased muscle capillarity in consistent with the increased mitochondrial oxygen demand, and the need to augment oxygen supply for contracting muscle.

The Molecular Mechanisms Involved in the Muscle Responses to Endurance Training Prolonged exercise is at the origin of homeostatic perturbations that are integrated into adaptive changes in the transcription level of target genes and messenger RNA (mRNA) diffusion, leading to translation into specific proteins by the ribosomal machinery [7]. Enhanced levels of specific mRNA would therefore support the synthesis of protein components that are at the origin of structural remodeling and functional adjustments in the long term. Current evidence demonstrates that exercise per se contributes to control the myofiber phenotype through the activation of specific signaling pathways and alterations of the transcription level of target genes. Responses of the Mitochondrial Network The nuclear genome encodes most of the genes for the mitochondria. However, mitochondria possess an independent genome consisting of 37 genes, 13 of which encode for essential proteins in the electron transport chain. That is why mitochondrial biogenesis requires a closed and fine-controlled coordination between transcription of the nuclear and mitochondrial genomes (mtDNA). The transcription of genes encoding mitochondrial proteins is primarily regulated through the transcriptional coactivator peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) (Fig. 4.1) [21]. Once activated in response to exercise, PGC-1α acts as a transcriptional coactivator and relies on

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Exercise

2+

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oxidation

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p38 MAPK AMPK

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ATF-2

NRF1

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PPARα/δ ERRα

PGC-1α PPARα/δ

PGC-1α NRF1

Nucleus PGC-1α ERRα

FIGURE 4.1 Sustained and repeated muscle contractions are associated with increased intracellular calcium, and then p38 MAPK and CaMK activations. The p38 MAPK stimulates PGC-1α promoter activity through engagement of the downstream transcription factor ATF-2. PGC-1α coactivates several transcription factors involved in mitochondrial function and density. Members of the PPAR nuclear receptor transcription factor family, such as PPAR-α and PPAR-δ, involved in the expression of genes encoding fatty acid oxidation enzymes are activated by PGC-1α. PGC-1α also coactivates ERRs to enhance the expression of fatty acid oxidation enzymes. Finally, PGC-1α contributes to increase expression of nuclear-encoded enzymes involved in OXPHOS through the NRF1 transcription factor. Expression of the mitochondrial genome (mtDNA) is specifically regulated by the Tfam, under the control of NRF1. ERR, estrogen-related receptor; NRF1, nuclear respiratory factor-1; PGC-1α, peroxisome proliferator activated receptor γ coactivator 1α; ATF, activating transcription factor; Tfam, mitochondrial transcription factor A.

transcription factors such as estrogen-related receptor-α and nuclear respiratory factor (NRF)-1 to control the nuclear transcription of genes encoding mitochondrial proteins [22]. Interactions between PGC-1α and NRF1 are potentiated by exercise in the nucleus, and such functional interactions increase the transcription of nuclear genome-encoded genes for electron transport chain proteins [22]. In addition, PGC-1α/NRF1 also increases the transcription of nuclear genome-encoded mitochondrial transcription factor A (Tfam). Endurance exercise enhances the PGC-1α translocation to the mitochondria where it interacts with Tfam, promoting the transcription of the mitochondrial genome-encoded genes [23]. Therefore, functional interactions of PGC-1α with existing transcriptional factors (i.e., NRF1, Tfam) allow the orchestrated regulation of the two independent genomes to meet the transcriptional requirements of the mitochondrial biogenesis. In the context of endurance exercise, the transcription level of the PGC-1α gene and biological activity of PGC-1α are regulated by multiple upstream intracellular factors [21]. Activation of the p38 MAPK likely through the elevation of the cytosolic Ca21 leads to activation of the activating transcription factor (ATF)-2, one of the p38 downstream targets, and then to increased PGC-1α gene expression in skeletal muscle in response to

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exercise (Fig. 4.1) [24]. Moreover, p38 MAPK also activates the myocyte enhancer factor (MEF)-2 that together with ATF-2 plays a critical role for the exercise-induced PGC-1α gene expression [25]. Elevated AMP and/or ADP in recruited myofibers during sustained muscle contraction activate the 50 AMP-activated protein kinase (AMPK). This enzymatic complex is often referred as the metabolic master switch and is now viewed as playing a central role in cellular energy metabolism. AMPK activity is sensitive to fluctuations in the AMP/ATP ratio and may act in concert with p38 MAPK to transcriptionally regulate mitochondrial biogenesis through PGC-1α, and then to contribute to maintain energy balance during sustained physical exercise (Fig. 4.1). In addition to mitochondrial biogenesis, repeated endurance exercises lead to dynamic remodeling of the mitochondrial reticulum. Previous experimental results showed that in endurance-trained healthy subjects, the improvement in mitochondrial function was accompanied by a coordinated increase in the expression of mitochondrial mRNAs encoded either by the nuclear or by the mitochondrial genome on the one hand, and gene expression of proteins involved in the mitochondrial fusion [14]. Animal models subjected to chronic contractile activity displayed increased expression of proteins involved in the outer mitochondrial membrane fusion (mitofusin 2), and in the inner membrane fusion (optical atrophy 1) [26]. A decrease in the expression of proteases involved in mitochondrial degradation (Lon protease), as well as in mitochondrial fission (dynamin-related protein 1), is also reported, providing a molecular basis for the drive toward upregulation of the fusion process which contributes to enhance reticular organelles. Responses of the Microvascular Network As discussed above, one of the well-known responses to ET is the expansion of the skeletal muscle microvascular network, which contributes with the mitochondrial network, to enhance the substrate availability in skeletal muscle (i.e., oxygen and fuel sources). The cellular and molecular mechanisms of capillary growth have been mostly examined in animal models. The exercise-induced increase in capillary density results from both sprouting and splitting angiogenesis, two complementary angiogenic modes that differ significantly in their initiation and implementation. Sprouting includes a sequence of events initiated by the activation of capillary endothelial cells (ECs), their proliferation, and migration into the interstitium [27]. Enhanced capillarity requires also the proteolytic breakage of the basement membrane of capillaries by higher levels of matrix metalloproteinases, thereby permitting the abluminal outgrowth of new capillary segments [28]. In contrast, splitting angiogenesis is achieved by the intraluminal splitting of capillaries and is triggered by the increase in capillary shear stress [29]. In response to shear stress, ECs send cytoplasmic projections to the opposite sides of the capillary lumen, until they form a transluminal pillar that increases by the deposition of connective tissue. The vascular segment is then separated into two independent microvessels. Interestingly, while abluminal sprouting is strongly dependent from capillary ECs proliferation, intraluminal splitting does not immediately depend on ECs proliferation [30]. However, whether the exercise-induced angiogenesis in humans results from vascular sprouting and/or splitting remains unanswered [31].

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These complex cellular events involve several signaling pathways that must be tightly coordinated to increase muscle capillarity. The exercise-induced angiogenesis is the result of a balance between factors that promote and those that inhibit vascular growth. Vascular endothelial growth factor (VEGF) is a peptide growth factor known to stimulate ECs proliferation and migration, promote EC survival and differentiation, as well as inducing capillary permeability [32]. Both VEGF and its receptors (i.e., VEGFR1, Flt-1; VEGFR2, Flk-1/KDR; VEGFR3, Flt-4) are essential for normal angiogenesis with individual but complimentary roles [32]. Regulation of VEGF production is complex and involves cytokines, growth factors, and/or hypoxia. VEGF is the product of a gene whose promoter contains an HRE (hypoxic-response element) that binds to HIF-1 (hypoxia-inducible factor 1), a transcription factor sensitive to hypoxic stimulus [33]. The myofiber intracellular PO2 falls from approximately 30 mmHg at rest to approximately 34 mmHg during moderate to heavy exercise, and such finding supports the role played by increased VEGF transcription in skeletal muscle capillary growth [34]. An interesting question concerns which cells within skeletal muscles are responsible for generating VEGF. We previously showed that within whole muscle, myofibers are a source of production of VEGF mRNA at the end of exercise and that the exercise-induced increase in VEGF mRNA occurred mainly in fasttwitch myofibers, which are large in size and predominantly glycolytic [35]. These findings are consistent with the suggestion that inadequate tissue cell oxygenation may be one of the triggers of exercise-induced capillary growth. These findings support the notion that myofibers generate the majority of measurable VEGF in muscle. However, whether this source of VEGF is effective in promoting angiogenesis remains discussed. But, VEGF within muscle is essential for muscle function, and, without this growth factor, exercise capacity is greatly diminished [33]. Taken together, these results suggest that reduced oxygen tension could be one of possible stimuli of capillary growth during exercise in human. However, VEGF alone may be insufficient to explain the muscle angiogenic response to exercise. Basic fibroblast growth factor (bFGF or FGF-2) and angiopoietins are growth factors involved in the exerciseinduced vascular remodeling [36]. Moreover, the expansion of the skeletal muscle microvascular network with ET results from an upregulation of angiogenic factors (i.e., mainly VEGF), suggesting that a combination of increased contractile activity, low O2 tension, and metabolic activity serves as stimuli for capillary growth. Responses of the Myosin Heavy Chain (MHC) Composition by Endurance Exercises It is now clear that in humans, ET leads to a shift toward a greater reliance on oxidative metabolism to provide energy for active myofibers. Adaptation of the contractile phenotype to ET is only subtle and consists mainly in a transition between fast MHC isoforms (i.e., between type IIa and type IIx MHC isoforms). A signal mechanism involving calcineurin, a cyclosporin-sensitive phosphatase, has been suggested to promote the myofiber-type conversion induced by increased motor nerve activity [37]. The serine/threonine phosphatase activity of the calcium-activated calcineurin leads to the dephosphorylation of a nuclear factor of activated T cells (NFAT). Dephosphorylated NFAT is then translocated to the nucleus, where it binds to specific nucleotide recognition sequences and stimulates the transcription of target genes, in synergistic interaction with other transcription factors [37]. Results of several studies suggest a

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specific role for calcineurin in the control of myofiber specialization [38]. In addition, MEF2, CaMK, PGC-1α, and PPARβ/δ signaling play a key role in controlling muscle fibertype-specific gene programs, providing links between motor nerve activity and selective changes in gene expression by molecular mechanisms [19].

THE MUSCULAR ADAPTATION TO RESISTANCE TRAINING (RT) Maintaining skeletal muscle mass and function is critical for autonomy, mobility, quality of life, and tertiary prevention of many chronic diseases. Skeletal muscle mass is also one of the determining factors for athletes involved in strength/power disciplines or who desire to improve esthetic appearance. The major adaptive response to RT concerns the increase in muscle size.

The Training-Induced Increase in Muscle Mass The rate of increase in muscle cross-sectional area, a marker representative of muscle growth, has been estimated to be approximately 0.15% day21 [39]. The balance between synthesis and breakdown of muscle proteins governs muscle mass accretion. If muscle protein synthesis (MPS) exceeds muscle protein breakdown (MPB), an increase in skeletal muscle mass can occur. Many previous studies showed that repeated bouts of resistance exercise induce cumulative periods of positive net protein balance which are at the origin of increases in the myofibrillar volume of individual myofibers, suggesting that muscle growth with RT is primarily dictated by the balance between synthesis and breakdown of muscle proteins [40]. It is commonly accepted that MPB changes much less than MPS in response to resistance exercise, and MPB is not generally considered to be a process that contributes significantly to explain the gains in muscle mass [41]. Moreover, resistance exercise is associated with an increase in MPS for up to 48 hours postexercise [42]. Thus, accretion of proteins in skeletal muscle and then muscle growth during RT are primarily dictated by the regulation of MPS. The control of MPS through the modulation of translation initiation is a particularly important regulatory step in response to resistance exercise. Repeated bouts of resistance exercise (i.e., RT) can significantly increase muscle size and muscle fiber hypertrophy. Current guidelines on RT suggest that loads higher than 70% 1-repetition maximum (1RM) contribute to maximize gains in muscle growth and strength [43]. However, a recent systematic review of the current scientific literature showed that gains in 1RM strength were significantly greater in response to high-load, versus low-load RT protocols, while changes in muscle mass were similar between conditions [44]. The findings clearly suggest that gains in maximal strength are maximized with the use of heavy loads while muscle hypertrophy can be equally achieved across a spectrum of loading ranges. There is now a large body of evidence to support the importance of increased MPS at the end of resistance exercise to explain the RT-induced muscle hypertrophy. Recent findings suggest that the large interindividual variance in MPS accounts, at least partly,

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Progress (% baseline)

Strength

Muscle hypertrophy

FIGURE 4.2 The respective roles played by neural and enhanced muscle mass on strength/ power gains during RT. In the early phase of RT, neural adaptations predominate. Thereafter, strength/power gains mainly depend from the extent of muscle hypertrophy. RT,resistance training.

Neural adaptation

time

for the large variability observed in muscle growth. The response of MPS at the end of a single bout of exercise likely changes with repeated bouts of resistance exercise, and we have now evidence that the amplitude and duration of the exercise-induced increase in MPS are modulated by the training status. Moreover, it has been shown that the acute measures of MPS following the first bout of resistance exercise were not correlated with muscle hypertrophy following 16 weeks of training [45]. It is therefore difficult to predict the hypertrophic response to a specific training program by only measuring MPS for a few hours after the first exercise. An interesting point of concern is related to the respective roles played by neural adaptation and changes in muscle mass to explain the training-induced improvement in strength performance. Strength gains during RT are related not only to the adaptive responses of the quantity and quality of muscles but also the extent to which skeletal muscles are activated. It is now commonly accepted that the early increase in voluntary strength reported during RT (i.e., training ,6 weeks) is mainly associated with neural adaptation, whereas muscle mass contributes to explain strength adaptations with training programs lasting more than 610 weeks [46] (Fig. 4.2).

Expansion of the Skeletal Muscle Microvascular Network RT also leads to an increase in skeletal muscle vascularization. Since repeated resistance exercises increase the mean fiber cross-sectional area, expansion of the microvascular network is required to ensure the delivery of oxygen and amino acids required for muscle growth. An increase in the capillary-to-fiber ratio is commonly reported following prolonged RT [47], and this adaptive response contributes to maintain, but not increase, perfusion capacity, oxygen, and substrate delivery. A recent study clearly demonstrated that myofiber hypertrophy and increased capillarization occurred in tandem during RT [48]. Because the amino acid availability within myofibers is required for protein synthesis, it could be speculated that angiogenesis develops during RT likely to preserve the oxygen and substrate diffusion capacity, and then muscle hypertrophy may be facilitated by the expansion of the muscle microvasculature.

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The Molecular Mechanisms Involved in the Muscle Responses to RT One of the most widely recognized mechanisms at the origin of RT-induced enhancement in muscle mass involves mechanical tension. Mechanical stimuli are known to regulate the rate of protein synthesis, and then to regulated muscle mass, but how skeletal muscles convert mechanical information into biochemical events remains poorly defined. The training-mediated increase in skeletal muscle size is mainly due to an increase in the cross-sectional area of each myofiber. Some experiments in animal models showed that muscle mass hypertrophy was associated with a slight increase in the number of individual myofibers. However, there is only little evidence that hyperplasia occurs in response to RT in human and could contribute to explain the increased muscle mass.

The Control of Ribosomal Protein Synthesis The increase in muscle mass mainly results, first, from enhanced translational efficiency related to the number of ribosomes binding to a single mRNA, and second, from enhanced translational capacity after upregulation of the ribosomal content [49]. It is generally thought that the increase in the rate of protein synthesis in response to a single bout of resistance exercise primarily results first from enhanced translational efficiency and that repeated resistance exercises lead to ribosome biogenesis to generate more ribosomes, and then to increase the muscle translational capacity. Initiation (i.e., starting off), elongation (i.e., adding on to the protein chain), and termination (i.e., finishing up) steps of mRNA translation into proteins mainly determine the translational efficiency. The preinitiation complex, which includes activated eukaryotic initiation factors 2 and 3 (eIF2, eIF3), as well as the recognition capacity of the mRNA, which involves activation of several eIF4 subunits, are needed for translation to get started. The phosphorylation rates of eIFs are then involved in the translation efficiency of mRNA, and the subsequent enhanced MPS. The translational capacity of myofibers is mainly determined by the quantity of ribosomes. The ability of myofibers to produce many proteins from one mRNA via polyribosomes has provided evidence that ribosome biogenesis is essential for increasing protein synthesis and ensure myofiber growth [49]. There is now evidence that ribosome biogenesis contributes to increase protein synthesis, thereby leading to skeletal muscle hypertrophy, especially during RT. Skeletal MPS is regulated through a multiprotein phosphorylation cascade which involves the protein B (Akt) and its downstream target mTORC1 (Fig. 4.3) [50]. The kinase mTORC1 has a central role in the control of ribosome biogenesis by promoting the translation of the mRNAs encoding the ribosomal proteins and the transcription of ribosomal RNAs. Moreover, mTORC1 acts as one of the main regulators of anabolic signaling via phosphorylation of several downstream signaling molecules including the 70-kDa ribosomal S6 kinase (p70S6k), eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein S6 that regulate MPS and muscle mass (Fig. 4.3). Activation of this signaling pathway contributes to control mRNA translation initiation, elongation, and termination, increasing the translational efficiency, and then enhancing MPS [51].

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p70

S6k

FIGURE 4.3 Simplified model of the mTORC1 signaling pathway, involved in the control of muscle protein synthesis. Protein synthesis is regulated by the successive activations of PI3K, protein kinase B (Akt), leading to mTORC1 phosphorylation, and activation of its downstream targets p70S6K, and 4EBP1. PA concentration may also promote mTORC1 activation. PA can be synthesized by various classes of enzymes, including PLD. Arrows indicate activating events, whereas perpendicular lines indicate inhibitory events. The solid lines represent direct activation, whereas dashed lines represent putative direct or indirect activation. mTORC1, mammalian target of rapamycin C1; PI3K, phosphoinositide 3kinase; p70S6K, p70S6 kinase; 4EBP1, eIF4Ebinding protein 1; PA, phosphatidic acid; PLD, phospholipase D.

4EBP-1 Protein synthesis

We have now a large body of evidence to consider that mTORC1 acts as a control center integrating inputs on energy stress, hormonal status, and amino acids, through its downstream targets, to control the rate of MPS by both translational efficiency and capacity. Potential Mechanisms Involved in the Mechanotransduction Signaling by mTORC1 can be regulated by various stimuli, including growth factors, nutrients, and mechanical signals. To date, signaling through the PI3K/Akt/mTORC1 pathway is the only molecular pathway that has been identified as being necessary for mechanically induced muscle growth (Fig. 4.3) [52]. However, a number of studies showed that activation of the PI3K/Akt signaling pathway is sufficient, but not necessary for mTORC1 activation in response to mechanical stimuli. It has been suggested that mechanical stimuli induce an increase in phosphatidic acid (PA) concentration, and PA has now emerged as a potentially direct regulator of mTORC1 signaling. Based on several experimental observations, it can be argued that phospholipase D activity might contribute to the early increase in PA, in response to mechanical stimuli. However, most of potential regulators of PA/mTORC1 signaling remain to be determined, and their roles in the mechanical activation of mTORC1 clarified. Activation of Satellite Cells and the Control of Muscle mass Several studies provided compelling evidence to support the concept that myofiber hypertrophy is facilitated by satellite cell activation, proliferation, and the addition of new myonuclei to existing myofibers [53]. At least some initial myofiber expansion is possible before the recruitment of nuclei from satellite cells. Primary regulators of satellite cell activation include members of the myogenic regulatory factor (MRF) family [e.g., myogenic

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differentiation (MyoD) and myogenin transcription factors]. Increased MRF mRNA abundance, especially MyoD, provides molecular landmarks for the activation and proliferation of satellite cells. It also has been suggested that individuals with a greater basal presence of satellite cells showed a high ability to expand the satellite cell pool, incorporate new nuclei, and achieve a high level of muscle growth [53].

THE SPECIFIC RESPONSES TO CONCURRENT TRAINING The integration of endurance exercises together with resistance exercises in the same session of a training program is referred to as CT [54]. This training modality is now a popular strategy to develop simultaneously strength/power gains and endurance capacity.

The Physiological Responses to CT Several studies showed that performing strength and endurance exercises concurrently can interfere with long-term adaptations, in comparison to when they are performed alone [55]. Indeed, the increased muscle strength and myofiber hypertrophy in response to resistance exercises are considered as negatively affected by the inclusion of ET, when compared to isolated RT. The reduced gains in muscle performances after a CT program seem to be dependent on several factors, such as exercise frequency, volume, duration of the recovery period, and type of exercise, as well as muscle groups trained. The sequence of exercises (i.e., strength exercise performed prior to endurance exercise, or the reverse) might influence the outcome of CT, in comparison with ET or RT alone. When one type of exercise is performed immediately prior to the second type of exercise, local or systemic fatigue, as well as interactions between signaling pathways, would negatively affect the long-term adaptations to physical training. A recent meta-analysis confirmed that when endurance exercises immediately preceded strength exercises, local or systemic fatigue, or negative interactions between intracellular signaling pathways, interfere the long-term strength adaptations [55]. Strength/power performances are compromised for at least 68 hours following endurance exercise [56]. In contrast, when strength exercises preceded endurance exercises, no significant difference was shown on the long-term strength/power adaptations. Likewise, the training sequence had no detectable impact on aerobic capacity. However, recent studies failed to evidence the interference of endurance exercises on muscle hypertrophy induced by resistance exercises [57]. It should be emphasized that it might difficult to compare CT studies due to marked differences in the training programs. Therefore, the CT effects on physical performances, compared with either endurance or RT programs, remain a matter of debate. Further studies are clearly required to clarify the roles played by the characteristics of endurance exercises on the gains in muscle mass in response to resistance exercises.

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The Molecular Responses to Concurrent Training Endurance exercise is associated with a reduction in the availability of muscle cell energy (i.e., ATP/AMP ratio), at the origin of AMPK activation. Some studies attributed the reduced gains in muscle mass and strength on the inhibitory effects of activated AMPK on mTORC1 phosphorylation, downregulating its downstream targets, and thus hampering anabolic processes and MPS [58]. This hypothesis has been examined in a rodent model in which muscle fibers were electrically stimulated for prolonged periods at low frequency (to mimic ET) or for short periods (to mimic RT) [59]. Using this exercise model, the authors observed a reciprocal relationship in the activation of AMPK and Akt pathways. During low-frequency stimulation, increased AMPK activity was associated with an inhibition of the Akt/mTOR signaling pathway. Results of this experiment led to the proposal of a mechanism for the negative interaction between endurance and strength exercises (Fig. 4.4). However, recent studies failed to support the AMPK-induced inhibition of the mTORC1 signaling. For instance, prior activation of AMPK by high-intensity cycling exercise did not inhibit the increased activation of mTORC1 signaling measured at the end of subsequent resistance exercise [60,61]. Therefore, the effects of CT on intracellular signaling and then on the long-term responses of skeletal muscles likely depend on the sequence, mode, type, intensity of exercises, and individual variability. In conclusion, mammalian skeletal muscles have a remarkable capacity for accommodating changes in demand. Skeletal muscle adaptations in response to increased use occur from (1) cumulative effects of transient changes in gene expression following acute bouts of exercises, and/or (2) enhanced translational efficiency and capacity, related to the number of ribosomes binding to a single mRNA, and the increased ribosomal content, FIGURE 4.4

PI3-K

Glycogen depletion AMP ATP

Putative adaptive pathways in response to the concomitant integration of endurance and resistance exercises in a training program. The figure highlights the cross talk between AMPK and mTORC1 pathways. mTORC1, mammalian target of rapamycin C1; AMPK, 50 AMP-activated protein kinase.

Akt AMPK activation TSC2 PGC-1α mTOR

p70S6k Increased protein synthesis

4EBP-1 Mitochondrial biogenesis

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respectively. Regardless of the mechanism involved, the enhanced translation efficiency and capacity, as well as activation and/or repression of specific pathways and subsets of genes, require an increase in energy and amino acid availability.

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[48] Holloway TM, Snijders T, van Kranenburg J, vanLoon LJ, Verdijk LB. Temporal response of angiogenesis and hypertrophy to resistance training in young men. Med Sci Sports Exerc 2018;50:3645. [49] Chaillou T, Kirby TJ, McCarthy JJ. Ribosome biogenesis: emerging evidence for a central role in the regulation of skeletal muscle mass. J Cell Physiol 2014;229:158494. [50] Gonzalez AM, Hoffman JR, Stout JR, Fukuda DH, Willoughby DS. Intramuscular anabolic signaling and endocrine response following resistance exercise: implications for muscle hypertrophy. Sports Med 2016;46:67185. [51] Drummond MJ, Fry CS, Glynn EL, Dreyer HC, Dhanani S, Timmerman KL, et al. Rapamycin administration in humans blocks the contraction-induced increase in skeletal muscle protein synthesis. J Physiol 2009;587:153546. [52] Hornberger TA. Mechanotransduction and the regulation of mTORC1 signaling in skeletal muscle. Int J Biochem Cell Biol 2011;43:126776. [53] Petrella JK, Kim JS, Mayhew DL, Cross JM, Bamman MM. Potent myofiber hypertrophy during resistance training in humans is associated with satellite cell-mediated myonuclear addition: a cluster analysis. J Appl Physiol 2008;104:173642. [54] Coffey VG, Hawley JA. The molecular bases of training adaptation. Sports Med 2007;37:73763. [55] Murlasits Z, Kneffel Z, Thalib L. The physiological effects of concurrent strength and endurance training sequence: a systematic review and meta-analysis. J Sports Sci 2017;7:18. [56] Robineau J, Babault N, Piscione J, Lacome M, Bigard AX. Specific training effects of concurrent aerobic and strength exercises depend on recovery duration. J Strength Cond Res 2016;30:67283. [57] De Sousa. Molecular adaptations to concurrent training. Int J Sports Med 2013;34:20713. [58] Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 2002;277:2397780. [59] Atherton PJ, Babraj JA, Smith K, Singh J, Rennie MJ, Wackerhage H. Selective activation of AMPK-PGC1alpha or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation. FASEB J 2005;19:7868. [60] Kazior Z, Willis SJ, Moberg M, Apro´ W, Calbet JA, Holmberg HC, et al. Endurance exercise enhances the effect of strength training on muscle fiber size and protein expression of Akt and mTOR. PLoS ONE 2016;11 (2):e0149082. [61] Apro´ W, Moberg M, Hamilton DL, Ekblom B, van Hall G, Holmberg HC, et al. Resistance exercise induced S6K1 kinase activity is not inhibited in human skeletal muscle despite prior activation of AMPK by highintensity interval cycling. Am J Physiol Endocrinol Metab 2015;308:E47081.

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The Role of Specific Nutriments in Sarcopenia Associated With Chronic Diseases: A Focus on Cancer Sami Antoun Emergency Unit, Ambulatory Care, Gustave Roussy Cancer Campus, Villejuif, France

SARCOPENIA IN CHRONIC DISEASES Nutritional disorders are a serious and often underrecognized consequence of many chronic diseases. A combination of decreased food intake and metabolic abnormalities can lead to nutrition-related disorders which in turn are associated with delayed recovery, as well as increased morbidity and mortality. A number of guidelines and definitions describing the relationships between chronic diseases and metabolic and nutritional effects have been published over the last few years [1 5]. However, there remains a need for standard terminology, and several terms including malnutrition, cachexia, and sarcopenia are widely used without precise definitions. The classification of nutrition-related disorders proposed in the recent ESPEN guidelines is unique, since it separates the general diagnosis of malnutrition according to etiology-based types of malnutrition [6]. They describe disease-related malnutrition with or without inflammation, as well as situations with malnutrition but without disease. The classification also differentiates between acute diseases, injury-related malnutrition, and chronic disease-related malnutrition with inflammation. In oncology literature, several terms have appeared including precachexia and refractory cachexia [7]. Between the guidelines and the evolving literature, the definitions and concepts are becoming progressively more precise and are increasingly taking into account the mechanisms of the nutritional disorders, differentiating between acute and chronic disease and severity of symptoms. Most definitions are based on similar criteria: weight loss, decreased energy intake, and body mass index (BMI). While including inflammatory markers such as C-reactive protein (CRP) in the definition of malnutrition is controversial, almost all definitions now incorporate the concept of skeletal muscle (SM) wasting or

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sarcopenia [3,7,8]. Nonetheless, there is no doubt that a high level of confusion remains in terms of the diagnostic criteria for cachexia and malnutrition. The controversy for diagnosing sarcopenia is even more resounding than that over the definition of cachexia. This term was originally used to define age-related SM disorders; however, it has evolved and is now used to describe SM decline in chronic diseases states and several other conditions including starvation, denervation, immobilization, and physical inactivity [9]. The main area of divergence in the definition of sarcopenia revolved around the inclusion of SM function or strength impairments. In the elderly and for multiple international organizations, sarcopenia is defined as functional impairment combined with low muscle mass [9], while in the cancer field cachexia specialists focus on SM mass loss alone [7,10]. This difference in definitions may be explained by the expected consequence of the muscle disorders in each chronic illness, with a given group including in their definition of sarcopenia, the most relevant item(s) for their setting. In the elderly, autonomy is a preoccupying issue and for gerontologists, sarcopenia includes not only loss of muscle mass but also muscle functional impairment, with sarcopenia being an element of the larger syndrome of frailty [11 15]. For oncologists, studies of the body composition of cancer patients reveal that it is specifically the loss of SM that predicts risk of postoperative complications, chemotherapy toxicity, and mortality [16,17]. Much remains to be clarified in terms of defining sarcopenia, whether it be in the definition of muscle function impairment, the best test to assess it (walking speed or grip strength), or in the threshold values for muscle wasting (dependent on ethnic groups or initial BMI). Given the differences in the contexts of the definitions of sarcopenia, it is in turn difficult to have a unique criterion to evaluate the effects of a treatment and particularly the effects of a specific nutritional support. The aim of using a specific nutriment approach is dependent on the context; it may be to improve the balance between skeletal muscle protein synthesis (MPS) and muscle proteolysis when the focus is the underlying metabolic mechanisms, to increase muscle mass in a setting where improving disease outcome is the important, or improving muscle function or strength in the context of frailty in the elderly.

IMPACT OF SARCOPENIA ON DISEASE OUTCOMES IN THE CHRONIC SETTING Chronic Diseases Other Than Cancer In the past, in chronic diseases, nutritionists focused on weight loss and the link between low BMI or weight loss and outcomes. However, focusing on weight is no longer the optimal approach as weight changes can be linked to water or adipose tissue changes, and SM has now become the “Holy Grail.” The last few years have thus seen extensive research into the association between SM dysfunction and outcomes in chronic diseases. Almost all studies have shown an association between sarcopenia (irrespective of whether it was assessed by muscle mass, muscle strength, or physical performance) and mortality, as reported for chronic kidney disease [18,19], heart failure [20 22], respiratory disease [23 25], and liver cirrhosis [26 28]. Sarcopenia has also been associated with the impact of muscle impairment on specific disease symptoms; for example, in chronic heart failure,

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patients with muscle wasting had lower left ventricular ejection fraction [22] and reduced peak VO2 [21]. In chronic respiratory diseases, SM plays a vital role providing the mechanical basis for breathing, and SM dysfunction is one of the most common extrapulmonary manifestations [29], while in liver cirrhosis, sarcopenia is a predictor of minimal hepatic encephalopathy [30].

Cancer In cancer patients, in addition to the role of muscle mass which has been widely explored, muscle density and muscle strength are associated with mortality. Studies have highlighted the impact of low muscle mass on outcome; patients with low muscle mass have decreased overall survival, increased susceptibility to chemotherapy toxicity, and an increase in postoperative complications. A prominent article by Prado et al. published in 2008 highlighted the value of studying body composition parameters as prognostic factors for cancer outcomes [16]. The authors showed that for obese colorectal and lung cancer patients, overall survival in patients with low SM mass was 10 months shorter compared to those without sarcopenia. Since then, many other studies have confirmed this link between sarcopenia and worse prognosis [17,31]. In another important study carried out in 1473 lung and gastrointestinal cancer patients, Martin et al. showed that higher weight loss, low muscle mass, and low muscle density were independent prognostic factors of survival [31]. Patients with all three of these parameters had a median survival of 8.4 months compared to those with none of these variables whose median survival was 28.4 months. This study was one of the first to show the prognostic role of low muscle density in cancer, which was one of the three strongest prognosis factors analyzed. Similar results with muscle density have been reported in patients with metastatic renal cancer [32], with median overall survival in patients with low muscle density half that of patients with high muscle density (14 vs 29 months, respectively). On the other hand, for muscle function (6 minutes’ walk test, handgrip strength test), one of three components of muscle assessment tested, few studies have shown a link with survival in advanced cancer [33,34]. The first study to show an association between sarcopenia and susceptibility to chemotherapy toxicity was published by Pr Carla Prado and the Edmonton team in colon cancer patients [35]. The group showed that patients experiencing dose-limiting toxicity (DLT) received a mean 5-fluourouracil dose of 17.9 mg/kg of lean body mass compared to 16.3 mg/kg in patients without DLT. Many other studies have since followed [36 38], including in advanced medullary thyroid carcinoma patients treated with vandetanib, in which patients with low SM mass had a higher probability of DLT (73% vs 14%) and higher vandetanib serum concentrations (1037 vs 745 ng/mL; P 5 0.04) [38]. One hypothesis to explain this is that the mechanism of this toxicity involves an overdose of the chemotherapy when adjusted to muscle mass. We have known for several years that there is a link between weight loss and postoperative complications. Many groups have shown that sarcopenia predicts postoperative complications for a broad range of surgical interventions [39 41]. Given that many studies have shown that sarcopenia alone is not the optimal predictor of postoperative

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complications, the concept of “frailty” including muscle function should be included in the muscle mass assessment [39,42]. Thus, in chronic diseases, a decrease in mass, density, strength, or function of SM, whether assessed alone or included in a more complex concept of frailty, is linked to decreased survival. Muscle wasting may be associated with disease-specific outcomes, such as increased susceptibility to chemotherapy toxicities in cancer, postoperative complications for surgery, decreased left ventricular ejection fraction in heart failure patients, and increased encephalopathy for liver cirrhosis.

MUSCLE ANABOLISM IN CANCER PATIENTS Retrospective Computed Tomography (CT) Studies While there is clearly a link between SM dysfunction (loss of muscle mass, decreased muscle function or strength) and outcome, the main issue patients and clinicians face concerns the possibility of reducing or correcting these muscle disorders, and some interesting studies have been published in cancer patients. Tan et al. published some surprising results [17]. Computed tomography (CT) imaging was used to demonstrate that muscle anabolism can occur during cancer treatment, with increases in muscle mass. They showed that in pancreatic cancer patients, one-third had an increase in muscle mass, one-third had a stable muscle mass, and one-third had decreased muscle mass, while all but one patient lost adipose tissue. Prado et al. conducted a study based on CT images evaluating muscle loss and gain in a cohort of 368 advanced cancer patients [43]. They showed that 60% of patients gained or stabilized muscle mass, notably in patients with long survival, with the majority of cases of muscle gain (84%) occurring at least 3 months before death. Possible causes of muscle gain have not been well studied. In addition, the Prado et al. study showed that among the top 5% of patients with the largest quantitative muscle gain, 89% were considered by the oncologist as having stable disease. These retrospective studies offer important data showing that gaining or maintaining muscle mass is possible for cancer patients and that tumor response is a principal factor in muscle gain. Currently, few studies have evaluated other potential factors which may favor a positive net balance between protein synthesis and protein breakdown.

Protein Anabolism: A Balance Between Protein Synthesis and Protein Breakdown The concept of SM homeostasis has historically been viewed as the net balance between the two separate processes of MPS and muscle protein breakdown (MPB) [44]. A more recent view suggests that these two biochemical processes are not independent of each other but are in fact precisely coordinated by a web of intricate signaling networks [45,46]. A review by Biolo et al. elegantly demonstrated that in chronic diseases, the rate of protein turnover also influences protein loss, with conditions of low protein turnover typically associated with protein preservation [47].

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Muscle protein anabolism results from the stimulation of synthesis or the suppression of proteolysis, with a combination of these two giving a maximal response. While it has been demonstrated in healthy humans that increasing food and protein intake can stimulate protein synthesis, this is not necessarily the case in catabolic situations. Reduced protein synthetic response to food intake has been termed anabolic resistance. Many interconnected and overlapping intracellular pathways regulate MPS [48]. Amino acids (AAs), insulin, and contraction appear to converge and stimulate mammalian target of rapamycin (mTOR) activation which is a central signaling pathway controlling protein synthesis. Administration of specific AAs could be of value because in addition to being a substrate, they are potential modulators of protein turnover. There are also solid data demonstrating the capacity of leucine to modulate protein metabolism by increasing substrate availability, increasing the secretion of anabolic hormones such as insulin, and directly modulating anabolic signaling pathways by stimulating protein synthesis and reducing protein breakdown [49]. The following section addresses the role of AAs in promoting muscle anabolism, highlighting leucine and branched chain AAs (BCAAs).

THE ROLE OF TOTAL AND SPECIFIC AMINO ACIDS IN PROMOTING MUSCLE ANABOLISM IN CANCER We have long known that in cancer cachexia, MPB produces AAs needed for synthesis of inflammatory proteins. This was first demonstrated by Lundholm et al. in 1978 in a study of the metabolic changes in SM s and liver tissue from cancer patients and tumorbearing mice [50]. Both mice and patients showed a significantly increased incorporation rate of leucine into liver proteins, while in SM tissue, the incorporation rate of leucine was significantly decreased. The results were compatible with an increased protein breakdown rate of SM proteins, since radioactivity disappeared significantly more rapidly in muscles from tumor-bearing mice. Since this study, many others have been published and the mechanisms involved in muscle wasting appear highly complex. Data concerning the relationship between AA administration and muscle protein anabolism are lacking. Few data are available, obtained from very disparate clinical studies from a wide variety of settings, including both highly sophisticated protein kinetic studies and clinical trials.

Short-Term Interventions: Amino Acid Kinetic Studies in Cancer Patients Studies using isotopic tracer methodology are of interest because they directly explore the processes of MPS and MPB that are affected by the administration of AAs. These sophisticated studies provide information about the mechanisms involved, although it is not yet proven that repeated AA administration gives similar results as single administration. Six kinetic studies have been published to date, three focusing on global protein turnover [51 53], while three studied muscle protein metabolism from muscle biopsies [54 56]. It is currently difficult to draw conclusions from these protein kinetic data given the small number of studies published. In addition, they involve a very small number of patients, different types of cancer, different nutritional status, and different study

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TABLE 5.1 Patient Characteristics and Study Design in Amino Acid Kinetic Studies—Studies Assessing Whole-Body Protein Synthesis and Protein Balance Reference (n patients per treatment) Winter et al. (n 5 10)

Type of cancer

Nutritional status Systemic inflammation Energy intake

Study design Perfusion vs oral intake AA vs AA 1 EAA

Nonsmall cell lung

WL: 7.8%; BMI: 22.060.9, CRP: 12.7

• Hyperinsulinemic-isoAA: Control: AA unknownExperimental: PB k Ca and Co, k PB in Ca. k PB in Co, PS unchanged in Ca and CO, protein 30 g net balance k Ca . k Co

Energy intake: 1891676

IV continuous, 150 min

WL: 8.4%; BMI: 26.561 CRP: 9.8

CaCo: AA 70 g (14 g PS and net balance mCaE group . EAA/non-EAA), CaE: CaCo group (P , 0.001) AA 70 g (14 g EAA 50% Leu)

Energy intake: 19446215

oral 5 min, 250 mL

WL: 24.3%; BMI: 20.0 CRP: 8.3

Control and cancer : 480 mL, (casein 50 g; Leu 5.25 g)

• Baseline: PB in Ca . PB in Co, PS in Ca equal to baseline, PS m Co, net balance m Ca and Co

Energy intake unknown

oral 4 h, 480 mL

• Ingestion: PB k Ca and Co PS in Ca equal to baseline, PS m Co, net balance m Ca and Co

Clin Nutr 2012

Engelen (n 5 13)

Nonsmall cell lung

Ann Oncol 2015 Van Dijk (n 5 8) JCSM 2015

pancreas

Results

• Hyperinsulinemic-hyperAA: PB in Ca and Co unchangedPS m in Ca and CoNet balance m in Ca and Co (similar)

WL, weight loss (% of 6 months prior weight); BMI, body mass index (kg/m2); CRP, C-reactive protein (mg/L); energy intake (kcal/day); Ca, cancer patients; Co, control group; E, experimental group; AA, amino acids; EAA, essential AA; PB, protein breakdown; PS, protein synthesis.

protocols. The interpretation of the results is all the more difficult as the method of AA supplementation (parenteral administration or oral feeding), the quantities of AAs, and the administration of specific AAs (particularly BCAAs) are different from one study to another. In addition, patients had a range of tumor stages, which is important given the growing evidence for a link between tumor evolution and potential anabolic resistance to AA administration. The translation of acute anabolic effects into gains in muscle mass and function need to be confirmed by long-term intervention trials. An overview of the six studies assessing whole-body protein synthesis and whole-body protein balance is provided below. Tables 5.1 and 5.2 summarize the studies for cancer type, the nutritional status, and, when reported, the mechanisms involved (energy intake and systemic inflammatory parameters, e.g., CRP), along with the study design, the administration of specific AAs, and the effects compared to a group control. • Winter et al. [51] assessed whole body and glucose intake kinetics in 10 nonsmall cell lung cancer (NSCLC) patients matched to a control healthy group during a euglycemic, hyperinsulinemic clamp under conditions of isoaminoacidemia followed by

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TABLE 5.2 Patient Characteristics and Study Design in Amino Acid Kinetic Studies—Studies Assessing Muscle Protein Synthesis and FSR Author (nbre in treated group)

Type of cancer

Williams (n 5 13)

Colorectal WL unknown; BMI 27.661.1, CRP 8.8

Am J Clin Nutr 2012 Dillon (n 5 6)

Clin Nutr 2011

Study design Perfusion vs oral intake AA vs AA 1 EAA

Results

IV continuous (bolus 30 mg/kg, continuous 102 mg/kg)

• Muscle PS m in Co; muscle PS no change in Ca • Muscle PB m in Co and Ca

WL .10%; BMI 22.063.0, CRP normal

Prot 40 g (EAA and non-EAA)

Energy intake unknown

Oral 540 mL, 4 h

Compared to basal: muscle PS m, Muscle PB unchanged, net balance m (from negative to zero)

WL 2.9%62.2%; BMI 25.163.3 CRP 28.7

CaCo: 24 g prot (Leu 2.0) CaE: 40.1 (leu 7.5)

Energy intake unknown

Oral, 30 min

Energy intake unknown Ovarian

Clin Nutr 2007 Deutz (n 5 10)

Nutritional status Systemic inflammation Energy intake

Colon lung

CaCo: FSR no change CaE: FSR increase 40% (P 5 0.027)

FSR, fractional synthetic rate; WL, weight loss (% of 6 months prior weight); BMI, body mass index (kg/m2); CRP, C-reactive Protein (mg/L); energy intake (kcal/day); Ca, cancer patients; Co, control group; E, experimental group; AA, amino acids; EAA, essential AA; PB, protein breakdown; PS, protein synthesis.

hyperacidemia. NSCLC patients showed severe peripheral insulin resistance of glucose concurrent with a blunted net whole protein anabolic response. With added hyperaminoacidemia, NSCLC patients achieved normal protein anabolism. • Because pancreatic cancer is often accompanied by severe muscle wasting, van Dijk et al. studied eight cachectic pancreatic cancer patients and seven control subjects [52]. Patients received oral treatment (480 mL) with 50 g/L casein (isoleucine 2.85 g/L and leucine 5.25 g/L). This study demonstrated that whole-body protein turnover is higher in cancer patients than in controls. Both groups generated a similar anabolic response to feeding; cancer patients achieved this by reducing protein breakdown, whereas controls increased their protein synthesis. • Engelen et al. [53] measured protein synthesis and whole-body protein breakdown in 13 NSCLC patients compared to healthy controls after oral intake of 14 g of high leucine level vs an AA mixture containing essential and nonessential AAs. The presence of essential AAs resulted in a high anabolic response. In both groups, a highly significant relationship was present between net protein anabolism and dietary essential AA intake. This relationship in NSCLC patients was still present even taking into account recent weight loss or survival (,6 vs .12 months). The design of these three studies differs in terms of the type of cancer, the mode of administration (perfusion vs oral intake), the type of AAs (essential vs nonessential), and the amount of systemic inflammation and of total daily energy intake. Nevertheless they

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all show that cancer patients have the potential for whole-body protein anabolism despite anticancer therapy and regardless of the status of tumor evolution, by increasing protein administration with or without essential AAs. These results are consistent with Bozzetti and Bozzetti’s recommendation to perfuse cancer cachexia patients with high-protein solutions having a high proportion of BCAAs [57]. The limiting factor of these three studies is that they assessed whole-body protein turnover and not SM protein anabolism which is a key aspect of nutritional support. The increase in whole-body protein turnover could be associated with a reprioritization of nitrogen away from peripheral tissue (muscle) toward increased hepatic production of acute phase proteins, as described in 1978 by Lundholm et al. [50]. The main question to address is the potential anabolic response in muscle and the increase of MPS secondary to nutritional support. The “gold standard” for the assessment of MPS is the measurement of the fractional synthetic rate by muscle biopsies. Given the difficulties of designing such studies, few data are available [54 56]. • The study by Dillon et al. was conducted in six ovarian cancer patients [54]. Interestingly, these patients were still receiving chemotherapy and were not in a terminal stage of their cancer. The nutritional support was based on the infusion of high amounts of AAs. Patients received 40 g of AA ingested as a 30 mL bolus every 10 minutes for 3 hours. Protein synthesis increased significantly from basal levels with AA support, while protein breakdown remained unchanged. • The design of the Deutz et al. study was similar to that of Dillon, using radiolabeled phenylalanine to study MPS [55]. Key aspects were that (1) the experimental group not only received high amounts of protein as in Dillon study but also the addition of leucine and fish oil; (2) the study was conducted in cancer patients with or without a small amount of weight loss and with a normal or overweight BMI showing that at early stages of cancer and before the observation of malnutrition, there was a resistance to muscle anabolism, as seen in the control group; and (3) the main result was that after feeding, MPS was increased in early stages of malnutrition. • More recently, Williams et al. studied the effect of cancer and tumor resection on protein metabolism under fasted and intravenous fed conditions [56]. They studied patients before and after tumor resection and compared them with a healthy agematched control group. Increases in MPS after feeding were apparent in healthy control subjects; however, there was no change in fed protein synthesis before surgery when the tumor had not yet been removed. After surgery, MPS increases were once again possible in the fed state. There was a trend for increases in MPB under both fasted and fed conditions in cancer patients before surgery. After surgery, fasted and fed rates of breakdown were reduced in cancer patients. Despite the differences in the results of these six studies, a number of general conclusions can again be drawn: 1. Early in tumor evolution with low or normal protein intake, there is a decrease in muscle protein anabolism (two of the three studies with muscle biopsies). This anabolic resistance is well described in the Deutz study [55]. Of note, anabolic resistance was observed early with or without very low weight loss (about 2%).

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2. Interestingly, patients in most of the studies had low inflammatory systemic syndrome. CRP levels were normal or near normal in five of the six studies while in the sixth study, the mean CRP value was 28.7 mg/L (which is still relatively low). Energy intake was normal in all three studies where this information was available which may reflect that patients were at an early disease stage. 3. With increasing protein intake, irrespective of whether or not it contained leucine, five of the six studies showed the potential for increasing the net protein balance, including the three studies that explored whole protein turnover and two of the three studies that explored the fractional synthetic rate in muscle biopsies. However, in the Williams study [56], after the removal of the cancer, the fed state increased MPS was seen again.

Branched Chain Amino Acids: Clinical Cancer Studies Human interventional studies with specific AA administration are rare and few studies assessing muscle mass or muscle function in cancer patients have been published. Among them, BCAAs were the most frequent AA studied [58]. A study by May et al. in 2002 patients who had weight loss of at least 5% gave promising results [59]. Patients received a control mixture of nonessential AAs or an experimental treatment containing β-hydroxyl-β-methylbutyrate, L-arginine, and glutamine. The authors reported that 35% of the patients withdrew before the evaluation at 4 weeks and also observed a significant increase in fat-free mass (FFM) in the AA-supplemented group (1.1 kg) compared to the control patients who lost 1.3 kg of FFM (P 5 0.02). While this FFM gain was an encouraging outcome, unfortunately a more recent study with a similar design and many more patients did not find a similar positive effect of essential AAs on FFM [60], with no statistically significant difference in the 8-week lean body mass, weights, or quality of life scores. An important point that should be highlighted is that in both of these studies, many patients withdrew before the end of the protocol. The lack of benefit of the nutritional support could be related to the status of disease progression, and the treated patients could have been in a refractory cachexia setting where nutritional support is pointless. In terms of the link between specific nutriments and tumor evolution, a number of small and diverse clinical trials, notably in hepatocellular carcinoma, have investigated the benefit of BCAA supplementation in terms of tumor evolution and cancer incidence. Despite the potential beneficial results, the role of specific nutriment supplementation remains to be clarified given the inconsistencies of the findings, patient heterogeneity, small sample size, and the poor study design [58,61,62].

Other Amino Acids: Clinical Cancer Studies A recent review summarizes the observations on the role of AAs in promoting protein anabolism in human cancer, addressing the role of arginine and citrulline [63]. Experimental studies have shown that these two AAs could be an alternative to BCAA for MPS [64]. Few human studies, and most conducted in patients undergoing surgery, are available [65,66], so there is currently little evidence to support a beneficial effect of arginine and citrulline as a treatment for muscle wasting.

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ROLE OF TOTAL AND SPECIFIC AMINO ACIDS IN PROMOTING MUSCLE ANABOLISM IN CHRONIC DISEASE OTHER THAN CANCER Given the wide variety of chronic diseases and the pathophysiologic processes involved in muscle wasting, the mechanisms of the imbalance between MPS and MPB clearly differ, not only between chronic diseases themselves (liver cirrhosis vs chronic obstructive pulmonary disease vs chronic heart failure, etc.) but also between chronic diseases vs acute illnesses or age-related muscle wasting. In chronic disease states, despite various degrees of inflammation and intensities of prolonged inactivity, there are nonetheless some similarities with anabolic resistance to protein and AA administration. All of these mechanisms ultimately lead to an increase in muscle protein wasting. The role of protein or specific AA intake is in attenuating or preventing the loss of SM is important for functional status and overall outcome. Few studies have focused on the effect of adding leucine alone or in combination with other ingredients to the diet. A brief summary of available studies is provided below. • In chronic kidney disease, two specific mechanisms for protein loss have been observed: inadequate AA intake because of a protein-restricted diet and loss of AAs into the dialysate with each hemodialysis cession [19]. A prospective randomized study showed that oral administration of essential AAs induces a significant improvement in the serum albumin concentration in hemodialysis but not in peritoneal dialysis. In addition, improvements were also seen in grip strength and mental health score although not in physical health score [67]. Interestingly from a mechanistic perspective, experimental studies in chronic uremic rats have shown that a chronic uremic state impairs basal signaling via the mTOR anabolic pathway and that leucine can stimulate this signal, although its effectiveness is partially attenuated [67]. • A recent review of cardiac cachexia concludes that, to this day, the treatment of body wasting remains an unresolved challenge [68]. Although in animal models some nutritional interventions can counteract body wasting and muscle loss, there is no evidence of proven effects in humans. Furthermore, in a randomized trial in heart failure patients, improvements in physical and functional capacities were attributed to resistance exercise but not to BCAA supplementation [69]. • A multidisciplinary task force created by the European Respiratory Society summarizes current knowledge in nutritional therapy in chronic obstructive pulmonary disease [70]. Overall, a review of the literature shows that nutritional intervention is probably effective in undernourished patients and the effect is greatest when combined with an exercise program. There was a significant improvement in FFM measures, in muscle functional tests, and in respiratory muscle strength. A limited number of trials investigated the impact of specific nutriments including essential AA, whey protein (rich in BCAA), and polyunsaturated fatty acids. The considerable heterogeneity of the studies and the fact that most were underpowered does not allow for formulation of any clinical recommendations. • A variety of mechanisms contribute to muscle wasting in cirrhosis. SM autophagic proteolysis and myostatin plasma concentrations (that inhibit PS through impaired mTOR) are increased in cirrhosis and contribute to anabolic resistance [26]. Hyperammonemia due to impaired ureagenesis and portosystemic shunting seems to

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be the underlying mechanism that induces both upregulation of myostatin and autophagy [26]. Leucine could be a candidate to stimulate mTOR because the carbon skeleton from leucine provides a pathway for ammonia detoxification. In six wellcompensated stable cirrhotic patients, oral administration of BCAA enriched with leucine not only activated molecular pathways that stimulate MPS but also resulted in similar fractional synthetic rates of muscle in cirrhosis and control. The impaired mTOR signaling and the increase in autophagy are reversed by BCAA with leucine [71]. Despite many similarities, the mechanisms underlying the development of sarcopenia in chronic diseases are variable and it is naı¨ve to imagine that a unique therapeutic strategy or a specific nutriment would prevent or minimize SM wasting for the different clinical situations of muscle wasting in chronic diseases. BCAA and leucine are theoretically good nutriment candidates; however, the methodology and the quality of the studies are too varied to make any firm recommendations at this time.

ROLE OF OMEGA-3 FATTY ACID SUPPLEMENTATION IN PROMOTING MUSCLE ANABOLISM IN CANCER PATIENTS Muscle anabolism may be favored by two additional parameters: omega-3 fatty acid intake and physical exercise. Omega-3 fatty acids are well known for their inhibition of the inflammatory process, leading to a decrease in protein breakdown. Their anabolic effect is less studied, although they improve insulin sensitivity and increase protein intake, and a few studies have shown that they activate the mTOR pathway and increase cell membrane fluidity, both of which could be beneficial [72,73]. Few clinical studies with lean body mass or muscle mass assessments have been published in cancer patients. The results of two studies in NSCLC patients support omega-3 fatty acid intake [74,75] (Table 5.3). In the Murphy TABLE 5.3 Characteristics of NSCLC Patients and Study Design in Omega-3 Fatty Acids Studies Author

Design Lean body mass or muscle mass assessments

Murphy et al.

Open-label, single arm with cotemporaneous control group

Cancer, 2011

Muscle mass by computed tomography analysis

Van der Randomized controlled, Meij et al. blinded J Nutr 2010

Fat-free mass by bioelectrical impedance analysis

Type of cancer

Results

40 NSCLC receiving chemotherapy • n 5 24 control • n 5 16 intervention

Intervention group • Weight change: 0.561.0 kg • Muscle rate change %/100 days: 0.161.6 • 65% gain or maintain muscle mass Control group • Weight change: 22.360.9 kg • Muscle rate change %/100 days : 26.862.6 • 29% gain or maintain muscle mass

33 NSCLC receiving chemotherapy • n 5 14 control • n 5 19 intervention

• Weight: intervention group better weight maintenance than control group • Fat-free mass decreased in both groups but less in intervention than in control group

NSCLC, nonsmall cell lung cancer.

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et al. study [74], the intervention group maintained weight and muscle mass, while in the control group a decrease in weight (about 2.3 kg) and a decrease in muscle mass (about 1 kg) were seen. Similar results were observed in the van der Meij et al. study [75]. The interventional group showed better outcomes in terms of weight and FFM maintenance compared to the control group. These data suggest that omega-3 fatty acids could potentially counteract anabolic resistance and muscle wasting. However, further research should be performed to define the optimal dose and timing of administration.

CONCLUSION Whether the definition of sarcopenia includes SM function or SM strength impairments is not a crucial question. What is important is that it is essential that SM disorders be assessed in chronic diseases, with each group of researchers defining the most pertinent parameters to evaluate. In chronic diseases, it is clear that decreases in mass, density, strength, or function of SM are linked to decreased survival and outcome. For cancer patients, muscle wasting is associated with susceptibility to chemotherapy toxicities, worse progression-free and overall survival, and more postoperative complications. The question of the possibility of increasing muscle mass or minimizing SM disorders is still up for debate. Under physiologic conditions, SM is characterized by protein breakdown in the fasting state and MPS in the postprandial state. The net balance at the end of the day is a stable muscle mass. In chronic disease and especially in cancer patients, the ability of AAs to stimulate protein synthesis is reduced. Considering nutritional intervention, this anabolic resistance could be in part counteracted by increasing protein, AA quantities, and administration of specific AAs (notably leucine). In cancer patients, tumor evolution stage and, in other chronic diseases, severity of the symptoms are crucial parameters for anabolic resistance. Few high-quality AA kinetic studies have highlighted the role of leucine in increasing MPS. However, the translation of acute anabolic effects into gains in muscle mass and function are yet to be confirmed. Further research is needed to confirm the conclusions of Deutz and Wolf that there are no upper limits to the anabolic response to protein or AA intake [76].

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[48] Drummond MJ, Dreyer HC, Fry CS, Glynn EL, Rasmussen BB. Nutritional and contractile regulation of human skeletal muscle protein synthesis and mTORC1 signaling. J Appl Physiol 2009;106:1374 84. [49] Ham DJ, Caldow MK, Lynch GS, Koopman R. Leucine as a treatment for muscle wasting: a critical review. Clin Nutr 2014;33:937 45. [50] Lundholm K, Edstrom S, Ekman L, Karlberg I, Bylund AC, Schersten T. A comparative study of the influence of malignant tumor on host metabolism in mice and man. Cancer 1978;1978(42):453 61. [51] Winter A, MacAdams J, Chevalier S. Normal protein anabolic response to hyperaminoacidemia in insulinresistant patients with lung cancer cachexia. Clin Nutr 2012;31:765 73. [52] van Dijk DP, van de Poll MC, Moses AG, Preston T, Olde Damink SW, Rensen SS, et al. Effects of oral meal feeding on whole body protein breakdown and protein synthesis in cachectic pancreatic cancer patients. J Cachexia Sarcopenia Muscle 2015;6:212 21. [53] Engelen MP, Safar AM, Bartter T, Koeman F, Deutz NE. High anabolic potential of essential amino acid mixtures in advanced nonsmall cell lung cancer. Ann Oncol 2015;26:1960 6. [54] Dillon EL, Volpi E, Wolfe RR, Sinha S, Sanford AP, Arrastia CD, et al. Amino acid metabolism and inflammatory burden in ovarian cancer patients undergoing intense oncological therapy. Clin Nutr 2007;26:736 43. [55] Deutz NE, Safar A, Schutzler S, Memelink R, Ferrando A, Spencer H, et al. Muscle protein synthesis in cancer patients can be stimulated with a specially formulated medical food. Clin Nutr 2011;30:759 68. [56] Williams JP, Phillips BE, Smith K, Atherton PJ, Rankin D, Selby AL, et al. Effect of tumor burden and subsequent surgical resection on skeletal muscle mass and protein turnover in colorectal cancer patients. Am J Clin Nutr 2012;96:1064 70. [57] Bozzetti F, Bozzetti V. Is the intravenous supplementation of amino acid to cancer patients adequate? A critical appraisal of literature. Clin Nutr 2013;32:142 6. [58] Kawaguchi T, Shiraishi K, Ito T, Suzuki K, Koreeda C, Ohtake T, et al. Branched-chain amino acids prevent hepatocarcinogenesis and prolong survival of patients with cirrhosis. Clin Gastroenterol Hepatol 2014;12:1012 18. [59] May PE, Barber A, D’Olimpio JT, Hourihane A, Abumrad NN. Reversal of cancer-related wasting using oral supplementation with a combination of β-hydroxy-β-methylbutyrate, arginine, and glutamine. Am J Surg 2002;183:471 9. [60] Berk L, James J, Schwartz A, Hug E, Mahadevan A, Samuels M, et al. A randomized, double-blind, placebocontrolled trial of a β-hydroxy-β-methylbutyrate, glutamine, and arginine mixture for the treatment of cancer cachexia (RTOG 0122). Support Care Cancer 2008;16:1179 88. [61] Choudry HA, Pan M, Karinch AM, Souba WW. Branched-chain amino acid-enriched nutritional support in surgical and cancer patients. J Nutr 2006;136:314S 8S. [62] Yoshiji H, Noguchi R, Ikenaka Y, Kaji K, Aihara Y, Douhara A, et al. Combination of branched-chain amino acid and angiotensin-converting enzyme inhibitor improves liver fibrosis progression in patients with cirrhosis. Mol Med Rep 2012;5:539 44. [63] Chevalier S, Winter A. Do patients with advanced cancer have any potential for protein anabolism in response to amino acid therapy? Curr Opin Clin Nutr Metab Care 2014;17:213 18. [64] Jonker R, Engelen MP, Deutz NE. Role of specific dietary amino acids in clinical conditions. Br J Nutr 2012;108:S139 48. [65] de Luis DA, Izaola O, Terroba MC, Cuellar L, Ventosa M, Martin T. Effect of three different doses of arginine enhanced enteral nutrition on nutritional status and outcomes in well-nourished postsurgical cancer patients: a randomized single blinded prospective trial. Eur Rev Med Pharmacol Sci 2015;19:950 5. [66] de Luis DA, Izaola O, Cuellar L, Terroba MC, de la Fuente B, Cabezas G. A randomized clinical trial with two doses of a omega 3 fatty acids oral and arginine enhanced formula in clinical and biochemical parameters of head and neck cancer ambulatory patients. Eur Rev Med Pharmacol Sci 2013;17:1090 4. [67] Chen Y, Sood S, McIntire K, Roth R, Rabkin R. Leucine-stimulated mTOR signaling is partly attenuated in skeletal muscle of chronically uremic rats. Am J Physiol Endocrinol Metab 2011;301:E873 81. [68] Loncar G, Springer J, Anker M, Doehner W, Lainscak M. Cardiac cachexia: hic et nunc: “hic et nunc”—here and now. Int J Cardiol 2015;201:e1 e12. [69] Pineda-Jua´rez JA, Sa´nchez-Ortiz NA, Castillo-Martı´nez L, Orea-Tejeda A, Cervantes-Gayta´n R, Keirns-Davis C, et al. Changes in body composition in heart failure patients after a resistance exercise program and branched chain amino acid supplementation. Clin Nutr 2016;35:41 7.

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[70] Schols AM, Ferreira IM, Franssen FM, Gosker HR, Janssens W, Muscaritoli M, et al. Nutritional assessment and therapy in COPD: a European Respiratory Society statement. Eur Respir J 2014;44:1504 20. [71] Tsien C, Davuluri G, Singh D, Allawy A, Ten Have GA, Thapaliya S, et al. Metabolic and molecular responses to leucine-enriched branched chain amino acid supplementation in the skeletal muscle of alcoholic cirrhosis. Hepatology 2015;61:2018 29. [72] Eltweri AM, Thomas AL, Metcalfe M, Calder PC, Dennison AR, Bowrey DJ. Potential applications of fish oils rich in omega-3 polyunsaturated fatty acids in the management of gastrointestinal cancer. Clin Nutr 2017;36:65 78. [73] Di Girolamo FG, Situlin R, Mazzucco S, Valentini R, Toigo G, Biolo G. Omega-3 fatty acids and protein metabolism: enhancement of anabolic interventions for sarcopenia. Curr Opin Clin Nutr Metab Care 2014;17:145 50. [74] Murphy RA, Mourtzakis M, Chu QS, Baracos VE, Reiman T, Mazurak VC. Supplementation with fish oil increases first-line chemotherapy efficacy in patients with advanced nonsmall cell lung cancer. Cancer 2011;117:3774 80. [75] van der Meij BS, Langius JA, Spreeuwenberg MD, Slootmaker SM, Paul MA, Smit EF, et al. Oral nutritional supplements containing n-3 polyunsaturated fatty acids affect quality of life and functional status in lung cancer patients during multimodality treatment: an RCT. Eur J Clin Nutr 2012;66:399 404. [76] Deutz NE, Wolfe RR. Is there a maximal anabolic response to protein intake with a meal? Clin Nutr 2013;32:309 13.

Further Reading Eustace JA, Coresh J, Kutchey C, Te PL, Gimenez LF, Scheel PJ, et al. Randomized double-blind trial of oral essential amino acids for dialysis-associated hypoalbuminemia. Kidney Int 2000;57:2527 38.

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Sarcopenic Obesity Andrea P. Rossi, Sofia Rubele and Mauro Zamboni Department of Medicine, Geriatric Division, University of Verona, Verona, Italy

INTRODUCTION The prevalence of obesity in older adults has increased in recent years; for example, in the United States, nearly 30% in men and in women aged 60 years and over, with increase also in extreme degree obesity [1]. High prevalence of obesity has been observed in Europe [2,3]. With aging, a progressive increase in fat mass, which normally peaks at about age 65 years in men and later in women is observed [4]. Aging is associated also with body fat distribution changes, with visceral abdominal fat increase and subcutaneous abdominal fat decrease [5]. Moreover in the elderly, ectopic fat deposition within nonadipose tissue such as the skeletal and cardiac muscle, liver, and pancreas has been observed [6,7]. This phenomenon occurs even without significant changes in body mass index (BMI) or body weight. On the counterpart, age-associated muscle mass and strength loss occur even in relatively weight stable healthy individuals [8].

SARCOPENIC OBESITY (SO) DEFINITION In 2000, Baumgartner introduced the term of sarcopenic obesity (SO), a condition characterized by coexistence of low muscle mass and a high body fat mass [9]. By the time, more definitions of SO have been proposed (Table 6.1 shows different SO definition). 1. The definition proposed by Baumgartner et al. [9] may underestimate sarcopenia in overweight and obese subjects, thus leading to an underdiagnosis of SO. In general, all the proposed SO definitions raise some concern. The main limitations of both are listed below. 2. All definitions do not take into account quality of muscle parameters and in particular fat infiltration and fibrosis. It has been documented that the amount of triglycerides in

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TABLE 6.1 Sarcopenic and Dynapenic Obesity Definitions and Prevalence Study

Definition of sarcopenic obesity

N

Mean age (SD)

Prevalence

New Mexico Aging Process Study [9]

• Sarcopenia: skeletal muscle mass 22 SD below mean of young population or ,7.26 kg/m2 in men and ,5.45 kg/m2 in women • Obesity: percentage body fat greater than median or .27% in men and .38% in women

831

60 and over

M: 4.4%, F: 3.0%

NHANES III [10]

• Sarcopenia: two lower quartiles of muscle mass (,9.12 kg/m2 in men and ,6.53 kg/m2 in women) • Obesity: two higher quintiles of fat mass ( . 37.2% in men and .40.0% in women)

2982

M: 76.3 (1.7), F: 77.3 (2.2)

M: 9.6%, F: 7.4%

Verona Health ABC Study, Italy [11]

• Sarcopenia: two lower quintiles of muscle mass (,5.7 kg/m2 in women) • Obesity: two higher quintiles of fat mass ( . 42.9% in women)

167

F: 71.7 (2.4)

F:12.4%

InChianti, Italy [12]

• Impaired strength: lower gender-specific tertile of handgrip strength (,32 kg for men and ,18 kg for women) • Obesity BMI $ 30 kg/m2

856

74.3 (6.9)

M: 6.3%, F: 8.7%

LASA [13]

• Impaired strength: lower gender-specific tertile of handgrip strength (,33 kg for men and ,20 kg for women) • Obesity BMI $ 30 kg/m2

1189

75.8 (7.2)

M: 5.1%, F: 8.9%

EXERNET Study, Spain [3]

• Sarcopenia: two lower quintiles of muscle mass (,8.61 kg/m2 in men and ,6.19 kg/m2 in women) • Obesity: two higher quintiles of fat mass ( . 30.3% in men and .40.9% in women)

3176

M: 72.4 (5.5), F: 72.1 (5.2)

M: 17.7%F: 14.0%

Health 2000 Survey, Finland [14]

• Impaired strength: lower gender-specific tertile of handgrip strength (,322 N for men and ,176 N for women) • Obesity BMI $ 30 kg/m2

1413

75.8 (7.1)

M: 6.1%, F: 11.0%

InChianti, Italy [15]

• Sex-specific lowest tertile of handgrip strength (,33 kg in men and ,19 kg in women) • Visceral obesity: sex-specific cutoff based on waist circumference tertiles (99 cm in men and 95 cm in women)

846

74.5 (6.9)

M: 8.1%, F: 12.6%

Verona Health ABC Study, Italy [16]

• Sex-specific lowest tertile of leg strength (15.33 kg in men and ,8.33 kg in women) • Visceral obesity: sex-specific cutoff based on waist circumference tertiles (100 cm in men and 87 cm in women)

262

71.8 (2.2)

12.2%

BMI, body mass index; LASA, Longitudinal Aging Study Amsterdam.

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muscle increases with both ageing and obesity [17,18]: mid-thigh low-density lean tissue, a surrogate of muscle fat infiltration, as evaluated by computed tomography, has been observed to be directly associated with age and adiposity [19]. In fact, both Dual Energy X-ray Absorptiometry (DXA) and Bioelectrical impedence analysis (BIA), the body composition methods usually recommended for definition of sarcopenia, are not able to recognize neither myosteatosis (MS) nor myofibrosis (MF) [13]. 3. SO definition doesn’t take into account muscle function in terms of strength and performance. This raises great concern because it is known that both muscle strength and performance decline quicker than muscle mass with aging. The above-mentioned SO definition does not fit with sarcopenia definition proposed by the European and the International Working Group on Sarcopenia in Older People [20,21], who recommended to consider both, low muscle mass and low muscle function as criteria for the diagnosis of sarcopenia. 4. SO definition uses BMI to quantify the amount of fat, but the most appropriate indices and cutoff of overweight and obesity in the elderly population are still under debate [22]. As with aging, an increase in abdominal fat and in particular in visceral abdominal fat can be observed, even without changes in BMI, the use of indices of fat distribution may be more representative than those of fat mass quantity [22]. Thus, measurements of abdominal circumferences, widely considered good surrogate of fat distribution and in particular of visceral abdominal adipose tissue, may be more adequate than BMI in order to discriminate a population at higher risk for unfavorable outcomes [16]. To bypass this problem, it has been recently suggested to use the ratio between appendicular skeletal mass (ASM)/h2, as evaluated by DXA, and visceral abdominal adipose tissue, as evaluated by computed tomography, as tool for SO definition [23], but this definition is still rising concern in relation with the above-mentioned points 1, 2, and 3 and then cannot be used in clinical practice. More recently, translational definitions of SO have been proposed considering muscle impairment, as expressed by muscle strength, rather than muscle mass, associated with central fat distribution as evaluated with waist circumference, introducing the concept of dynapenic abdominal obesity (DAO). This condition has negative effects on physical function and on the risk of developing mobility disability [24,25]. Separately dynapenia and abdominal obesity have shown associations with important outcomes in older adults, such as worsening disability and mortality [12,24,26 30], but only a few studies have investigated health risk associated with the simultaneous presence of both conditions [14,25,31]. When the definition of SO and DAO has been applied to the same population, the prevalence was, respectively, of 2.8% and 10.6% (Fig. 6.1). It must be noted that only two subjects belonged to both groups [15], Fig. 6.1).

PATHOGENESIS OF SO The age-related reduction in muscle mass and fat mass increase is strictly related with each other from a pathogenic point of view.

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6. SARCOPENIC OBESITY

FIGURE 6.1

Sarcopenic obese Thigh CT Lowest tertile BMI>30 kg/m2

n = 24 (2.8%)

Sarcopenic obese and dynapenic abdominal obese n=2

Dynapenic abdominal obese

Prevalence of sarcopenic obese and dynapenic abdominal obese subjects.

Handgrip 87 cm for females

n = 90 (10.6%)

The age-related decrease in muscle mass and strength may lead to reduced physical activity. A reduction in muscle mass and physical activity reduces total energy expenditure and may lead to weight gain [32]. Moreover, obesity is associated with subacute low-grade inflammation, resulting from the secretion of many adipokines that may contribute to the progression of sarcopenia, acting both at a local and at systemic level. In fact, several adipokines, in particular tumor necrosis factor (TNF)-alpha, IL-6, and leptin, may lead to progressive loss of muscle mass and further increase in fat mass [33]. Skeletal muscle aging is associated with an impairment in muscle quality characterized by a progressive replacement of muscle by fibrous connective (MF) and adipose tissue (MS) [34]. A number of predictors of MS have been found. MS increases with both, age and adiposity, and has been shown to be associated with the degree of insulin resistance as well as to serum leptin levels [34]. In the Health ABC Study population, thigh intermuscular fat was significantly associated with higher levels of inflammatory markers [35]. Moreover, IL-6 gene expression in subcutaneous adipose tissue near the muscle was positively related to MS deposition suggesting a prevalent role of local tissue inflammation [34]. MF has not been yet extensively studied even though functional consequences of MF are relevant. MF is associated with a decrease in muscle strength, elasticity, and blood supply of muscle fibers, further increasing muscle atrophy [36]. Collagen accumulation increases with age in mice [36]. Muscle stem cells from aged mice tend to convert more to a fibrogenic lineage compared to cells from young muscles [37]. In healthy elderly men undergoing elective vertebral surgery, our study group showed that not only the degree of MS but also that MF was associated with increased adiposity, central fat distribution, and a worse metabolic profile [13].

CLINICAL IMPLICATIONS OF SO Previous studies indicate that when obesity and muscle impairment coexist, they act synergistically on the risk of developing multiple health-related outcomes.

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In a cross-sectional study participants in the New Mexico Aging Process Study cohort, [9] the odds ratios for disability in sarcopenic, obese, and sarcopenic-obese groups, relative to the group of seniors with a normal body composition, were 2.07, 2.33, and 4.12, respectively. A more recent study by Rolland et al. [11] confirmed these findings showing that compared with women with a healthy body composition, those with SO had a 2.60 higher odds of having difficulty climbing stairs, 2.35 higher odds of having difficulty going down stairs, and 1.54 higher odds of having moving difficulties. Baumgartner et al. [38], in a 8-year follow-up of the New Mexico Aging Process Study, showed that subjects with SO at baseline were two or three times more likely to develop instrumental disability than lean sarcopenic or nonsarcopenic obese subjects. Previous studies evaluated the joint effect of obesity and low muscle strength. Increased fat mass percentage and decreased muscle strength in the Finnish Health 2000 Survey were shown to be associated with higher prevalence of walking limitation compared to those with only high fat percentage or low muscle strength [28]. Association between obesity, metabolic alterations, and cardiovascular diseases has been observed even in older ages [16,22]. Some evidence show association between Sarcopenia, both defined as low muscle mass or low muscle strength, and metabolic alterations, in particular diabetes. Only a few studies evaluated the association between SO and metabolic alterations and their findings are not conclusive. In the cross-sectional analysis of the New Mexico Aging Process Study [38], subjects with SO did not show higher incidence of congestive heart disease and hip fracture. Despite the fact that type 2 diabetes was more frequent in SO subjects, the incidence of type 2 diabetes was not affected by the SO status. Aubertin-Leheudre et al. [39] in a small study sample of postmenopausal women did not observe any difference in cardiovascular and metabolic risk factor profile between obese old women with a normal muscle mass and those with SO. Stephen in 3000 older adults reported that the low muscle mass associated with abdominal obesity was not associated with an increased risk for the development of cardiovascular disease over an 8-year follow-up period [40]. More recently, association between SO, evaluated as ASM to visceral abdominal fat ratio, metabolic syndrome, and arterial stiffness has been observed in 526 apparently healthy adults enrolled in the Korean Sarcopenic Obesity Study, an ongoing prospective observational cohort study [23]. By multiple logistic regression analysis, the odds ratio for metabolic syndrome was 5.43 higher in subjects with SO [23]. These findings seem to suggest that if fat distribution is taken into account in the definition of SO, its association with metabolic syndrome and vascular damage may reach evidence.

CLINICAL IMPLICATIONS OF DAO Dynapenia and obesity also have independent, additive, and negative effects on physical function.

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In a cross-sectional study of 2039 men and women aged 55 years and older, where leg extension strength was measured with a dynamometer, 12% of the nondynapenic and nonobese group had walking disability, compared with 18% of those with obesity alone, 24% of those with dynapenia, and 36% of those with dynapenic obesity [26]. Interestingly, they observed that dynapenic obese subjects had a walking speed of approximately 0.14 m/s so that the nondynapenic and nonobese subjects determine a difference of 2.8 seconds every 20 m distance [26]. A study published in 2010 of 904 men and women aged 67 84 years reported that combining handgrip strength and fat mass is the best predictor of a low physical function score, compared with any other body composition and strength markers [41]. DAO also has shown unfavorable consequences on mobility and autonomy. In a crosssectional study of 2039 men and women aged 55 years and older, where leg extension strength was measured with a dynamometer, Bouchard et al. observed that dynapenic obese subjects had a lower walking speed compared with nondynapenic and nonobese subjects [25]. In a population of 3594 adults ranging in ages between 50 and 91 followed up for 33 years, Scott et al. observed instead that both low handgrip strength and obesity independently predict the risk of death [31]. Moreover in a recent study, DAO showed increased falls risk compared to reference group [15]. Mortality risk associated with obesity coupled with poor strength has been evaluated [42] in a 30-year prospective study in initially healthy men. They observed that overweight persons in the lowest grip strength tertile had 1.39 times higher mortality risk compared to normal weight persons in the highest grip strength tertile. DAO subjects show an unfavorable metabolic profile, high cardiovascular risk, also when compared with sarcopenic obese subjects, suggesting in obese individuals low strength is a predictor of mortality stronger than low muscle mass [43]. DAO shows also an increased risk of worsening disability and mortality than subjects with dynapenia or central fat distribution only [16]. More recently, Rossi et al. in a larger population suggest that dynapenia is related to the risk of death regardless of the presence of central obesity whereas abdominal obesity strongly increases the risk of disability and hospitalization associated with low muscle strength. Identification of elderly subjects with central fat distribution and simultaneous low muscle strength could help, with easily available and inexpensive tools, to select a subgroup of subjects with the highest risk of functional decline and loss of independence [15].

TREATMENT Debate has been raised in the past about the hazard of obesity in the elderly and thus about the need of its treatment. However, since SO is associated with poor outcomes, its treatment should be considered. Weight loss is usually associated with decline in fat mass, as well as decline in fat free and bone mass [44]. However, it seems crucial that any treatment of SO should cause a reduction in fat mass, in particular abdominal fat mass, preserving muscle mass quality and function. Some evidence show that improvement of muscle quality after weight loss as well as of its function may be obtained if physical exercise is combined with dietary counseling and if weight loss is moderate ranging from 5% to 10% of initial body weight.

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TABLE 6.2 General Nutritional Recommendation for Sarcopenic Obesity Treatment in the Elderly • Moderate energy restriction (with a maximum energy deficit of 500 kcal/day) with 5% 10% weight loss and very low hypoenergetic diet should be avoided • Diet should enriched in high biological value protein (at least 1.1 1.2 g/kg/day) and supplementation with leucine-enriched amino-acid essential mixture, including whey protein and leucine, should be considered • Dietary treatment should be combined with short-term resistance training and aerobic exercise • Supplementation with vitamin D and calcium is mandatory

Villareal et al. showed that 6-month weight loss treatment aimed to be no more than 1.5% per week combined with exercise training three times per week (with each session lasting 90 minutes) ameliorates function and frailty in obese older subjects [45]. We observed that a moderate weight loss (nearly 5%) in a group of elderly women determines a significant improvement in insulin resistance, fat distribution, and more importantly of muscle lipid infiltration, with just a small decrease in appendicular lean tissue [46]. Exercise has beneficial effects on multiple aspects of SO with increase in muscle protein synthesis, reduction in myostatin expression, increase in intramuscular insulin-like growth factor (IGF)-1, enhanced mitochondrial function, and activation of skeletal muscle satellite cells [45]. Further, the reduction in lean mass associated with weight loss therapy is attenuated when combined with regular exercise and in particular resistance training [47]. Therefore, the most effective treatment strategy for SO should incorporate both, diet induced weight loss and regular exercise program including resistance training. A recent review of literature that aimed to assess studies investigating the effectiveness of exercise or nutritional interventions to improve the body composition, strength, or function in older adults with sarcopenia and obesity found that none of the included studies showed a significant decrease in body fat or increase in either skeletal muscle mass or lean mass [48]. Authors empathized a general lack of published data on this topic and the necessity for new research adopting universally accepted cutoffs for SO. Quality and amount of dietary protein should be relevant when trying to prevent fatfree mass during weight loss. Verreijen et al. in a double-blind randomized-controlled trial in 80 obese older adults found that a high whey protein-, leucine-, and vitamin D-enriched supplement compared to isocaloric control preserves appendicular muscle mass during hypocaloric diet and resistance exercise program and might therefore reduce the risk of sarcopenia [49]. However, some general recommendations should be considered and are summarized in Table 6.2.

CONCLUSION With aging, loss of muscle mass and gain in fat seem to be linked to each other and contribute, in the presence of positive energy balance, to the development of SO.

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Identification of elderly subjects with SO could help to identify a group of subjects at particularly high health risk, and the concept of SO may help to clarify the relation between obesity, morbidity, and mortality in the elderly. Recently, great effort has been done to improve sarcopenia definition in order to get it carried out in the clinical assessment of elderly people. Similar effort seems to be mandatory also for SO definition. Recently proposed DAO definition that requires two measurements that are relatively simple to obtain and interpret, especially in outpatient settings, seems a plausible alterative to the classic SO definition, useful in order to identify older subjects at high risk for unfavorable health outcomes.

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[17] Cree MG, Newcomer BR, Katsanos CS, Sheffield-Moore M, Chinkes D, Aarsland A, et al. Intramuscular and liver triglycerides are increased in the elderly. J Clin Endocrinol Metab 2004;89:3864 71. [18] Song MY, Ruts E, Kim J, Janumala I, Heymsfield S, Gallagher D. Sarcopenia and increased adipose tissue infiltration of muscle in elderly African American women. Am J Clin Nutr 2004;79:874 80. [19] Goodpaster BH, Carlson CL, Visser M, Kelley DE, Scherzinger A, Harris TB, et al. Attenuation of skeletal muscle and strength in the elderly: The Health ABC Study. J Appl Physiol 2001;90:2157 65. [20] Cruz-Jentoft AJ, Baeyens JP, Bauer JM, Boirie Y, Cederholm T, Landi F, et al. Sarcopenia: European consensus on definition and diagnosis. Age Ageing 2010;39:412 23. [21] Fielding RA, Vellas B, Evans WJ, Bhasin S, Morley JE, Newman AB, et al. Sarcopenia: an undiagnosed condition in older adults. Current consensus definition: prevalence, etiology, and consequences. International working group on Sarcopenia. J Am Med Dir Assoc 2011;12:249 56. [22] Zamboni M, Mazzali G, Zoico E, Harris TB, Meigs JB, Di Francesco V, et al. Health consequences of obesity in the elderly: a review of four unresolved questions. Int J Obes 2005;29:1011 29. [23] Lim KI, Yang SJ, Kim TN, Yoo HJ, Kang HJ, Song W, et al. The association between the ratio of visceral fat of thigh muscle area and metabolic syndrome: the Korean sarcopenic obesity study (KSOS). Clin Endocrinol 2010;73:588 94. [24] Stenholm S, Rantanen T, Helio¨vaara M, Koskinen S. The mediating role of C-reactive protein and handgrip strength between obesity and walking limitation. J Am Geriatr Soc 2008;56:462 9. [25] Bouchard SR, Janssen J. Dynapenic-obesity and physical function in older adults. J Gerontol Med Sci. 2010;1:71 7. [26] Visser M, Goodpaster BH, Kritchevsky SB, Newman AB, Nevitt M, Rubin SM, et al. Muscle mass, muscle strength, and muscle fat infiltration as predictors of incident mobility limitations in well-functioning older persons. J Gerontol A Biol Sci Med Sci. 2005;60:324 33. [27] Fantin F, Di Francesco V, Fontana G, Zivelonghi A, Bissoli L, Zoico E, et al. Longitudinal body composition changes in old men and women: interrelationships with worsening disability. J Gerontol A Biol Sci Med Sci. 2007;62:1375 81. [28] Angleman SB, Harris TB, Melzer D. The role of waist circumference in predicting disability in periretirement aged adults. Int J Obes. 2006;30:364 73. [29] Norman K, Stoba¨us N, Gonzalez MC, Schulzke JD, Pirlich M. Hand grip strength: outcome predictor and marker of nutritional status. Clin Nutr. 2011;30:135 42. [30] Koster A, Leitzmann MF, Schatzkin A, Mouw T, Adams KF, van Eijk JT, et al. Waist Circumference and mortality. Am J Epidemiol. 2008;167:1465 75. [31] Scott D, Sanders KM, Aitken D, Hayes A, Ebeling PR, Jones G. Sarcopenic obesity and dynapenic obesity: 5year associations with falls risk in middle-aged and older adults. Obesity. 2014;22:1568 74. [32] Nair KS. Aging muscle. Am J Clin Nutr 2005;81:953 63. [33] Schrager MA, Metter EJ, Simonsick E, Ble A, Bandinelli S, Lauretani F, et al. Sarcopenic obesity and inflammation in the InCHIANTI study. J Appl Physiol 2007;102:919 25. [34] Zoico E, Rossi A, Di Francesco V, Sepe A, Olioso D, Pizzini F, et al. Adipose tissue infiltration in skeletal muscle of healthy elderly men: relationship with body composition, insulin resistance, and inflammation at the sistemic and tissue level. J Gerontol Med Sci 2010;3:295 9. [35] Beasley LE, Koster A, Newman AB, Javaid MK, Ferrucci L, Kritchevsky SB, et al. The Health ABC Study. Inflammation and race and gender differences in computerized tomography-measured adipose depots. Obesity 2009;17:1062 9. [36] Huard J, Li Y, Fu FH. Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 2002;84: 822 32. [37] Brack AS, Conboy MJ, Roy S, Lee M, Kuo CJ, Keller C, et al. Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science 2007;317:807 10. [38] Baumgartner RN, Wayne SJ, Waters DL, Janssen I, Gallagher D, Morley JE. Sarcopenic obesity predicts instrumental activities of daily living disability in the elderly. Obes Res. 2004;12:1995 2004. [39] Aubertin-Leheudre M, Lord C, Goulet EDB, et al. Effect of sarcopenia on cardiovascular disease risk factors in obese postmenopausal women. Obesity 2006;14:2277 83.

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[40] Stephen WC, Janssen I. Sarcopenic-obesity and cardiovascular disease risk in the elderly. J Nutr Health Aging 2009;13:460 6. [41] Choquette S, Bouchard DR, Doyon CY, Se´ne´chal M, Brochu M, Dionne IJ. Relative strength as a determinant of mobility in elders 67 84 years of age. a nuage study: nutritions a determinant of successful aging. J Nutr Health Aging. 2010;14:190 5. [42] Rantanen T, Guralnik JM, Foley D, Masaki K, Leveille S, Curb JD, et al. Midlife hand grip strength as a predictor of old age disability. JAMA 1999;281:558 60. [43] Se´ne´chal M, Dionne IJ, Brochu M. Dynapenic abdominal obesity and metabolic risk factors in adults 50 years of age and older. J Aging Health. 2012;24:812 26. [44] Bouchonville MF, Villareal DT. Sarcopenic obesity—how do we treat it? Curr Opin Endocrinol Diabetes Obes. 2013;20:412 19. [45] Villareal DT, Banks M, Sinacore DR, Siener C, Klein S. Effect of weight loss and exercise on frailty in obese older adults. Arch Intern Med 2006;166:860 6. [46] Mazzali G, Di Francesco V, Zoico E, et al. Interrelations between fat distribution, muscle lipid content, adipocytokines, and insulin resistance: effect of moderate weight loss in older women. Am J Clin Nutr 2006;84: 1193 9. [47] Frimel TN, Sinacore DR, Villareal DT. Exercise attenuates the weight-loss induced reduction in muscle mass in frail obese older adults. Med Sci Sports Exerc. 2008;40:1213 19. [48] Theodorakopoulos C, Jones J, Bannerman E, Greig CA. Effectiveness of nutritional and exercise interventions to improve body composition and muscle strength or function in sarcopenic obese older adults: a systematic review. Nutr Res. 2017;43:3 15. [49] Verreijen AM, Verlaan S, Engherink MF, Swinkels S, de Vogel-van den Bosch, PJM Weijs. A high whey protein-, leucine-, and vitamin D-enriched supplement preserves muscle mass during intentional weight loss in obese older adults: a double blind randomized controlled trial. Am J Clin Nutr. 2015;101:279 86.

Further Reading Unger RH. Longevity, lipotoxicity and leptin: the adipocyte defense against feasting and famine. Biochimie 2005;87:57 64.

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Effects of Sarcopenic Obesity on Cardiovascular Disease and All-Cause Mortality Janice L. Atkins University of Exeter Medical School, Exeter, Devon, United Kingdom

INTRODUCTION Obesity, excess body fat, is a major public health problem, and in recent years its prevalence has increased dramatically; since 1980, the prevalence of obesity has more than doubled worldwide [1,2]. Obesity it well recognized as a major risk factor for cardiovascular morbidity and mortality in adult populations [3
6]. Obesity prevalence is also increasing with age; in an ageing population, the obesity epidemic represents a growing financial issue in regards to healthcare resources [7
10]. Body composition changes occur with age including a progressive increase in fat mass and redistribution of body fat — typically visceral abdominal fat increases and subcutaneous abdominal fat decreases [11,12]. Visceral fat increases the risk of developing metabolic disorders (hypertension, dyslipidemia, and insulin resistance) and cardiovascular disease (CVD). There is also an age-associated progressive loss of muscle mass and strength, known as sarcopenia, which is often associated with visceral obesity [10,13
15]. The etiology of sarcopenia is multifactorial and not fully understood. However, some of the underlying mechanisms of age-related muscle loss include neuronal and hormonal changes, physical inactivity, poor nutrition such as low protein intake and inflammation [10,16
19]. Sarcopenia is associated with cardiovascular risk factors and adverse health outcomes including physical disability and mortality [20
23]. Despite these changes in body composition with ageing, there may be no significant changes in an individual’s body weight or body mass index (BMI) [10]. Due to body composition changes that occur with ageing, sarcopenia may often coexist with increases in fat mass. Recently, a new body composition category — sarcopenic obesity — has emerged, which reflects the coexistence of sarcopenia and obesity [10,14,24].

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Therefore, four body composition phenotypes have been proposed in older populations: normal, sarcopenic, obese, and sarcopenic obese. Visceral fat and muscle mass are pathogenically related and share common inflammatory pathways [10,25]. The presence of both obesity and sarcopenia in older adults may therefore increase the impact on each other emphasizing their effect on CVD and mortality, possibly resulting in individuals with a sarcopenic obese body composition having the worse health outcomes [10,15,26
28].

DEFINING SARCOPENIC OBESITY The term “sarcopenic obesity” was first created by Baumgartner et al. [24] and combines the definitions of both sarcopenia and obesity. Obesity is most commonly defined as BMI, measured as weight divided by height squared, greater than or equal to 30 kg/m2 [29]. However, there have been questions regarding the validity of BMI in representing adiposity accurately, since BMI does not distinguish between fat mass and lean mass [30]. Alternative definitions of obesity have therefore focused on body fat distribution, with central or visceral obesity commonly being measured. Measures of central adiposity have also been shown to be better predictors of CVD and mortality than BMI in older adults [31,32]. Two such measures of central obesity are waist circumference ( . 102 cm for men and .88 cm for women) and waist-to-hip ratio ($0.90 cm for men and $ 0.85 cm for women), defined by the World Health Organization using sex-specific cut-points [33]. Sarcopenia has been defined using many different methods. Baumgartner et al. initially defined sarcopenia as appendicular skeletal muscle mass (ASM) two standard deviations below the sex-specific reference for a young healthy person, adjusted for height and assessed using dual x-ray absorptiometry (DXA) [17]. Another sarcopenia definition was developed by Janssen et al., using bioelectrical impedance analysis (BIA) to calculate skeletal muscle mass [34]. The European Working Group on Sarcopenia in Older People (EWGSOP) proposed a clinical definition of sarcopenia, for case finding in older adults, in 2009 [35]. This definition proposed including the presence of both low muscle mass and low muscle function (low strength and/or low physical performance). The EWGSOP algorithm for sarcopenia suggests measuring muscle strength using handgrip strength and measuring physical performance using gait speed [35]. The International Working Group on Sarcopenia proposed a similar definition in 2011, with the diagnosis of sarcopenia being based on a low appendicular or whole-body fat-free mass combined with poor physical functioning [21]. In 2014, the Foundation for the National Institutes of Health Sarcopenia Project recommended defining sarcopenia using specific cut points for low lean mass (appendicular lean mass adjusted for BMI ,0.789 for men and ,0.512 for women) and for muscle weakness (grip strength ,26 kg for men and ,16 kg for women) [36]. However, to date there is no universally accepted definition or classification for sarcopenia, or therefore for sarcopenic obesity [22,35]. Therefore, a wide variety of the aforementioned measures of both obesity and of muscle mass or function have previously been used to define sarcopenic obesity [28]. The cut-point values used in previous literature to define sarcopenia and obesity have varied greatly depending on the population, age, gender, and ethnicity [37]. This means that comparing findings between different studies of

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sarcopenic obesity is challenging, and prevalence estimates for sarcopenic obesity have differed significantly. Studies have shown that in older adults, the prevalence of sarcopenic obesity varies from 0% to 25% in older adults, with an approximate average prevalence between 5% and 10% [37].

SARCOPENIC OBESITY AND CARDIOVASCULAR RISK FACTORS IN OLDER AGE Many studies have examined the associations between sarcopenic obesity and established cardiovascular risk factors [26,27,38]. Several cross-sectional studies which have been carried out in Korean older adults have found that individuals with sarcopenic obesity have the worse cardiovascular risk; sarcopenic obesity (defined by skeletal muscle mass assessed by DXA and obesity assessed by either DXA, BMI, or waist circumference) was associated with up to eight times the risk of the metabolic syndrome, lower cardiorespiratory fitness, an increased risk of hypertension, dyslipidemia and insulin resistance, and higher fasting glucose levels compared to non-sarcopenic, nonobese individuals [39
46]. Comparably, sarcopenic obese Taiwanese older adults (defined by BIA measured muscle mass and BMI) also had the highest risk of the metabolic syndrome, with a 12-fold increase in risk, compared to the non-sarcopenic, non-obese group [47]. Sarcopenic obese individuals (defined by BIA and BMI) have also been shown to have the highest risk of dysglycemia and insulin resistance in a large cross-sectional study of 14,000 adults from the National Health and Nutrition Examination Survey [48]. However, not all studies show that sarcopenic obese individuals have the worst cardiovascular risk profiles; some cross-sectional studies have suggested that obese older adults may have higher levels of cardiovascular risk factors than sarcopenic obese individuals. Older adults from the New Mexico Aging Process Study, aged 60 years or over, found that the prevalence of the metabolic syndrome and hypertension was highest in the nonsarcopenic obese group, followed by the sarcopenic obese group (assessed using DXA measurements) [49]. Studies in postmenopausal women have also shown that sarcopenic obese individuals did not show an unfavorable metabolic profile compared to non-sarcopenic obese individuals [50] and that glucose level, lipid profile, and blood pressure were not significantly different between sarcopenic obese and non-sarcopenic, non-obese individuals [51]. Despite inflammation being strongly associated with both sarcopenia and obesity, conflicting results have been found regarding the relationship between inflammatory and hemostatic markers and sarcopenic obesity. Cross-sectional analysis of older adults from the “Invecchiare in Chianti” (InCHIANTI) study, aged 65 years and older, showed that sarcopenic obesity (based on grip strength and waist circumference measurements) was associated with higher levels of inflammatory markers including C-reactive protein (CRP) and interleukin 6 (IL-6) [52]. Comparably, sarcopenic obese adult Korean women had the highest CRP levels [46]. However, analysis of baseline data from the Trial of Angiotensin Converting Enzyme Inhibition and Novel Cardiovascular Risk Factors study found there were no significant interactions between sarcopenia and obesity with CRP, IL-6, or plasminogen activator inhibitor [53], and another study found that CRP levels were not significantly different between sarcopenic obese and non-sarcopenic, non-obese postmenopausal women [51]. II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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SARCOPENIC OBESITY AND CARDIOVASCULAR DISEASE IN OLDER AGE Despite the growth of literature on the relationships between sarcopenic obesity and cardiovascular risk factors over the last decade, to date very few studies have examined the prospective associations between sarcopenic obesity and CVD risk in older people [26,27,38,54]. Table 7.1 summarizes studies that have examined the associations of sarcopenic obesity with risk of CVD and mortality in older people. Cross-sectional analysis of baseline data from the New Mexico Aging Process Study compared CVD prevalence in older adults, aged 60 years and over, between sarcopenic obesity groups (defined by ASM and percentage body fat from DXA) [49]. The prevalence of CVD was not higher in sarcopenic obese individuals (11.5%) compared to nonsarcopenic, non-obese individuals (13.7%). A cross-sectional study of male and female participants, aged 65 years or older, from the Korea National Health and Nutrition Examination Survey found that although the sarcopenic obesity group (based on ASM from DXA and BMI $ 25 kg/m2) showed a slightly higher prevalence of CVD (12.3%) compared to non-sarcopenic obese (10.0%), this difference was non-significant [55]. There are a very limited number of prospective studies that have examined the association between sarcopenic obesity and CVD risk. Stephen et al analyzed data from the Cardiovascular Health Study, a moderately large prospective study of 3366 communitydwelling older men and women (aged 65 years or above) over 8 years of follow-up [56]. They found that compared to the normal body composition group, the risk of CVD events was not significantly elevated in the sarcopenic obese groups, when defined using BIA-measured muscle mass and waist circumference. However, when sarcopenic obesity was determined by grip strength and waist circumference, CVD risk was increased by 23% in the sarcopenic obese group. These results imply that muscle strength could be more important than muscle mass when assessing the association between sarcopenic obesity and CVD risk. Results from a prospective study of men, aged between 60 and 79 years, from the British Regional Heart Study, also showed no association between sarcopenic obesity (when defined by a measure of muscle mass, midarm muscle circumference, and waist circumference), over 11 years of follow-up [57]. This study did not, however, include muscle strength when defining sarcopenic obesity. Overall, it seems that research to date from these cross-sectional and prospective studies do not provide sufficient evidence to reach a conclusion regarding the association between sarcopenic obesity with CVD risk.

SARCOPENIC OBESITY AND ALL-CAUSE MORTALITY IN OLDER AGE In recent years, there has been a growth in the literature of studies that have prospectively examined the association between sarcopenic obesity and all-cause mortality risk in healthy older people [26,58]. These studies are summarized in Table 7.1. A small

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TABLE 7.1 Summary of Studies Examining the Associations Between Sarcopenic Obesity and Cardiovascular Disease or Mortality in Older Adults Author, Year

Study Design

Rantanen, 2000 Prospective cohort (30 year [64] follow-up)

Subjects

Sarcopenic Obesity Measurement Outcomes

N 5 6040

Grip strength; BMI

Mortality

Overweight men (BMI $ 25 kg/m2) in the lowest grip strength tertile had 1.39 times the risk of mortality compared to normal weight men in the highest tertile

MAMC; BIA (FM; FFM); WC; WHR

Mortality

A composite measure of WC and MAMC most effectively predicted mortality. Sarcopenic obesity, based on high WC ( . 102 cm) and low MAMC (lowest quartile), showed a 55% increase in mortality risk compared to non-sarcopenic, non-obese

Healthy adults from Hawaii Men Aged 45
68 years

Wannamethee, 2007 [60]

Prospective cohort (6 year follow-up)

N 5 4107 Participants from the British Regional Heart Study Men Aged 60
79 years

Prado, 2008 [59]

Prospective cohort (followup unknown)

N 5 250 Canadian patients with respiratory or gastrointestinal cancers

Main Findings

CT (muscle crossMortality sectional area); BMI $ 30 kg/m2

Patients with sarcopenic obesity had a higher rate of mortality compared to those with obesity alone

CT (calf skeletal muscle; calf FM); BMI

Mortality

No significant difference reported in mortality risk across six sarcopenic obesity groups

BIA (skeletal muscle mass); grip strength; WC

CVD; CHD

Sarcopenic obesity, based on muscle strength but not muscle mass, was modestly associated with a 23% increased risk of CVD

Men and women Aged 35
88 years Cesari, 2009 [62]

Prospective cohort (6 year follow-up)

N 5 934 Participants from the InCHIANTI study Men and women Aged $ 65 years

Stephen, 2009 [56]

Prospective cohort (8 year follow-up)

N 5 3366 Participants from the Cardiovascular Health Study Men and women Aged $ 65 years

(Continued)

TABLE 7.1 (Continued) Author, Year

Study Design

Subjects

Atkins, 2014 [57]

Prospective cohort (11 year follow-up)

N 5 4111 Participants from the British Regional Heart Study

Sarcopenic Obesity Measurement Outcomes MAMC; BIA (FM; FFM); WC

Mortality; CVD mortality; CVD events; CHD events

Sarcopenic obese group, based on WC ( . 102 cm) and MAMC (lowest 2 quintiles), had the highest risk of allcause mortality but not CVD mortality. Sarcopenic obesity, based on BIA, was not associated with CVD or mortality

BIA (Skeletal muscle mass; % body fat)

Mortality

Women with sarcopenic obesity had a higher mortality risk than those without sarcopenia or obesity. For men, the risk of mortality associated with sarcopenic obesity was not significant

Grip strength; BMI

Mortality

Both obesity and low handgrip strength, independent of each other, predicted the risk of mortality with an additive pattern

Men Aged 60
79 years Batsis, 2014 [61]

Prospective cohort (14 year follow-up)

N 5 4652 Participants from the National Health and Nutrition Examination Survey III

Main Findings

Men and women Aged $ 60 years N 5 3594 Stenholm, 2014 Prospective cohort (17.9 year [65] follow-up) Participants from the MiniFinland Health Examination Survey

Among 50
69 year olds, the highest mortality risk was among obese participants with low handgrip strength

Men and women Aged 50
91 years Hamer, 2017 [66]

Prospective cohort (8.1 year follow-up)

N 5 6864 Participants from the English Longitudinal Study of Ageing Men and women Mean age 66.2 years (SD 6 9.5)

Grip strength; BMI

Mortality

Sarcopenic obesity did not confer any greater risk of mortality than sarcopenia alone

Hirani, 2017 [63]

Prospective cohort (7 year follow-up)

N 5 954 Participants from the Concord Health and Ageing in Men Project

DXA (appendicular lean mass: BMI ratio; % body fat)

Mortality

There was no significant association between sarcopenic obesity and mortality in adjusted analysis

Grip strength; WC

Mortality

Mortality risk was significantly higher in participants with sarcopenia (but not sarcopenic obesity) compared to the normal body composition group, after adjustment for confounders

DXA (ASM; % body fat)

Instrumental activities of daily living disability

At baseline, subjects with sarcopenic obesity did not show a higher prevalence of CVD

DXA (ASM); BMI $ 25 kg/m2

CVD

Sarcopenic obese group showed higher prevalence of CVD (12.4%) compared to non-sarcopenic obese (10.1%), but difference was non-significant

Men Aged $ 70 years Rossi, 2017 [67] Prospective cohort (11 year follow-up)

N 5 846 Participants from the InCHIANTI study Men and women Aged 65
95 years

Baumgartner, 2004 [49]

Chin, 2013 [55]

Cross-sectional analysis at baseline of a prospective cohort (8 year follow-up)

N 5 451

Cross-sectional

N 5 1578

Participants from the New Mexico Aging Process Study Men and women Aged $ 60 years

Participants from the Korea National Health and Nutrition Examination Survey Men and women Aged $ 65 years

Table updated from Atkins et al. [26]. Studies arranged by type of evidence presented (prospective, followed by cross-sectional) and by date. ASM, appendicular skeletal muscle mass; BIA, bioelectrical impedance analysis; BMI, body mass index; CHD, coronary heart disease; CT, computerized tomography; CVD, cardiovascular disease; DXA, dual energy x-ray absorptiometry; FM, fat mass; FFM, fat free mass; MAMC, midarm muscle circumference; WC, waist circumference; WHR, waist-to-hip ratio.

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prospective study in 250 patients, with gastrointestinal or respiratory cancers, found that sarcopenic obese patients had a significantly higher risk of mortality compared to those with obesity alone [59]. Comparably, an increased risk of all-cause mortality in sarcopenic obese individuals has also been found in a large community-based study. Over 4000 men, aged 60
79 years, from the British Regional Heart Study with a high waist circumference ( . 102 cm) and in the lowest quartile of midarm muscle circumference, had a 55% increase in mortality risk compared with non-sarcopenic, non-obese individuals over 6 years of follow-up [60]. More recently, this same population was examined again after 11 years of follow-up, and sarcopenic obese men had the highest all-cause mortality risk, with a 72% increase in risk after adjustment for lifestyle and cardiovascular risk factors [57]. The risk of mortality was assessed in relation to sarcopenic obesity in the National Health and Nutrition Examination Survey, in over 4000 participants, aged 60 years or above, followed for 14 years [61]. Sarcopenic obese women (based on BIA measured skeletal muscle mass and body fat) had a 29% significantly increased risk of mortality compared to those without sarcopenia or obesity, after adjustment for age, gender, ethnicity, and cardiovascular risk factors [61]. However, the risk of mortality with sarcopenic obesity was not significant in men in this cohort. The InCHIANTI study of 934 males and females aged 65 years categorized participants into six different sarcopenic obesity groups, based on the presence of sarcopenia (calf skeletal muscle) and whether participants were obese, overweight, or normal body weight according to BMI [62]. Over 6 years of follow-up, no significant difference in mortality risk across the six sarcopenic obesity groups was found. A recent study in 954 men aged 70 years and above, classifying sarcopenic obesity based on DXA measured lean mass and body fat, also found no significant association between sarcopenic obesity and mortality in adjusted analysis, over 7 years of follow-up [63]. Some prospective studies have also used a measure of muscle strength instead of muscle mass to define sarcopenic obesity in relation to mortality risk. In over 6000 adult men aged 45
68 years, with over 30 years of follow-up, there was a significant increase in risk of mortality by 39% in men with sarcopenic obesity (BMI $ 25 kg/m2 and those in the lowest grip strength tertile) [64]. Another study in 3594 participants from the Mini-Finland Health Examination Survey over 17 years of follow-up defined sarcopenic obesity using BMI and grip strength and found that obesity and sarcopenia each independently predicted mortality, with an additive pattern [65]. However, two other prospective studies which defined sarcopenic obesity using BMI or waist circumference and grip strength found that individuals with sarcopenic obesity did not have any greater risk of mortality than those with sarcopenia alone [66,67]. A recent meta-analysis (which searched for literature up to September 2014) found that in pooled analyses, sarcopenic obesity was associated with a 24% increase in all-cause mortality risk compared to individuals without sarcopenic obesity, particularly in men [58]. Also, sarcopenic obesity, defined by both midarm muscle circumference and muscle strength significantly increased the risk of mortality by 46% and 23%, respectively. The risk of all-cause mortality did not differ considerably based on geographical region of study or length of follow-up.

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CONCLUSIONS Sarcopenic obesity is a new class of obesity in older adults in which low skeletal muscle mass is coupled with high levels of adiposity. The evidence suggests that older adults with sarcopenic obesity may have higher levels of cardiovascular risk factors and an increased risk of mortality. However, there is heterogeneity in these observed associations, which may be due to the differences in sarcopenic obesity definitions used between studies. To date, there is no universally accepted definition or classification for sarcopenia, or therefore for sarcopenic obesity. However, in 2016, sarcopenia was recognized as a disease and assigned an International Classification of Disease, tenth revision (ICD-10) code [68]. This increased recognition of the importance of sarcopenia, and hence sarcopenic obesity should help to progress research.

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Skeletal Muscle in Obesity and Chronic Overfeeding Christelle Guillet Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France

In situation of obesity, beside metabolic alterations, functional limitations are frequently observed and are illustrated by reduced ability to move and lower level of physical activity due to joint pain and limited respiratory capacities [1,2]. Extremely obese individuals have impaired postural stability even in young individuals, increasing thus the risk of falls in this population. Those alterations are improved after weight loss [3,4]. According to the degree of obesity and thus the severity of these limitations, the capacity in the accomplishment of basic daily activities might be reduced and concurred to a lower quality of life, as well as overall increased risk of disability [5 7]. The substantial change in body composition occurring in obese individuals, consisting in a disproportionate increase in fat mass relative to fat-free mass (FFM), might impact the functional abilities of skeletal muscles during locomotion and other specific actions of life, by interfering significantly with both aerobic and anaerobic activities and lead to disability [8]. It is known that skeletal muscle has a remarkable plasticity illustrating by its abilities to adapt to different external stimuli such as habitual level of contractile activity (e.g., endurance exercise training), loading conditions (resistance exercise, microgravity), substrate supply (nutritional interventions), or environmental factors [9 11]. In obesity, excess body weight leading to additional loading, increased visceral adipose tissue, high and unsuitable nutritional intakes, and metabolic derangements might induce modifications in skeletal muscle mass, composition, and function which contribute to reduce mobility and quality of life in obese individuals. In spite of the considerable functional limitations associated with body mass excess, the estimation of skeletal muscle in obese or overweight individuals is mostly based on indirect evaluation of fat-free or lean soft tissue mass and thus the effects of increased adiposity and obesity on muscle characteristics are still undefined and controversial.

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MUSCLE STRENGTH Investigations focused on the effects of obesity on muscle function have described that obese people elicited higher absolute muscle strength and power than normal-weight individuals [8,12,13]. For instance, in obese females the strength of knee extensors, back extensors, and oblique abdominals were stronger than the lean counterparts [12]. However, no impact of obesity was found on handgrip strength. The results of this study suggest that additional weight from high levels of fat mass occurring in obese individuals might elicit a positive training stimulus for the loaded musculature. The work of Bosco et al. has contributed to strengthen this hypothesis. In their study, normal weight subjects wore a weighted vest during 3 weeks to stimulate hypergravity and thus simulate the loaded antigravity musculature of the lower limbs faced by obese individuals during daily activities [14]. The authors reported increases in muscle power in antigravity muscles in normal weight subjects wearing the weighted vest. The explanations given by these authors for the increase in performance were essentially based on specific neural adaptations which occur in the initial phase of the loading. This extraloading might be assimilated to resistance exercise. However, due to the gradual and sustained increases in fat mass that an obese individual would experience, the adaptations to skeletal muscle to body weight excess may differ from that of a healthy individual undertaking loaded resistance exercise, notably in relation with comorbidities that can be associated with obesity. Gender differences in body composition, muscle strength, and power have been reported in morbidly obese adults [8]. Generally, obese young men are significantly stronger in both upper and lower limbs and more powerful than the obese young women. Beside the increase in fat mass, obesity induces an increase in fat-free and muscle mass not in proportion with body weight increase and at a lower rate in women than in men [8]. Thus, the differences in muscle strength between men and women are attributed to greater FFM in the men. Indeed, when isotonic strength was normalized to FFM, all differences disappeared between genders in both the obese and normal-weight participants [8]. Hulens et al. have also observed in their obese cohort that when FFM was normalized, maximum knee extensor strength was significantly 6% 7% lower than normal-weight subjects [12]. Independently from the mechanical factors related to body mass excess, metabolic abnormalities frequently associated with obesity have a negative impact on muscle function during obesity [15]. Insulin resistance and type 2 diabetes (highly prevalent among obese individuals) have been shown to be associated with abnormal adipose tissue infiltration in skeletal muscles [16], or appearing as increased localized intermuscular adipose tissue [17]. These features are associated with a diminution of power output and strength per unit of muscle mass, an indicator of muscle quality and impaired physical function of the individual. In the calf of obese subjects with diabetes, Hilton et al. evidenced a marked increase of intermuscular adipose tissue which inversely correlated with muscle power, strength, and physical performance scores [18]. Interestingly, also in nonobese subjects, body adiposity was significantly related to poor muscle quality in both genders [19]. A similar effect of obesity-related adipose infiltration in lower limb muscles on quadriceps performance was reported in older adults [20], providing further evidence that lipid infiltration has a role in modulating the relationship between muscle mass and function.

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MUSCLE MASS Increased body mass index and adiposity are associated with increased skeletal muscle volume in young obese but not in older obese subjects [21]. This discrepancy is also present between male and female. It can be established that overall leg muscle volume increases on average by about 2 4 kg every five BMI units in man and about 1 3 kg in women, when variations due to age are accounted for [22]. Interestingly, in genetic models of obesity deficient in leptin or leptin receptors, a lower muscle mass has been reported [23,24], whereas in diet-induced obesity animal models in general, muscle mass is unchanged despite a higher fat mass [25,26]. The duration of diets inducing obesity is between 30 days and 16 weeks. The weight of the leg muscles showed an increase in the mass of these muscles after 30 days of cafeteria diet [27]. During the establishment of obesity in a rat model of prolonged diet-induced obesity, a gradual increase in body weight was observed during the first 16 weeks of high-fat diet, followed by a stabilization of the body weight for the following 8 weeks, suggesting a transition between two phases of obesity development [28]. In parallel with this change in body weight, an increase in muscle mass was reported during the first phase of obesity development, whereas after switching to the second phase, muscle mass decreased. This suggests that the onset of obesity might be an anabolic stimulus to skeletal muscle, because of increased body weight and exposure to greater mechanical loads which may have a physical training effect and may promote anabolism together with hyperinsulinemia. However, when obesity continues, chronic exposure to exogenous and endogenous lipid overload, probably due to the saturated storage capacity of adipose tissue, seemed to be harmful for muscle mass. Animal studies have also highlighted that age influence the effect of chronic feeding or obesity on muscle mass. Indeed, a decrease in muscle mass is observed in old rats fed a high fat diet during 10 weeks, whereas it is not the case in young rat fed the same diet [29]. Since aging is associated with a progressive loss of muscle mass and function, known as sarcopenia, aging might be considered as specific situation which might be more sensitive to deleterious impact of obesity on muscle. Moreover, chronic high-fat diet appeared sufficient to generate muscle fiber atrophy and impaired muscle function in mice that are at an age preceding sarcopenia [30].

SKELETAL MUSCLE PROTEIN METABOLISM AND OBESITY Skeletal muscle is composed by about 18% 20% of proteins, which are permanently synthesized and degraded at a rate of 1% 2% per day. Thus, changes in protein metabolism participated to the maintenance of protein quantity and quality and thus contribute strongly to muscle mass, composition, and function. In obese Zucker rats, reduction in muscle mass arises also from changes in the anabolic process of muscle protein accretion [23,31,32]. In these studies, the lower muscle mass is associated with a significant decrease in muscle protein synthesis rate [23,32] or an impairment of amino acid incorporation in muscle tissue [31]. In a rat model of diet-induced obesity, changes in muscle protein mass are dependent upon whether the animals are in the dynamic phase of obesity

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development or in the static phase of obesity [28]. More specifically, it was found that during weight gain, muscle mass is increased together with fat mass and is associated with an increase in muscle protein synthesis rate. However, when obesity is chronically installed, i.e., period of weight stabilization, muscle mass is decreased in association with protein synthesis rate [28]. Therefore, the onset of diet-induced obesity in this rat model seems to be an anabolic period for skeletal muscle, possibly because of increased weight bearing and exposure to greater mechanical loads which may have a physical training effect together with changes in anabolic hormones production. However, as obesity continues to develop, chronic exposure to exogenous and endogenous lipid overload, probably due to the saturated storage capacity of adipose tissue, seems to be harmful for muscle mass and muscle protein metabolism [28]. These observations therefore highlight the importance of characterizing the changes occurring in skeletal muscle protein metabolism during both the dynamic and static phases of obesity. In obese human, the turnover rate of skeletal muscle proteins has been reported to be reduced in postabsorptive state, illustrated by a much lower protein synthesis [33] and a reduced protein breakdown [34] rates than lean subjects. These two observations emphasize that protein kinetics at the muscle level is affected by adiposity but the mechanisms are still unknown. Protein turnover is highly regulated by nutritional intakes, hormones, and exercise and is affected in different situations, notably during obesity [35 37]. Insulin is an important regulator of protein metabolism since this hormone together with amino acids are key factors for the regulation of body protein mass [38]. The main in vivo effect of insulin and amino acids on whole-body and skeletal muscle protein metabolism is to inhibit protein breakdown and to stimulate protein synthesis [39,40]. In obesity, the control of protein metabolism by these factors has been shown to be modified like in other situations of insulin resistance (type 2 diabetes, aging) [36]. In obese animals, several studies have observed a diminished ability of insulin to stimulate protein synthesis and/or to inhibit protein degradation in muscle. In obese Zucker rats with diabetes induced by alloxan injection and treated with insulin for a short period of time, a lesser increase in muscle protein synthesis rate together with a higher protein degradation rate were observed compared to the lean animals [41]. The results of the study suggested a resistance of protein metabolism to the anabolic action of insulin in this model of obesity. Furthermore, another study has shown that in nondiabetic obese Zucker rats, in situ insulin infusion in hind limbs induced a greater stimulation of muscle protein synthesis than in the lean, despite a lower muscle mass [42], suggesting also that obese Zucker rats develop resistance to insulin’s antiproteolytic actions. Diet-induced obesity in mice impairs the activation of skeletal muscle protein synthesis in response to nutrient ingestion [43]. Interestingly, excess energy intake combined with adequate protein intake induces a stimulation of muscle protein synthesis rates in rats [26,27]. In obese subjects, the combination of insulin and amino acids infusion during 4 hours mimicking a fed state induced a normal stimulation of skeletal muscle protein synthesis rate [33]. However, in these subjects, the response of mitochondria protein synthesis to insulin and amino acids was blunted. In addition, anabolic response of myofibrillar protein synthesis to meal intake is blunted in overweight and obese adults [44]. All of these studies have shown disturbances and/or dysregulation in muscle protein metabolism during overfeeding or obesity that might participate to the changes in skeletal muscle mass and quality observed in these contexts.

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MUSCLE TYPOLOGY Skeletal muscle is composed of different types of fibers dependent of myosin heavy chain isoforms that condition metabolic and contractile properties of the muscle. Skeletal muscle fibers are characterized as one type of slow-twitch fiber (type I) and three types of fasttwitch fibers (type IIa, type IIx/d, and type IIb). Although the fiber type specification varies between species, it is generally accepted that type I and type IIa fibers are oxidative, whereas type IIx and type IIb fibers are primarily glycolytic [45]. Oxidative-type fibers are involved mainly in aerobic oxidative metabolism of fatty acids and glucose and are resistant to fatigue, leading to the performance of prolonged physical activities. Glycolytic-type fibers are using mainly glucose in anaerobic conditions and are adapted for rapid contractions but are sensitive to fatigue. In obesity, most of the studies demonstrated a decrease in type 1 fibers, and an increase in proportion of type 2 fibers. However, there are some discrepancies on that changes in fiber type distribution with some studies reporting no differences in muscle fiber composition between obese and lean subjects [46]. A relationship between muscle fiber type and obesity characterized by fewer type 1 (oxidative) and/or more type 2b (glycolytic) muscle fibers have been observed in obese subjects compared to lean individuals [47 49]. Other researches have reported a negative relationship between adiposity and the proportion of type 1 muscle fibers [50,51]. More precisely, the negative correlation is predominant between the percentage of type 1 fibers in skeletal muscle and central adiposity in the body [48,50,51], whereas no correlation was found when compared with subcutaneous fat [50,52,53]. Altered skeletal muscle fiber types in obesity might be explained by two different mechanisms, one metabolic and another one more mechanical. For the metabolic hypothesis, the obesity-related disorders such as inflammation, oxidative stress, and insulin resistance might be incriminated in the changes in the metabolism of muscle fibers. For instance, some evidence showed that slow-type myosin heavy chain is sensitive to inflammation [54]. In addition, type 1 fibers are known to be more sensitive to insulin than type 2b fibers which could explain the changes in muscle typology due to obesity-mediated insulin resistance [48]. In rodents, the induction of hyperinsulinemia with insulin infusion resulted in an increased percentage of type 2b muscle fibers at the expense of type 1 fibers [55]. The mechanical hypothesis is based from one side on the increased workload in weight-bearing muscles with obesity, simulated a resistance exercise which enhanced glycolytic-type muscle modification [56], and from the other side to the reduced abilities to perform physical activity, notably walking [57]. Indeed, walking ability is associated with an increase in muscle oxidative-type fibers, particularly in lower extremities [58]. Changes in muscle typology could have potential consequences on metabolic capacity of skeletal muscle in obesity. Indeed, skeletal muscle from obese individuals show markedly lower oxidative capacity and mitochondrial content [59,60]. These modifications are associated with a decrease in lipid oxidation [51,61] and thus an increase in fat storage within skeletal muscle but also in adipose tissues, explaining the association between adiposity and typological features related to obesity [47,50,51]. To illustrate this hypothesis, rodents that gained the most weight with high-fat feeding possessed significantly fewer type I fibers than littermates that gained little to no weight with the same diet [62]. This is indirectly suggested by data in humans indicating that subjects with higher respiratory quotient (i.e., lower fat oxidation) display a more rapid weight gain [63].

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CONCLUSION Obesity and overfeeding have deleterious impact on skeletal muscle illustrating by poor muscle performance and lower muscle quality. Clearly, obesity induces numerous structural and metabolic alterations within skeletal muscle ultimately resulting in decreased relative strength and power. However, further studies are needed to understand deeply the specific adaptations of skeletal muscle in the presence of chronically elevated adiposity. The maintenance of skeletal muscle is necessary for locomotion, gait, and substrates homeostasis. Strategies of weight loss, combining most of the time nutritional aspects and physical activity programs, need to be developed with the consideration of improvements of muscle size and quality. Nevertheless, attention should be focused on rapid, acute, and important weight loss, occurring for instance in bariatric surgery, that is accompanied by a noteworthy muscle mass loss.

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Skeletal Muscle Mitochondrial, Obesity, and High-Fat Feeding Christelle Guillet Clermont Auvergne University, INRA, Human Nutrition Unit, CRNH Auvergne, Clermont-Ferrand, France

INTRODUCTION Skeletal muscle plays a major role not only in the regulation of whole body energy homeostasis but also in the regulation of whole body insulin-resistance. However, the mechanisms underlying the relationship between obesity and skeletal muscle insulin resistance are yet a matter of debate. The prevailing theory is based on the inability of adipose tissue to store the excess energy, which results in an elevated plasma free fatty acids from fat depots to other tissues including skeletal muscle [1]. The excessive lipid content within this tissue causes metabolic dysregulation, including insulin resistance. The theory also suggests that mitochondria are key players in the insulin resistance development, but there are different views about the mechanisms by which mitochondria contribute to insulin resistance pathogenesis. It has been proposed that a decrease in mitochondrial fatty acid oxidation caused by mitochondrial dysfunction and/or reduced mitochondrial content leads to the accumulation of increased levels of intracellular fatty acyl-CoA and diacylglycerol, which interfere with the insulin signaling [2]. Chronic high consumption of dietary lipids and calories and/or obesity exerts a high metabolic load for mitochondria in skeletal muscle to oxidize substrates and thus initiate and maintain mitochondria derangements which progressively lead to the development of insulin resistance. These impairments are developed in this chapter and concern mainly mitochondria content, morphology, and oxidative capacities.

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MITOCHONDRIAL CONTENT Biochemical markers can provide information on mitochondrial content: mitochondrial to nuclear DNA copy numbers, cardiolipin expression, or enzymatic markers such as citrate synthase activity. Mitochondrial content can be also evaluated by measuring the mitochondrial relative to whole cell area by transmission electron microscopy imaging [3]. However, regarding the methodologies used to assess mitochondrial content, contradictory findings have been produced. For instance, citrate synthase activity was found to be lower in obesity in some studies [4 6], whereas other studies, however, have shown no significant reductions [7,8]. Enzyme activity measurements are sensitive to variations in assay protocols and rely on assumptions of enzyme activity being uniformly proportional to mitochondrial content. In skeletal muscle, mitochondria can be difficult to isolate from the cytoskeleton, and differences in the purity or yields of mitochondrial isolates add variability to measurements. Likewise, mtDNA content has produced conflicting findings. Insulin resistance was associated with less mtDNA in some studies [9,10] but not in others [11]. Studies using transmission electron microscopy imaging to calculate mitochondrial content have observed a reduced content of muscle mitochondria in obese subjects concerning mainly intermyofibrillar (IMF) mitochondria [3]. Indeed, within skeletal muscle, mitochondria are divided in two populations regarding their subcellular localization: subsarcolemmal (SS) and IMF mitochondria. Mitochondria that are clustered in proximity to the sarcolemma are termed SS mitochondria, and those embedded among the myofibrils are called IMF mitochondria. SS mitochondria provide energy for membrane-related processes, including signal transduction, ion exchange, substrate transport, and substrate activation (steps clearly relevant to insulin action), whereas the IMF mitochondria more directly support muscle contraction [12]. In obesity, the size and morphology of mitochondria is also altered characterized by B35% reduction of mitochondria area [7]. Morphologically, mitochondria in muscle of obese individuals are enlarged and fragmented, signing likely increased apoptosis. Altered morphology of mitochondria is a hallmark of a number of different myopathies known to result in disturbances of biochemical function of mitochondria, suggesting thus that mitochondria function in skeletal muscle is impaired in obesity [13]. In addition, the size of mitochondria is correlated with insulin sensitivity such as it has been well described in type 2 diabetes [7]. In insulin-resistant subjects, the underlying factor explaining the relationship with lower mitochondrial content and reduced insulin sensitivity might be intramuscular lipid overload and attendant mitochondrial adaptations leading to long term to mitochondria dysfunction.

MUSCLE OXIDATIVE CAPACITIES One of the striking physiological characteristics of skeletal muscle is metabolic flexibility expressed by the capacity of skeletal muscle to switch from high rates of fatty acid uptake during fasting conditions and predominantly lipid oxidation to the suppression of lipid oxidation and increased glucose uptake, oxidation, and storage under insulin-stimulated conditions [14]. This metabolic “switch” is impaired in obesity, characterized by a lower

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fasting rate lipid oxidation than in lean counterparts and an inability to change substrate oxidation during insulin infusion [15]. The inability to modify fuel oxidation in response to changes in nutrient availability, so-called metabolic inflexibility, has been implicated in the accumulation of intramuscular lipids (triglycerides, diacylglycerol, and ceramides) and insulin resistance [16,17]. The reduced reliance on lipid oxidation seen in obese individuals might be due to defects at several levels in the catabolic process for lipids in skeletal muscle mitochondria [6]. More precisely, the oxidation of both long (palmitate and palmitoyl carnitine) and medium (octanoate) chain fatty acids is reduced in skeletal muscle homogenates from obese subjects [6]. Muscle carnitine palmitoyltransferase-1 (CPT-1) activity, essential step in the beta-oxidation of long chain fatty acids, is reduced with obesity, which may at least partially explain the reduction in palmitate oxidation. The decrease in palmitoyl carnitine, however, implies a post-CPT-1 defect such as a reduction in oxidative capacity via reduced mitochondrial content, as illustrated by lower muscle citrate synthase and β-hydroxyacyl CoA dehydrogenase activities [6]. Results have evidenced that in rats, high-fat or high-sucrose intake, and more importantly overfeeding (which effects have to be related to those observed with obesity), are major factors associated with decreased muscle mitochondrial oxidative phosphorylation (OXPHOS) activity, but in a musclespecific way [18]. Thus, the mitochondrial OXPHOS activities within oxidative muscles, resistant to fatigue and dependent on mitochondrial activity for adenosine triphosphate (ATP) production, are more affected than within glycolytic muscles. The main changes included a reduction in the respiratory chain activity with a concomitant decrease in mitochondrial ATP production [18]. Decreased mitochondrial respiration rates [19] and reduced expression of genes involved in mitochondrial oxidative capacity [20] have been reported in diet-induced obese rats. These changes may be mediated by decreased expression of PPARγ coactivator 1α (PGC1α) and nuclear respiratory factor 1 genes, both of which control mitochondrial biogenesis. Interestingly, high-fat diets downregulated PGC1α and PGC1β as well as genes coding for proteins of the electron transport chain in human skeletal muscle [21], which suggests that excess dietary fat could alter mitochondrial functions. Generally, skeletal muscle of obese subjects is characterized by low aerobic-oxidative capacities (citrate synthase and cytochrome c oxidase) together with increased anaerobic and glycolytic capacities (phosphofructokinase, glyceraldehyde phosphate dehydrogenase, hexokinase) [22,23]. The activity of skeletal muscle NADH oxidoreductase, corresponding to complex I on the mitochondria respiratory chain, is reduced in obese compared with lean volunteers [7]. The reduction in the activity of marker enzymes of oxidative pathways correlates with the severity of insulin-resistant glucose metabolism [24]. Mitochondrial dysfunction may be further compromised in type 2 diabetes obese skeletal muscle, as type 2 diabetes skeletal muscle exhibits decreased respiration rates and lower oxidative enzyme levels compared to obese, nondiabetic or lean muscle [24,25]. These findings raise the possibility of impaired mitochondrial function in obesity as an additional aspect of the pathogenesis of insulin resistance. Indeed, the degree of severity of mitochondria dysfunction in obesity is mainly dependent of the insulin sensitivity state of the individuals. Chanseaume et al. have explored the association between abdominal obesity, insulin sensitivity, and muscle mitochondrial content and function in middle-aged healthy sedentary men recruited according to waist circumference [26]. Insulin sensitivity index was negatively linearly correlated to waist circumference. Increased abdominal

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obesity was not associated with alterations in muscle mitochondrial content, whereas intrinsic mitochondrial function [adenosine diphosphate (ADP)-stimulated respiration, ATP, and reactive oxygen species (ROS) production] is significantly affected [26]. Muscle mitochondrial function first positively adapts to fat mass gain to protect against the metabolic conditions that can induce insulin resistance. Thereafter, metabolic alterations related to obesity (such as lipotoxicity, oxidative stress, and/or low-grade inflammation) that are responsible for the induction of insulin resistance may be involved in modifying muscle mitochondrial function. Taken together, studies on men are mainly consistent with the association between insulin resistance and mitochondrial impairment, although in general, they do not discriminate between cause and effect. In addition, in human studies, it is difficult to control parameters such as energy intake, quality of the diet, and level of activity. Many studies have also been carried out in animal models of obesity and diabetes, mainly rats and mice. The results obtained in these animal models are mostly in agreement with those obtained in human studies. The most used protocol to induce insulin resistance is overfeeding with high-fat/high-sugar diets in rats [18,27] or in mice [28,29]; however, models of genetic obesity are also used [30]. However, other observations report discrepant results, showing that high-fat feeding in rats is associated with no variation [31,32] or even a higher mitochondrial capacity [33], and insulin resistance could be related to incomplete intramitochondrial β-oxidation [34]. Several studies show that insulin resistance arises when mitochondrial function is unaffected or even improved [35 38], or conversely that impaired mitochondrial function alone does not cause insulin resistance [39]. These latter findings data seem to indicate that, at least in rodents, consumption of highfat diet is not always accompanied by mitochondrial dysfunction but rather leads to improved mitochondrial oxidative capacity and/or biogenesis [40,41], even in the presence of insulin resistance. The duration of high-fat feeding also seems to differently affect mitochondrial function in skeletal muscle. In fact, OXPHOS was found increased after shortterm (2 3 weeks) dietary treatment but decreased after long-term treatment [42 44]. Even if there are some discrepancies between studies, it is noteworthy that mitochondria oxidative capacities are impaired in obesity which leads to reduced fat oxidation within skeletal muscle and been associated with the development of insulin resistance.

FACTORS INVOLVED IN MITOCHONDRIA DYSFUNCTION Oxidative stress in skeletal muscle is probably one of the major determinants of mitochondrial alterations. This is supported by a study showing that an increase in muscle ROS production occurs specifically after 16 weeks of high-fat, high-sucrose diet when mice are hyperglycemic and hyperlipidemic [42]. In this work, ROS production is also associated with mitochondrial alterations in the muscle of hyperglycemic streptozotocin-treated mice. In addition, incubation of cultured muscle cells with high glucose or high lipid concentrations induced ROS production and altered mitochondrial density and functions [42]. Other factors might be involved in mitochondrial defects related to obesity. Mitofusin 2 (Mfn2) is a mitochondrial protein, highly abundant in skeletal muscle, heart, and brain [45], that regulates the generation of the mitochondrial network in mammalian cells [45 47]. Thus, partial or total elimination of Mfn2 expression causes a reduction of the

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mitochondrial network in cells [45,48]. In addition, Mfn2 controls mitochondrial function and modulates mitochondrial metabolism [45]. Its expression level regulates mitochondrial membrane potential, fuel oxidation, and the OXPHOS system [45,49]. Thus, in myoblasts with a limited oxidative capacity, Mfn2 gain of function causes an increase in the rate of glucose oxidation and a parallel increase in mitochondrial membrane potential, which indicates augmented pyruvate oxidation in mitochondria and enhanced Krebs cycle and OXPHOS [49]. In addition, Mfn2 repression leads to a decrease in the oxidation rates of glucose, pyruvate, and palmitate and reduces mitochondrial membrane potential in myotubes [45,49]. Expression of Mfn2 mRNA and protein is reduced in obese men and women [45,50]. Mfn2 expression in muscle is inversely proportional to the BMI and directly proportional to insulin sensitivity. TNFα or interleukin-6 downregulate Mfn2 expression; these factors may participate in the dysregulation of Mfn2 expression detected in obesity or type 2 diabetes. Mfn2 mRNA levels is an additional level of regulation for the control of mitochondria muscle metabolism and could provide a molecular mechanism for alterations in mitochondrial function in obesity or type 2 diabetes.

CONCLUSION In obesity, mitochondria alterations mainly characterized by impairment in mitochondria oxidative enzyme activities and reduced fat oxidation constitute inherent functional defects in skeletal muscle. Reduced mitochondrial OXPHOS is associated with insulin resistance, as well as mitochondria content and morphology. However, further studies are necessary to confirm the link between obesity, mitochondria derangements, and insulin resistance in order to understand the sequential steps of these alterations. In a general view, mitochondria impairments occurring during obesity might contribute to reduced contractile and metabolic capacities in muscle leading to decreased locomotor abilities and appearance of metabolic disorders.

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Muscle Immune Cells, Obesity, and High-Fat Feeding Carla Domingues-Faria, Nicolas Goncalves-Mendes and Marie-Chantal Farges Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France

INTRODUCTION After skeletal muscle injury, a regeneration process takes place to repair muscle. Skeletal muscle recovery is a highly coordinated process involving cross talk between immune and muscle cells. However, the physiological activities of both immune cells and muscle stem cells (MSC) are modified with obesity or high-fat feeding, thereby blunting the capacity of skeletal muscle to regenerate. The first part concerns the impact of obesity and diet-induced obesity (DIO) on skeletal muscle inflammation. The second part deals with immune and muscle cell cross talk involved during the physiological regeneration process, and how obesity and DIO likely impair muscle regeneration repair.

OBESITY, HIGH-FAT FEEDING, MUSCLE IMMUNE CELL INFILTRATION, AND ACTIVATION Skeletal muscle is a major organ for whole body glucose homeostasis and it accounts for a large part of the insulin-stimulated whole body glucose capture and disposal in the physiological state [1]. Skeletal muscle insulin resistance in obesity is multifactorial and results not only from intermyocellular nonesterified-free fatty acid and triglyceride depots and the formation of subsequent metabolites [2], mitochondrial dysfunction [3], and endocrine effects of adipokines [4] but also from in situ inflammation mediated by immune cells [5]. Most investigations of obesity-linked inflammation and insulin resistance have

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focused on the activation of the innate immune system particularly in adipose tissue [6]; however, current evidence suggests an important role for the adaptive immune system [7,8]. Despite the evidence that inflammation occurs in skeletal muscle in obesity and results mainly in increased immune cell infiltration and pro-inflammatory activation, the underlying mechanisms are less studied in muscle than in adipose tissue.

Immune Cell Infiltration Into Skeletal Muscle Among the immune resident cells present in skeletal muscle, the leukocyte population, cells expressing the cluster of differentiation 45 (CD451 cells), is predominant. Macrophages (F4/801) and nonmyeloid (F4/802) cells in particular T CD41 and T CD81 lymphocytes witch represent more than 50% and 25% of leukocytes, respectively [9]. Histologically, if few macrophages and T lymphocytes are located between myofibers, they are mainly found in the extramyocellular adipose depots and more precisely in the intermuscular adipose tissue (IMAT), located between myocytes, and in the perimuscular adipose tissues (PMAT), located around large muscle [10]. As obesity develops and progresses along with development of visceral and subcutaneous adipose tissue, ectopic fat depots expand between muscle fibers and surrounding muscle and highly correlate with insulin resistance [10]. As observed in adipose tissue, increasing evidence suggests that immune cells accumulate into skeletal muscle and contribute to the in situ inflammation. A short-term high-fat diet (HFD), high-caloric diet inducing insulin resistance in skeletal muscle of healthy lean subjects, results in an upregulation in the expression of general macrophage markers such as CD68 and CD14, as well as the expression of pro-inflammatory M1 macrophage markers such as macrophage receptor with a collagenous structure (MARCO), CD11b and CD11c, and mannose receptor C type 1 [11]. Previously, Varma et al. showed that the skeletal muscle from obese subjects contains 2.5-fold higher CD681 macrophage numbers compared to lean subjects [12]. Consistent with previous studies [9,13], macrophage marker CD11b was significantly upregulated in skeletal muscle of obese humans compared to lean control [10]. In addition to macrophage infiltration, an elevated T-cell accumulation occurs as shown by the increased expression of T-cell markers, such as CD4, CD8, and regulated on activation, normal T cell expressed and secreted (RANTES) in obese humans compared to lean subjects [10]. Similarly, in mice, DIO associated with insulin resistance results in skeletal muscle accumulation in macrophages, T cells, and neutrophils [9,10,14]. Among the T-cell population, both CD41 and CD81 cell numbers increase in skeletal muscle of obese mice while among the CD41 cells, the proportion of CD251FoxP31 (forkhead box P3) T regulator (Treg) was reduced [10,15,16]. Moreover, during the obesity setup, the increase in macrophage numbers in IMAT occurs during the early stage and precedes T-cell accumulation [10]. Conversely to what is observed in adipose tissue, changes in other immune cells such as B, natural killer (NK), and NKT cells have not been explored in skeletal muscle.

Chemokines, Adhesion Molecules Involved in Immune Cell Infiltration Into Skeletal Muscle The recruitment of monocytes into skeletal muscle requires chemokines and adhesion molecules such as monocyte chemoattractant protein 1 (MCP-1), chemokine (C-X3-C motif) II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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ligand 1 (CX3CL1), chemokine (C-X-C motif) ligand 1 (CXCL1). Mice quadriceps gene expression of MCP-1, also called chemokine (C-C motif) ligand 2 (CCL2), and its receptor chemokine (C-C motif) receptor type 2 (CCR2) are upregulated after only 1 week of HFD and in presence of glucose intolerance [9]. Conversely, quadriceps from CCL2-knockout mice placed on DIO do not accumulate macrophages and maintain their insulin sensitivity [9]. MCP-1 expression is also upregulated in skeletal muscle of both ob/ob and long-term HFD mice [14]. Interestingly, skeletal muscle-specific overexpression of MCP-1 in transgenic mice under normal calorie diet (NCD) induces local CD68 and CD11c macrophage recruitment, RANTES, tumor necrosis factor (TNF)-α, and interleukine (IL)-1β expression and altered local insulin signaling in association to an elevated plasmatic MCP-1 levels [14]. T-cell recruitment into skeletal muscle is mediated by a combination of chemokines and adhesion molecules such as MCP-1, RANTES, and CD11a [17
20]. Compared with obese wild-type, obese CD11a 2 / 2 mice as well as obese mice treated with CD11a-neutralizing antibody, skeletal muscle extramyocellular adipose tissue T-cell content was reduced, including both CD41 and CD81 T cells without affecting the macrophage number [10]. So, CD11a appears to be essential to the T-cell transmigration into skeletal muscle. Moreover, reduced total T and αβ T cells in HFD-fed TCRβ 2 / 2 mice were accompanied by decreased macrophage accumulation in skeletal muscle, suggesting a potential role of skeletal muscle T cells in macrophage recruitment [16]. Chemokine and adhesion molecule production by skeletal muscle results not only from immune resident and infiltrated cells (CD451) but also from other cell types (CD452) including myocytes, endothelial cells, and adipocytes. If RANTES is restrictively expressed in the CD451 cells, the mRNA levels of CX3CL1, MCP-1, and CXCL1 are detected in both muscle CD451 and CD452 fractions in wild-type mice and their expressions are significantly increased in ob/ob mice in both fractions [14]. Obesity-driven chemokine secretion is upregulated by INF-γ and TNF-α, the main T helper type 1 (Th1), and M1 macrophage pro-inflammatory cytokines [21]. In obesity, elevated levels of circulating free fatty acids, mainly derived from adipocyte lipolysis, and elevated levels of triglyceride-rich lipoproteins, lead to increased fatty acid influx into skeletal muscles. Fatty acids induce inflammation in immune cells and myocytes and contribute to insulin resistance. Palmitate induces the expression of both MCP-1 and RANTES in C2C12 differentiated myotubes and palmitate-treated C2C12 conditioned media display enhanced properties to recruit Raw264.7 macrophages [14]. Similarly, in vitro palmitate-stimulated monocytes express macrophage CD11b and CD11c markers [22] and secrete factors that enhance the protein levels of intracellular adhesion molecule 1 and E-selectin [23]. Mice fed a palm oil-enriched diet significantly increase skeletal muscle C681CD111 macrophage content [14]. In the context of obesity, the renin
angiotensin
aldosterone system is inappropriately activated in several tissues such as adipose tissue [24] and skeletal muscle [25]. Angiotensin II via nuclear factor kappa B (NF-κB) activation induces expression of MCP-1, TNF-α, vascular cell adhesion protein 1, and reactive oxygen species (ROS) production in monocytes, endothelial cells [26], and cultured L6 myotubes [27] and also participates in both leukocyte recruitment and activation. Inhibition of the angiotensin-converting enzyme reduces systemic and skeletal muscle expression of pro-inflammatory mediators such as MCP-1, IL-6 in HFD mice [28]. Both angiotensin-converting enzyme inhibitor and angiotensin II receptor blocker reduce the TNF-α content in skeletal muscle of high-fructose diet fed mice [29]. If angiotensin II has pro-inflammatory effects on skeletal muscle, conversely, angiotensin 1
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exerts anti-inflammatory properties and reverses HFD-induced skeletal muscle insulin resistance [30]. To date, angiotensin 1
7 impact on skeletal muscle leukocytes has not been studied in experimental model of obesity and HFD; however, it has been reported to reduce macrophage infiltration in dystrophic muscles [31].

Skeletal Muscle Immune Cell Activation Macrophages and T cells infiltrated into extramyocellular muscle adipose tissue polarize into pro-inflammatory phenotypes. Indeed, in the skeletal muscle microenvironment, pro-inflammatory cytokines such as TNF-α, interferon (IFN)-γ, IL-1β are markedly increased while anti-inflammatory cytokines such as IL-10 are reduced as reported both in human and mice in obese conditions [10,11,15] or in vitro cocultures of differentiated myotubes and macrophages [12,14,32]. In response to various signals, mainly IFN-γ and tolllike receptor ligands, macrophages undergo classical M1 pro-inflammatory phenotype [33]. The expression of pro-inflammatory macrophage markers such as CB11b and CD11c is increased in skeletal muscle of obese glucose intolerant humans [9,10,13]. Similarly, HFD induces a rise in inflammatory CD11c1 macrophage in wild-type mice muscle compared to the control diet [9]. At the same time, IL-10, a potent inhibitor of the proinflammatory cytokines and chemokines, is reduced both in human and mice in obese condition [11,15]. Its overexpression attenuates macrophage and cytokine response in skeletal muscle of HFD mice and prevents diet-induced insulin resistance [34]. Among the T cells infiltrated in skeletal muscle of obese mice, CD41 helper cells polarize into a pro-inflammatory Th1 phenotype under the activation of cytokines such as IFNγ, TNF-α, IL1-β, and IL-6. Indeed, gene expression of IFN-γ and TNF-α is upregulated in the PMAT [10] and IL-6 is overexpressed in the myocellular area [14]. In addition, Th1 cells antagonize Th2 immunosuppressive cell functions [35]. At the same time, myocytes may become inflamed and produce also pro-inflammatory mediators as reported in in vitro studies [12,14]. Khan et al. reported that Th1 cells through IFN-γ induce skeletal muscle myocyte inflammation and insulin resistance via activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway as demonstrated by in vitro and in vivo studies using JAK/STAT inhibitors [10,16]. To date, it is unknown if resident leukocytes, which are typically neutral or anti-inflammatory, expand and eventually adopt a pro-inflammatory polarization in the context of obesity [36]. In summary, increasing evidence suggests that inflammation occurs in skeletal muscle in obesity and is mainly attributed to macrophages and T-cell infiltration and activation in both IMAT and PMAT. By secreting pro-inflammatory mediators and in coordination with proinflammatory adipokines, they induce myocyte inflammation and metabolic dysfunction and contribute to in situ and whole body insulin resistance through paracrine and autocrine effects.

OBESITY, HIGH-FAT FEEDING, AND MUSCLE REGENERATION CAPACITY After skeletal muscle injury, a regeneration process takes place to repair muscle. Skeletal muscle recovery is a highly coordinated process involving cross talk between II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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immune and muscle cells. However, the physiological activities of both immune cells and MSC are modified with obesity or high-fat feeding, thereby blunting the capacity of skeletal muscle to regenerate.

Immune and Muscle Cell Cross Talk During Skeletal Muscle Regeneration Process Polynuclear neutrophils are among the immune cells that first infiltrate muscle lesions. They are involved in the development of necrosis phase and in the initiation of proinflammatory response following muscle injury [37,38]. Following neutrophil infiltration, circulating monocytes migrate from blood to the site of the lesion, where they differentiate into macrophages, gradually replacing neutrophils as the main population of immune cells [39,40]. Monocyte migration and infiltration to the lesion site start in response to chemokines such as IL-1, IL-8, IL-6, and MCP-1 [41,42]. These chemokines are secreted by muscle cells, resident macrophages, and infiltrated macrophages [41]. We thus note that macrophages potentiate their own infiltration at the injured site [43]. At this step, macrophages have a pro-inflammatory M1 phenotype. M1 macrophages lyse muscle cells and phagocyte cellular debris [37,44]. Furthermore, they attract satellite cells, i.e., MSC, to the injured site [45] and stimulate their proliferation [46]. Muscle cell proliferation is in part dependent on macrophage-secreted IL-6, IFN-γ, and TNF-α [43,47
49]. TNF-α also plays a chemoattractant role on satellite cells at the damaged site of the muscle [50]. After the pro-inflammatory stage, macrophage M1 phenotype is converted to an antiinflammatory M2 phenotype, a central process in muscle regeneration [51,52]. Several molecular and cellular processes are involved in macrophage phenotype transition, macrophage phagocytosis being one of them [51]. Macrophage-secreted cytokines also orchestrate M1/M2 conversion such as IFN-γ, TGF-β, and IL-10 [41]. At the cellular level, the M1/M2 transition is coordinated by mitogen-activated protein kinase phosphatase-1 (MKP-1), which regulates the activation of mitogen-activated protein kinase p38 and the activity of Akt (protein kinase B) [53]. Cyclic adenosine-monophosphate response [element binding protein (CREB) binding on the promoter of CCAAT/enhancer binding protein (C/EBPβ)] induces the expression of C/EBPβ, leading to the expression of M2 phenotyperelated genes such as IL-10 [54]. The involvement of adenosine 50 -monophosphate-activated protein kinase alpha (AMPKα) activation in the M1/M2 transition has also been demonstrated, as it induces CREB activation [54,55]. Anti-inflammatory M2 macrophages are involved in the pro-inflammatory response blockage and stimulate muscle cell differentiation, leading to myofiber formation or repair [41]. M2 macrophages-secreted insulin-like growth factor-1 (IGF-1) seems to contribute to muscle differentiation and repair [56]. Finally, M2 macrophages are essential for angiogenesis, contributing to efficient muscle repair [57]. The anti-inflammatory response has to cease at the end of the muscle repair process. A study has demonstrated that when regeneration is completed, M2 macrophages become inactive, and consequently anti-inflammatory cytokine production is interrupted [53]. Macrophages then revert to a quiescent state or are eliminated through apoptosis. Like the M1/M2 transition, inflammatory reaction is stopped by a coordinated action of p38/MKP-1 II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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and Akt [53]. To sum up, macrophages stimulate satellite cell proliferation and differentiation during muscle repair. In addition, they limit myotube and satellite cell apoptosis during muscle regeneration [58,59]. Neutrophil and macrophage functions during muscle repair have been thoroughly investigated. However, other types of immune cells also contribute to this process as mast cells, CD41 T cells, CD81 T cells, Treg cells, NK cells, or monocyte-derived dendritic cells known to stimulate satellite cell proliferation and/or differentiation [41].

Effect of Obesity and/or High-Fat Feeding on Skeletal Muscle Repair Several studies have demonstrated that high-fat feeding and/or obesity impair skeletal muscle repair capacity [60
63]. First, it has been demonstrated that obesity seems to increase the necrosis step. The initial necrosis phase is prolonged during the muscle regeneration process of DIO mice and contributes to the impairment of skeletal muscle repair [61]. This is probably due to the chronic infiltration of M1 macrophages in skeletal muscle observed in obesity [5]. On the one hand, the sustained secretion of proinflammatory cytokines and the sustained process of phagocytosis probably contribute to the increase of the necrosis period. On the other hand, the obese muscle-infiltrated M1 macrophages likely impair the normal response of satellite cells to them during muscle repair. D’Souza et al. demonstrated that satellite cells do not activate normally in DIO mice compared to control mice [61]. In physiological conditions, they activate and proliferate in response to M1 macrophages during muscle regeneration. With obesity, the sustained pro-inflammatory phenotype of macrophages in skeletal muscle likely inhibits satellite cell activation, contributing to the reduction of skeletal muscle repair capacity. Another explanation for impaired muscle repair capacity with obesity is the reduced skeletal muscle precursor cells in mice fed with a HFD compared to NCD leading to a muscle repair decrease of 44% [60]. Conversely, a more recent work demonstrates that satellite cell content is not modified in the skeletal muscle of DIO mice [61]. This discrepancy is certainly due to the differences in in vivo models used (HFD mice vs DIO mice), muscle samples studied (hindlimb muscles vs tibialis), and methods used to detect and quantify muscle cells (immunofluorescence vs flow cytometry) [60,61]. Interestingly, a reduced number of satellite cells as well as a reduction in their activation are characteristics found in aged skeletal muscle [41]. They contribute to the poor muscle repair capacity observed in aged people promoting sarcopenia development [41]. These observations suggest that obesity may increase the risk of developing sarcopenia, and thus sarcopenic obesity [64]. Interestingly, HFD does not alter the proliferative capacity of satellite cells during muscle repair in mice [60,65]. However, the differentiation process seems to be strongly impaired by obesity and/or HFD. Indeed, it has been demonstrated that expression of muscle differentiation markers, i.e., myoblast determination protein (MYOD) and Myogenin (MYOG), is delayed and reduced during the muscle regeneration process in obese mice [61,62]. In addition, embryonic myosin heavy chain expression, i.e., an immature form of myosin, is persistent [61,65]. In physiological conditions, macrophage conversion of M1 phenotype into M2 phenotype is essential to ensure a successful muscle repair

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process [41]. M1 macrophages promote satellite cell proliferation, whereas M2 macrophages stimulate muscle cell differentiation, i.e., from satellite cells to myofibers [41]. To our knowledge, little is known about M2 macrophages during muscle regeneration in obesity. However, knowing that this pathology is associated with chronic low grade inflammation, it can be assumed that the latter prevents the conversion of M1 macrophages to M2 phenotype. This would explain why muscle proliferation is not altered but that the effectiveness of muscle differentiation is decreased during muscle regeneration in an obesity context. Moreover, obesity and HFD promote fibrotic area development after muscle regeneration. The healing of muscle lesion areas is poor in HFD and obese mice as an increase of collagen deposition was observed at the end of muscle regeneration process [61,62,65]. At the mechanistic level, insulin/IGF-1 signaling exerts an important role in muscle repair [65]. However, this pathway can be inhibited by enhancing phosphatase and tensin homolog deleted from chromosome 10 (PTEN) expression, which consequently reduces the level of phosphatidylinositol 3,4,5-trisphosphate (PIP3) [65]. Thus, the phosphorylation of Akt and the activation of downstream products involved in muscle growth are reduced [65]. Interestingly, Hu et al. have demonstrated that HFD rose circulating fatty acids in mice stimulating PTEN expression [65]. Then, the growth of regenerating myofibers is impaired, myofiber maturation is delayed, and collagen deposition increases in HFD mice compared to control mice [65]. Interestingly, M2 macrophages secrete IGF-1 in the physiological muscle regeneration process, which stimulates muscle repair [56]. Thus, we can hypothesize that obesity and HFD impair macrophage and muscle cell cross talk: (1) by enhancing PTEN expression in muscle cells, and (2) by preventing M1/M2 conversion with the consequence of IGF-1-reduced secretion. Both of these effects could lead to a general inhibition of insulin/IGF-1-signaling pathway and thus of skeletal muscle repair in obesity. The activity of AMPKα1 is known for its regulatory role in energy metabolism and its implication in muscle regeneration has been demonstrated [63]. AMPKα1 knockout led to an important reduction of satellite cell proliferation and differentiation in mice [63]. In the obesity context, AMPKα1 activity is reduced leading to a muscle repair capacity that is importantly attenuated, rescued by AICAR, a drug that specifically activates AMPK [63]. Interestingly, AMPKα1 activity is also essential for M1/M2 macrophage conversion, as its deletion or its stimulation, respectively, limits or enhances macrophage phenotype conversion [54,55,66]. Metabolic disorders are associated with reduced AMPK activity [63]. In this context, low AMPKα1 activity alters macrophage and muscle cell cross talk, and thus muscle regeneration, by both limited M1/M2 conversion and limited muscle cell proliferation and differentiation. Leptin signaling is also essential for skeletal muscle regeneration. In fact, it has been highlighted that the lack of leptin signaling leads to the impairment of muscle regeneration, associated with the reduction of macrophage accumulation in skeletal muscle and myoblast activity in ob/ob and db/db mice [67]. Obesity is associated with leptin resistance [68]. Thus, leptin resistance might be one of the factors contributing to impaired muscle repair during obesity, probably through a reduction of macrophage accumulation in skeletal muscle and myoblast activity. This likely leads to the impairment of macrophage and muscle cell cross talk.

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Muscle accumulation of adipose tissue or collagen deposition could impair skeletal muscle repair in obesity as they could mechanically prevent or limit satellite cell migration, immune cell infiltration, and/or immune/muscle cell cross talk [61,62,65,69]. Finally, recent investigations have evidenced epigenetic and transcriptional changes of myogenic transcription factors, e.g., MYOD1, MYOG, myogenic factor (MYF) 5, MYF6 or paired-box protein (PAX) 7, during differentiation of primary obese human MSC compared to nonobese ones [70]. This result demonstrates that obese-induced metabolic changes likely impact gene expressions by an epigenetic mechanism, contributing to the impairment of muscle regeneration during obesity.

CONCLUSION Immune and muscle cell cross talk is essential for an optimal skeletal muscle regeneration process. Obesity is associated with increased inflammation in skeletal muscle resulting mainly from an enhanced immune cell influx and pro-inflammatory activation in extramyocellular adipose tissue which negatively regulate myocyte metabolic functions. Moreover, muscle accumulation of adipose tissue impairs skeletal muscle regeneration by reducing the number and the activity of satellite cells and decreasing muscle cell differentiation. During aging, reduced skeletal muscle cell repair enhances the risk of sarcopenia which is majored by obesity. Obesity and sarcopenia are frequently associated, a phenomenon named sarcopenic obesity. Whatever the context obesity, aging or both, exercise, body weight loss, or anti-inflammatory therapy may be a promising strategy to limit extramyocellular adipose tissue depot and improve skeletal muscle functions (i.e., insulin sensitivity and muscle regeneration process).

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Physiological Regulation of Skeletal Muscle Mass: Resistance ExerciseMediated Muscle Hypertrophy Joaquı´n Pe´rez-Schindler and Christoph Handschin Biozentrum, University of Basel, Basel, Switzerland

INTRODUCTION Skeletal muscle is one of the most abundant and metabolically active tissues in mammals, representing B40%
50% of total body mass and accounting for about 30% of whole body energy expenditure in humans at rest. Consequently, modulation in skeletal muscle mass and function can directly affect whole body health, exemplified by the increased morbidity and mortality in skeletal muscle atrophy and weakness in conditions such as cancer, type 2 diabetes, and aging [1
3]. The balance between protein synthesis and degradation is the main factor that determines skeletal muscle mass and function. While amino acids, circulating growth hormones, and exercise play a major role in stimulating anabolic processes, skeletal muscle disuse (e.g., due to an injury or a sedentary life style), illness, undernutrition, and aging are among the most common factors that boost catabolic processes and skeletal muscle wasting. Besides the decrease in the rate of protein synthesis, conditions associated with skeletal muscle atrophy are characterized by a robust increase in the rate of proteolysis of structural and contractile proteins in skeletal muscle [4]. Molecularly, the ubiquitin proteasome pathway, in which ubiquitin ligation of target proteins by E3 ubiquitin ligases represents a process that is highly sensitive to skeletal muscle load, is a key event in muscle atrophy [4]. Accordingly, the skeletal muscle-specific E3 ubiquitin ligases Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx; also known as atrogin-1) are strongly upregulated by skeletal muscle disuse, while genetic deletion of these genes confers a protective effect against skeletal muscle atrophy [5]. Interestingly, synergist ablation-mediated skeletal muscle hypertrophy in mice also increases proteasomal activity, but the E3 ubiquitin ligases MuRF1 and MAFbx are not linked to essential regulatory functions in this context [6]. In addition to reduced load,

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skeletal muscle atrophy is induced by other factors such as mitochondrial dysfunction and autophagy. Such catabolic stimuli are, however, primarily activated under pathological conditions and, thus, are beyond the scope of this chapter. Additional information about the molecular mechanisms involved in skeletal muscle atrophy can be found in excellent recent reviews [4,7,8]. Even though both protein degradation and synthesis can be activated by physical activity, the latter is the main physiological process that controls adaptive growth of skeletal muscle [9,10]. During development, muscle growth is mainly dependent on hormonal stimuli (e.g., testosterone), whereas postdevelopmental growth is primarily regulated by environmental cues among which nutrients and mechanical load play a central role [11]. Resistance exercise consists of a low number of bouts of muscle contractions (,12 repetitions) exerted against a high load [ . 70% of the 1 repetition-maximum (1 RM)], which represents one of the strongest stimuli to increase the rate of myofibrillar protein synthesis and, consequently, the cross-sectional area of individual skeletal muscle fibers. However, it has been proposed that low-load resistance exercise (#60% 1 RM) also efficiently increases skeletal muscle strength and size when performed to failure, in part due to the recruitment of a large number of muscle fibers, similar to that observed in high-loadresistance exercise [12]. Importantly, muscle strength, a key factor improved by resistance exercise, as well as whole body oxygen consumption, promoted by endurance training, negatively correlate with all-cause of mortality, foremost in subjects over 60 years of age [2]. In general, endurance training improves health by promoting oxygen consumption and cardiovascular function. Nevertheless, protein supplementation and resistance exercise (mainly in combination) are currently considered as the most efficient approaches to ameliorate the detrimental effects of disuse and sarcopenia on muscle mass and function in young and old individuals [3,10]. Importantly, besides enhancing muscle size and strength, resistance exercise elicits additional beneficial programs such as improved insulin sensitivity, neuromuscular function, and muscle vascularization, which further improve whole body health [13]. In this chapter, the molecular mechanisms that underlie resistance exercise-regulated postnatal skeletal muscle hypertrophy are reviewed.

RESISTANCE EXERCISE AND PROTEIN SYNTHESIS Among the different signal pathways modulated by resistance exercise, the activation of the multiprotein complex centered on the serine/threonine kinase mammalian target of rapamycin (mTOR) [14] plays a central and dominant role in the control of protein synthesis and adaptive growth in skeletal muscle and other cell types. This protein kinase was initially discovered as the target of the immunosuppressive drug rapamycin, which revealed the key function of mTOR in cellular proliferation and growth in yeast and mammals [15
17]. Of the two different mTOR complexes, complex 1 (mTORC1), comprising the regulatory protein associated with mTOR (raptor), the mammalian lethal with Sec13 protein 8 (mLST8), the Dishevelled, Egl-10 and Pleckstrin domain (DEP) domaincontaining mTOR-interacting protein, and the proline-rich Akt substrate of 40 kDa (PRAS40), integrates environmental cues and controls the molecular mechanisms regulating protein synthesis, cell growth, and metabolism [18]. In contrast to these well-documented

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effects of mTORC1 in skeletal muscle, for example, illustrated by skeletal muscle-specific knockout of raptor or mTOR that results in severe muscle dystrophy and impaired energy metabolism in mice, genetic inactivation of mTORC2 has a minimal impact on these aspects of muscle function [19,20]. Hence, both developmental and adaptive growthrelated signaling events most likely converge on mTORC1, which therefore represents a central hub in the regulation of skeletal muscle hypertrophy. The heterotrimer formed by the tuberous sclerosis complex 1 (TSC1), TSC2, and TBC1 domain family member 7, of which TSC2 harbors a GTPase activating protein domain, is an important upstream modulator of mTORC1 activity [18]. Activation of the TSC complex enhances the activity of the small GTPase protein ras homolog enriched in brain (Rheb), promotes GTP hydrolysis, and, thus, reduces the ability of Rheb to activate mTORC1 [18]. In skeletal muscle, the signal pathways regulated by the anabolic hormone insulin-like growth factor-1 (IGF-1) is associated with the activation of Akt (also known as protein kinase B) that subsequently phosphorylates and inhibits TSC2, resulting in the elevation of mTORC1 activity by GTP-bound Rheb [21,22]. The function of this small GTPase in skeletal muscle has recently been explored by Jacobs et al., who described that genetic deletion of Rheb in mouse skeletal muscle was sufficient to significantly blunt the positive effects of high-resistance mechanical load (in the form of eccentric contractions) on mTORC1 signaling [23]. Besides its effect on the TSC complex, Akt can activate mTORC1 by inhibiting PRAS40 and, consequently, abrogate the repressive effect of PRAS40 on mTOR [24]. However, despite the important role in developmental growth, the function of this signal pathway (insulin/IGF1) appears not to be essential for the adaptive growth of skeletal muscle induced by resistance exercise [25
27]. In general, mTORC1 activity corresponds to the cellular energy status, in which catabolic conditions promote mTORC1 inhibition to reduce costly anabolic processes and increase ATP synthesis [28]. The switch between anabolic and catabolic processes, and the corresponding inhibition of mTORC1 activity, is mainly regulated by the adenosine monophosphate (AMP)-activated protein kinase (AMPK) [28], which reduces mTORC1-dependent anabolism by enhancing TSC2 activity [29] and phosphorylation of the mTOR-interacting protein raptor [30]. Interestingly, AMPK has been proposed to mediate some aspects of the so-called training interference [31]. In this context, activation of mTORC1 by resistance exercise is blunted by subsequent endurance training through the activation of AMPK, which is reflected in lower functional adaptations to resistance exercise (e.g., muscle strength) [31]. Somewhat counterintuitive to this concept, the catalytic subunit α1 of AMPK is activated by chronic mechanical overload of mouse skeletal muscle in the synergist ablation model [32]. Notably, however, this specific experimental model is associated with a robust dysregulation of muscle energy metabolism [33]. Consistent with the antianabolic role of AMPK, genetic deletion of AMPK α1 enhances the hypertrophic response to chronic mechanical overload [34]. In contrast, neither knockout nor overexpression of the regulatory subunit γ3 of AMPK affects adaptive growth in mouse skeletal muscle [35]. Hence, additional work is required to further elucidate the possible function of AMPK in resistance exercise-mediated mTORC1 modulation and skeletal muscle hypertrophy. Besides the TSC heterotrimeric complex, changes in the amino acids levels in blood and tissues have emerged as a key stimulus regulating mTORC1 activity [18]. Amino acids promote the activation of Rag GTPase proteins, recruiting mTORC1 to the lysosome and,

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thus, facilitating its activation by Rheb [36
38]. Interestingly, resistance exercise increases postexercise amino acid transport in human skeletal muscle [39], while high-resistance electrical stimulation of rat skeletal muscle enhances the intracellular levels of the amino acid leucine [40]. These findings suggest a link between amino acid-mediated mTORC1 activation and skeletal muscle adaptive growth, which is further supported by the fact that consumption of essential amino acids enhances protein synthesis in human skeletal muscle after resistance exercise [41]. This synergistic effect of amino acids on resistance exercise-mediated skeletal muscle protein synthesis appears to be effective up to 24 hours of the postexercise period when skeletal muscle is more nutrient-sensitive [42]. Although the mTORC1 signaling pathway has been extensively characterized [18], the molecular mechanism by which mechanical overload of skeletal muscle enhances mTOR kinase activity remains elusive. Interestingly, Jacobs et al. have found that acute highintensity eccentric contractions of mouse skeletal muscle induces the translocation of mTOR from the cytosol to the lysosome, while TSC2 translocates in the opposite direction [43]. This effect on cellular localization was accompanied by a significant increase in TSC2 phosphorylation levels [23,43]. Moreover, under the same experimental conditions, skeletal muscle overload also induced an increase in raptor phosphorylation in a rapamycininsensitive way, which appears to be a key step in muscle contraction-mediated mTORC1 activation [44]. These data suggest that skeletal muscle contractions lower the inhibition of mTORC1 by TCS2 through changes in cellular localization and thereby enhanced activation of mTORC1 at the lysosome by amino acids (Fig. 11.1). Furthermore, another mechanism mediating mTORC1 activation and adaptive growth comprises the upregulation of G protein-coupled receptor 56 (GPR56) and the corresponding receptor ligand collagen III in response to mechanical overload of mouse and human skeletal muscle [45] (Fig. 11.1). While GPR56 and collagen III exert a synergistic effect on mTORC1 activation in muscle cells, GPR56 knockout is sufficient to blunt mouse skeletal muscle hypertrophy after chronic overload [45]. However, the mechanisms by which GPR56 regulates mTORC1 activity and whether this receptor is involved in resistance exercise-induced mTORC1 translocation to the lysosome remain to be investigated. Upon activation, mTORC1 can modulate a number of targets and biological processes such as lipid metabolism, autophagy, and protein synthesis, in which phosphorylation of the ribosomal protein S6 kinase B1 (S6K1) and the eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4EBP1) represents a central mechanism in the regulation of the rate of protein synthesis [18]. These mTORC1-dependent phosphorylation events result in an activation of S6K1 and an inhibition of 4EBP1 activity, which exert positive and negative effects on mRNA translation, respectively [18]. The requirement of the mTORC1
S6K axis for modulating skeletal muscle adaptive growth has been deduced from pharmacological (rapamycin) and genetic approaches, where disruption of mTORC1 activity or expression strongly blunts the effects of chronic mechanical overload on rodent skeletal muscle hypertrophy [21,46]. In this context of adaptive growth, inhibition of mTORC1 by rapamycin prevents the dissociation of the 4EBP1
eIF4E protein complex and the subsequent interaction of eIF4E with eIF4G induced by skeletal muscle overload [21]. Hence, mTORC1 can sense a number of environmental cues (e.g., energy metabolism and mechanical stress) and, consequently, promote cellular growth via an increase in the rate of protein synthesis mainly through the regulation of S6K1 and 4EBP1 (Fig. 11.2).

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Muscle unload

Muscle overload Collagen III Collagen III Collagen III

GPR56 GPR56

Cytosol

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TBC1D7

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TSC1

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TSC2

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?

mTOR Raptor mLST8

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TSC2

Lysosome

TBC1D7 TSC1

TSC2

mTOR mLST8 Raptor

mTOR Raptor mLST8

mTOR Raptor mLST8

Lysosome Rheb

FIGURE 11.1 Regulation of mTORC1 activity by resistance exercise. Under basal conditions (left panel), skeletal muscle experiences low levels of mechanical load, a state in which mTORC1 is mainly localized in the cytosol where it is inhibited by the TSC heterotrimeric complex. Moreover, skeletal muscle unload promotes the translocation of the TSC complex from the cytosol to the lysosome, which further inhibits mTORC1 activity. Conversely, increased skeletal muscle mechanical load (right panel) by resistance exercise induces the translocation of mTORC1 from the cytosol to the lysosome, while the TSC complex moves in the opposite direction. mTORC1 is activated at the lysosome by Rheb, where AAs exert a synergistic effect. Resistance exercise upregulates GPR56 and collagen III expression, which is an essential process in mTORC1 activation and skeletal muscle hypertrophy. Moreover, skeletal muscle overload enhances TSC2 and Raptor phosphorylation (yellow circles) events, but the precise molecular mechanisms that mediate resistance exercise adaptations and AA-mediated mTORC1 activation have not been fully elucidated. (mTORC1, mammalian target of rapamycin; TSC, tuberous sclerosis complex; Rheb, ras homolog enriched in brain; AA, amino acids; GPR56, G protein-coupled receptor 56.)

RESISTANCE EXERCISE AND RIBOSOMAL BIOGENESIS In addition to rise in the rate of protein synthesis, resistance exercise also promotes skeletal muscle hypertrophy by inducing ribosomal biogenesis and thus exerts a positive effect on the molecular machinery controlling mRNA translation. Ribosomes are macromolecular complexes comprising several ribosomal RNAs (rRNAs) and about 80 proteins, with the large 60S and small 40S subunits representing central components in humans. The

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Muscle overload

? TBC1D7 TSC1

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?

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40S mTORC1 TF

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3⬘ 40S

60S 40S

FIGURE 11.2 Resistance exercise-mediated adaptive growth of skeletal muscle. Upon activation by skeletal muscle overload, mTORC1 phosphorylates S6K1 and 4EBP1 (yellow circles) that consequently increase the rate of protein synthesis by the ribosome. Additionally, resistance exercise enhances the expression and activity of TF (e.g., Pol I) mediating the expression of rRNAs and, thus, inducing ribosomal biogenesis. Hence, resistance exercise has been proposed (dotted lines) to induced skeletal muscle hypertrophy via ribosomal biogenesis. However, it remains to be demonstrated if ribosomal biogenesis is mTORC1-dependent and whether this is an essential process in adaptive growth of skeletal muscle. (mTORC1, mammalian target of rapamycin; S6K1, S6 kinase B1; 4EBP1, 4E binding protein 1; TF, transcription factors; rRNAs, ribosomal RNAs; Pol I, polymerase I.)

biogenesis of this ribonucleoprotein complex is highly regulated at the transcriptional level to a large extent by the RNA polymerase I (Pol I), which specifically transcribes ribosomal DNA (rDNA) genes to pre-rRNAs that are further processed to mature rRNAs in nucleolus compartments within the nucleus. Moreover, Pol III regulates the transcription of 5S rRNA (a component of the 60S subunit), while a number of ribonucleoproteins are transcribed by Pol II within different nuclear compartments. Interestingly, besides its function in protein synthesis, the mTORC1
S6K1 axis has also been implicated in the transcriptional regulation of ribosomal biogenesis [47]. In fact, Pol I regulates gene transcription in a rapamycin-dependent manner [48], with mTOR being physically located at the promoter of a number of rDNA genes [49]. Furthermore, mTORC1 promotes the translation of mRNA molecules harboring a 50 -terminal oligopyrimidine tract (50 -TOP) motif, which is characteristic of all mammalian ribosomal proteins and several proteins involved in

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protein synthesis [47]. The regulation of 50 -TOP mRNAs requires the activation of the mTORC1
4EBP1 axis [50], whereas S6K1 activation seems to be dispensable in this context [47]. Hence, the positive regulation of ribosomal biogenesis represents an additional layer of complexity in the mechanisms by which the mTORC1 signaling pathway controls cellular growth (Fig. 11.2). Interestingly, acute and chronic resistance exercise induce a significant increase in the RNA content of both human and mouse skeletal muscle [25,51
54]. About 80% of total RNA represents rRNAs, thus suggesting a positive effect of resistance exercise on skeletal muscle ribosomal biogenesis. This effect of mechanical overload on skeletal muscle ribosomal biogenesis is an mTORC1-dependent mechanism [46,55] that, like skeletal muscle hypertrophy [56], appears to be attenuated by aging [57]. In fact, a link between anabolic resistance and ribosomal content has recently been investigated in humans by Brook et al., who found that old individuals (  69 years) show lower gains in protein synthesis, muscle function, and total RNA (mostly reflecting rRNA) compared to young subject (23 years) [58]. Importantly, the increase in total skeletal muscle RNA induced by mechanical overload is directly linked with the upregulation of 45S pre-rRNA, which is subsequently processed to generate the ribosome subunits 5.8, 18, and 28S [59]. The importance of transcriptional regulation in resistance exercise-induced ribosomal biogenesis was demonstrated in synergistic ablation and high-resistance electrical stimulation experiments, in which an increase of the expression of several Pol I-accessory genes (e.g., POL1RB and TTF1) and their upstream transcription factors (c-Myc and upstream-binding factor (UBF)) was found [59]. The recruitment of Pol I, c-Myc, and UBF to the rDNA promoters is significantly increased, an effect that is associated with higher levels of H3K9 acetylation (a marker of active transcription), while Pol I protein levels remain unchanged in these experimental paradigms [59]. Interestingly, West et al. have recently found that some aspects of resistance exercise-mediated ribosomal biogenesis, including c-Myc and TAF1B expression, are rapamycin-insensitive [55], implying that postexercise maintenance of ribosomal content might be regulated in an mTORC1-independent manner.

RESISTANCE EXERCISE AND SATELLITE CELLS Satellite cells are lineage-committed, adult muscle stem cells localized between the muscle sarcolemma and the basal lamina [60]. Under normal conditions, satellite cells remain in a quiescent state, whereas upon stimulation (e.g., muscle injury), these cells start to proliferate and, eventually, differentiate into myoblasts to form new muscle fibers (hyperplasia) or to fuse with existing muscle fibers (myonuclear accretion) [60]. Due to the asymmetry of the proliferation process, a subset of satellite cells remain in the inactivated state and thereby contribute to a self-renewal process that is essential to maintain a proper stem cell pool [60]. Besides skeletal muscle injury or damage, physical exercise represents a potent stimulus to promote satellite cell activation [61]. In human skeletal muscle, both acute and chronic resistance exercise induce an increase in satellite cell number and myonuclear content, closely correlating with skeletal muscle hypertrophy [61]. However, since such studies are mostly descriptive and correlative, a direct assessment of the function of satellite cells in resistance exercise-mediated skeletal hypertrophy in humans remains

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elusive. In mice, satellite cell depletion via a genetic knockout of Pax7 results in a lower number of muscle fibers, exacerbated atrophy, and impaired regeneration indicating the importance of an adequate pool of satellite cells for postnatal skeletal muscle growth [62]. However, it is unclear whether satellite cells play an equally important role in the modulation of postnatal adaptive growth of skeletal muscle. For instance, McCarthy et al. found that genetic deletion of Pax7-positive cells specifically in mature skeletal muscle was sufficient to ameliorate the effects of 2 and 6 weeks of synergist ablation on skeletal muscle hyperplasia and regeneration, but the hypertrophic response and the activation of mTORC1 were not impaired [63]. In line with these findings, in an unloading
reloading model of hindlimb suspension, skeletal muscle regrowth following unloading-induced atrophy is unaffected by satellite cell depletion in adult mice [64]. In contrast, Egner et al. found that synergist ablation-mediated skeletal muscle hypertrophy after 2 weeks is significantly attenuated by satellite cell depletion [65]. Different from the approach used by McCarthy et al., Egner et al. excluded regenerating fibers from the analysis and reported absolute changes in muscle mass, leading to the idea that satellite cell activation and subsequent fusion with muscle fibers is an essential process in skeletal muscle adaptive growth [65]. Satellite cell depletion has also been found to blunt skeletal muscle hypertrophy following 8 weeks of synergist ablation, but not at earlier time points in mice, suggesting that the relevance of these cells in adaptive growth is time-dependent [66]. Skeletal muscle hypertrophy during aging likewise depends on satellite cells, as reflected by the lower magnitude of hypertrophy and satellite cell density following 6 weeks of mechanical overload in aged (23.5 months old) compared to adult (7.5 months old) mice [67]. In summary, even though satellite cells seem to be required for skeletal muscle hypertrophy under some experimental conditions and in specific models, it remains to be defined whether these stem cells regulate resistance exercise-mediated skeletal muscle remodeling in humans.

CONCLUSIONS AND OUTLOOK Resistance exercise has emerged as a major physiological stimulus to enhance skeletal muscle mass and to improve both the contractile and metabolic properties of this tissue. Accordingly, resistance exercise in combination with adequate protein intake counteracts at least some of the detrimental effects of skeletal muscle disuse and sarcopenia, which consequently improves whole body health and the quality of life. At the molecular level, mTORC1 activation is an essential step in resistance exercise-mediated skeletal muscle hypertrophy. However, the upstream signal pathways controlling mTORC1 activation and cellular localization are poorly understood. A number of additional potential mechanisms by which resistance exercise regulates mTORC1 activity besides those summarized in this review have been proposed, including activation of focal adhesion kinase [68], diacylglycerol kinases ζ [69], vacuolar protein sorting mutant 34 [40], and extracellular signalregulated kinase [25] among others. Nonetheless, the requirement of such pathways for the adaptive response of skeletal muscle to resistance exercise remains to be experimentally determined. Although additional work is also required to clarify the relevance of ribosomal biogenesis and satellite cells in resistance exercise-mediated skeletal muscle

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hypertrophy, mTORC1-dependent and -independent activation of Pol I represents an attractive mechanism regulating such adaptive responses. The dissection of the molecular mechanisms controlling adaptive growth of skeletal muscle would allow the discovery of new molecules and/or biological processes with therapeutic potential for the treatment of skeletal muscle wasting-associated diseases. However, until new actionable knowledge has been gained, skeletal muscle overload remains the main strategy to enhance mTORC1 activity, which subsequently integrates the mechanical stimulus with nutritional and hormonal signals to further promote postnatal skeletal muscle growth.

KEY POINTS • Resistance exercise is the main physiological stimulus promoting postnatal skeletal muscle growth. • Amino acid supplementation is an efficient strategy to enhance the positive effects of resistance exercise on protein synthesis. • The activation of mTORC1 is a central process mediating adaptive growth of skeletal muscle. • Resistance exercise stimulates the translocation of mTORC1 to the lysosome to be activated by Rheb. • Resistance exercise boosts skeletal muscle hypertrophy by stimulating protein synthesis and ribosomal biogenesis, in part via mTORC1-dependent mechanisms. • Satellite cells depletion blunts the effects of chronic mechanical overload on skeletal muscle hypertrophy under some experimental conditions.

Acknowledgments Work in our laboratory is supported by the Swiss National Science Foundation, the European Research Council (ERC) Consolidator grant 616830-MUSCLE_NET, Swiss Cancer Research grant KFS-3733-08-2015, the Swiss Society for Research on Muscle Diseases (SSEM), SystemsX.ch, the Biozentrum and the University of Basel. JPS was supported by a Sir Henry Wellcome Postdoctoral Fellowship of the Wellcome Trust. Conflict of Interest The authors declare no conflict of interest related to this manuscript.

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Training, Changes in Nutritional Requirements and Dietary Support of Physical Exercise Ire`ne Margaritis Human Nutrition Risk Assessment French Agency for Food, Environmental and Occupational Health Safety (ANSES), Maisons-Alfort, France

INTRODUCTION INTO THE ROLE OF NUTRITION ON ADAPTIVE EFFECTS OF EXERCISE Scientific evidence is to date robust enough to consider the effects of physical activity in preventing the occurrence of more than 35 chronic noncommunicable diseases [1,2]. Considering either leisure or athletic sport, the benefits of physical activity (PA) arise from adaptive hormonal, metabolic, cellular, and molecular responses that rely on metabolic adaptations modulated by various behaviors or exposures (sleep, physical stress, mental stress, diet, etc.) among which diet plays a pivotal role. Physical activity is conventionally defined as any bodily movement produced by skeletal muscles, which results in an increase in energy expenditure above resting energy expenditure [3]. This can occur during leisure, work, sport, active transport (including walking, cycling, etc.), recreational sports, programed physical exercises [4], or military operations. Exercise is physical activity that is planned, structured, repetitive, and purposive in the sense that improvement or maintenance of one or more components of physical fitness is an objective [3]. Muscle contraction leads to changes in the metabolic demand, physiological stress, and nutritional needs, at acute or chronic (repeated) exercise. The physiological stress varies according to the duration, intensity, environment, and type of the set of PAs performed by both recreational and competitive athletes who are seeking for adaptation to improve or maintain performance. Physical performance can widely be considered without being restricted to sport and competition. Actually, sole high energy expenditure or strenuous muscle solicitation is

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likely to modify the requirements. Performance optimization presents adaptive and nutritional issues. So the most often nutrition for exercise is considered to be nutrition for sport. Focusing on performance, it could be forgotten in that performance is primarily based on healthiness to be achieved by synergy between training loads and nutrition. Whatever the context and whatever the level of performance targeted, the challenge is to satisfy the needs for both health maintenance—including “muscle health”—and maximize traininginduced adaptive effects. Both optimal training and nutrition are corequisites for peak performance achievement and overtraining eschewal. Regarding sport nutrition, the main issues are related to acute exercise-induced response and chronic training-induced adaptation optimization. Here will be given an overview on the main issues related to nutrition and physical activity combination, after several summarized reminders of training and adaptive theory to be linked with. The consistency of nutritional strategies seeking peak performance with averting risky behaviors will be exposed and discussed. More than an in-depth coverage of the topic, the issue of nutrition and physical exercise is addressed regarding the close relationship linking nutrition and short and long-term exercise-induced physiological adaptive effects. It will be focused on current trends in sport nutrition and several issues that remain controversial despite being well documented.

ACUTE AND CHRONIC EXERCISE: STRESS AND ADAPTATION Targeting the movement achievement by muscle effectiveness, based on an integrative approach including physiological functions, the double challenge of sport nutrition is to cater for nutritional needs, allow the most effective muscle function—as effectors—and promote plasticity to maximize the targeted training adaptations. Facing the increase in nutritional requirements underpinned by the need to adapt to stress and avoid inadequate intakes supposes the nutrient coverage to be ensured by adequate intakes (AIs) of energy, vitamins, and minerals.

Training: The Hormesis Theory Training principle is to generate physiological adaptations and thus improve the performance level. This can be reached by placing the athlete in an unusually physiological state of stress. In practice, exercise training involves the repeated exposure to an acute increase in metabolic, thermoregulatory, hypoxic, oxidative, and mechanical stress. This exposure to stressors is provoked by repeated bouts of exercise (training load) to stimulate compensatory physiological adaptations that improve tolerance to subsequent similar stressors. As physiological adaptations take place during recovery, intakes play a major role in this key interval period between two bouts. Training loads are increased over a clearly identified period: This is the overload principle targeting chronic adaptation.

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Training to be Based on Adaptive Theory Hans Selye’s theory of the general adaptation syndrome developed in 1936 [5] refers to three successive phases of stress which provoke (or fail to) adaptations, whatever the nature of the stressor. These phases include (1) an alarm reaction: preparation time, mobilization of resources to deal with stress (below the normal resistance level, then passing over); (2) resistance: use of resources (above level of normal resistance), and then (3) exhaustion: appearance of somatic disorders (phase of decline of the level of resistance of the previous phase to the normal level). In the context of training, a stress—the training bout—creates initial homeostatic perturbation and induces impairments (distress) that mobilize central and peripheral molecular regulators of stress response; these preconditions increase resistance to subsequent acute exposure to large doses of the same stressor (eustress). In training theory, the stressor is the load and eustress is the supercompensation which determines the readiness for forthcoming workloads. This theory perfectly illuminates the principle of the biphasic response to increasing amounts of a substance exposure which defines hormesis. In well-conducted training, overcompensation translates in improved performance. Today, the hormesis theory emerges to describe the training process. In muscle, evidence suggests that numerous signals are emitted from the mitochondria during exercise and have the potential to induce a nuclear transcription response [6]. In this process, mitochondrial adaptations are decisive. This is considered to date as the concept of mithormesis [7] with reactive oxygen species (ROS) being the primary candidate. Mithormesis Theory and the Key Role of ROS Aerobic organisms are continually exposed to ROS production. Physical exercise is well known since 1982 for being a generator of ROS [8] as a result of increases in mitochondrial oxygen consumption and electron transport flux in skeletal muscle. The increased flow rate of mitochondrial oxidative phosphorylations is related to intensity and duration of exercise and causes a proportional increase in ROS flow and their leakage to cytosol [8] which is better tolerated by trained subject [9]. The main sources of mitochondrial ROS production are related to the significant reduction of enzymatic complexes of the respiratory chain [10,11]. During the last decades, ROS had been mostly considered as playing a major role as mediators of skeletal muscle damage and inflammation after strenuous exercise. In the late 1990s, the discovery of specific genes and pathways affected by oxidants led to hypothesize that ROS serve as subcellular messengers in gene regulatory and signal transduction—first reviewed by Allen and Tresini in 2000 [12]. Many redox-sensitive enzymes are known to be involved in this process. Signaling molecules like Mitogen-activated protein (MAP) kinases, NFκB, p53, heat shock proteins [13], and others modulate muscle adaptation to exercise [14,15]. Proliferator-activated receptor gamma coactivator 1 alpha plays a role in the control of fat oxidation and angiogenesis [6] and is rapidly activated by endurance exercise. Goulev et al. [16] recently proposed a framework that explains the emergence of acquisition of long-standing phenomenon conserved across species from unicellular to mammals—tolerance—in the context of the H2O2 response discovering a mechanism for a long-known phenomenon for which a quantitative mechanistic

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understanding was missing. It is now widely recognized that oxidative compounds including hydrogen peroxide, even endogenous and exogenous antioxidants, by modulating gene expression and signaling pathways, play pivotal role in adaptive responses. The issue of the dose
response is crucial to articulate exercise-induced stress and nutritional intakes. Adaptation to Environmental Stress: An Additional Challenge More or less pronounced forms of the three-stage reaction represent the usual response of the organism to stimuli such as temperature changes, drugs, muscular exercise, etc., to which habituation or inurement can occur [5]. An additional stress to extreme ranges of environmental conditions during physical activity could hamper the exercise-induced adaptive effects by overloading. The most frequent are changes in PpO2, temperature, and in a lesser extent—regarding prevalence of exposition—pressure (diving), radiations, or microgravity, the latter being very specific and mostly referring to the effects of hypokinesia. Whatever the stress factor, acute and hormetic response to these exposures requires special metabolic mobilization and adapted nutritional intakes to cope with these stresses. In well-trained subjects, short bouts of intense exercise in the heat or the cold seem not to produce significant oxidative stress [17]. Regarding cold, energy supply is of utmost importance to avoid hypothermia; hydration can be too, particularly in altitude with the decrease in water tension and the risk of increased blood viscosity and frostbites. In hot environments, thermoregulation requires physiological responses to allow thermolysis which strongly depend on blood volume and fluidity. Dehydration is well-known to transitory decrease maximal oxygen uptake. Speed running is limited by the degree of hyperthermia for which hydration is a preventive factor before muscle glycogen availability becomes a significant contributor to the onset of fatigue [18]. However, ingested fluids has to contain the right minerals avoiding overdrinking as hyponatremia has occasionally been reported [19,20]. Whatever the conditions, nutrition has to enable athletes to cope with environmental stress while allowing hormetic effects of training. Hypoxia is an interesting model of environmental stressor and is moreover used as a training strategy. After 40 years of altitude training, several strategies of such training regimens have been proposed as “live high, train high”, “live high, train low”, or “intermittent hypoxic training” (IHT). Each of them combines the effect of acclimatization and different training protocols, which requires specific nutrition [20]. Nutrition in altitude refers to both situations of altitude training: under chronic oxygen deprivation frequently used to improve physical performance even at sea level and of long-term exposure (e.g., mountaineering). The decreased oxygen supply implies to deal with the increased need of blood oxygen transporter and cell uptake. The most important aspects of nutrition strategy for altitude training include proper hydration and optimal energy balance. A significant and classical element of altitude nutrition could relate to the monitoring of iron, [20] as frequently cited. In a larger extent, precise dietary recommendations are known to be difficult because of the heterogeneous modalities of exposure and of the great range of altitude of exposure which could vary from 2000 to more than 8000 m. Training adaptations can attenuate altitude/hypoxia-induced oxidative stress during long-term hypoxic exposure [21] by improving the defense capabilities, likely by helping

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the adaptive increase in the activities of the scavenger enzyme systems. However, homeostatic systems are intrinsically limited by the transcriptional response time, which may restrict a cell’s ability to adapt to unanticipated environmental challenges [16]. As exercise-induced oxidative stress is worsened by altitude exposure, it can be supposed that the additional hypoxic stress delays adaptive response in hypoxic conditions. So, increase in ROS overwhelming antioxidant defenses may lead to increased DNA damage, in the form of damaged bases or as single-strand or double-strand breaks, membrane damage, in the form of a variety of phospholipid oxidation products, damage to proteins and other compounds as well as cell apoptosis, immunosuppression, fatigued states, and underperformance. However, IHT at a moderate altitude does neither hamper the antioxidant defense system in competitive swimmers nor induce oxidative stress [22]. This confirms the role of time left to the physiological response: the recovery time which allows the adaptive effects to occur. Additionally, the targeted endogenous response depends partly from exogenous supplies. Indeed, decrease food intake has to be monitored, due to suppressed hunger and appetite (mostly up to 5000 m) that can lead to a negative energy balance [20] and micronutrient—including antioxidants—deficiencies. As the antioxidant systems are critical to provide a protective role against ROS produced during exercise, the balance between ROS generation and antioxidant defense is a critical point to determine the development of ROS-mediated tissue damage and probably ROS-induced mithormesis. To control the balance, both training and nutrition periods have to be considered and articulated regarding the overtime variations of training loads with periodization.

Training Periodization as a Necessity Specificity of Training Cycles, Principle of Supercompensation Based on the hormesis theory, in most sports the dose
response relationship between training and performance is widely accepted. “Modern sports” began to be considered in the 1980s [23] and have further been developed. On this basis, the concept has evolved mostly referring to the comprehensive, science-based theory of training periodization. The most recent work of Platonov [24] based on the development of the initial concept (P) adds significant knowledge to training periodization theory [25]. The core principle is that training load results in a degree of fatigue or depletion that is followed—provided that the recovery is adapted—by supercompensation (Fig. 12.1) which results from the physiological adaptive effect to the prior stress. Athletes in endurance sports generally require high training loads to develop the background necessary for success in high-level competition, during many years for elite athletes [26]. For more intense activities, as Olympic rowing, swimming, kayak, track running, track cycling events, the majority of training is generally aimed at increasing aerobic metabolic capacity [26]. In well-trained athlete, high-intensity interval training added to high training volume increases ability to oxidize fat [27]. A polarized approach to training, whereby 75% of total training volume is performed at low intensities, and 10%
15% is performed at very high intensities, has been suggested as an optimal training intensity distribution for elite athletes who perform intense exercise [26]. The metabolic adaptations

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Adaptation to proper training/recovery vs overreaching/overtraining with inadequate recovery Overreaching/overtraining

Proper training/recovery

6

Performance enhancement

Adaptation and performance index

5 4 3 2 1 0 –1 –2

Normal acute fatigue

Overtraining syndrome:

Functional overreaching (temporary performance decrements)

Nonfunctional overreaching

–3

(weeks/months of performance decrements)

Long-term downturn in performance; often with increased rates of illness, psychological distress

–4 Training and recovery cycles (periodization)

FIGURE 12.1 The path toward overreaching and the overtraining syndrome compared to performance enhancement. From Nieman DC and Mitmesser SC. Potential impact of nutrition on immune system recovery from heavy exertion: a metabolomics perspective. Nutrients 2017;9:513 with permission.

that occur with high-volume training and high-intensity training show overlap albeit regarding the molecular events that signal for these adaptations may differ [28,26]. Overreaching and Overtraining According to the general adaptation syndrome, stress causes a temporary decrease in function followed by an adaptation that improves function. In the training response, overload is the stress that causes fatigue (temporary decrease in exercise ability). Following the recovery from fatigue, improved performance is the more objective sign of adaptive response according to the overcompensation principle. Progressive overload is the foundation of all successful training. Since increase in either training volume or intensity are sudden or the overload poorly adapted to the athlete’s ability to recover, the mechanisms of adaptation are overwhelmed. The level of performance is durably reduced (exhaustion phase), as extensively described [29]. Without corrective measure, a maladaptation potentially results in overtraining syndrome (OTS), functional overreaching (FOR), or nonfunctional overreaching (NFOR). There is a continuum from NFOR to FOR which is training stress resulting in a short-term to long-term decrement in performance capacity. Overtraining is diagnosed in athletes by decreased performance and fatigue, with either physiological or psychological signs or symptoms [30,31] and triggered by metabolic,

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immune, hormonal, and other dysfunctions, sometimes without abnormalities in baseline biochemical markers [32], to allow an early detection. This includes additional pressure on several physiological functions as immune function [33,29] which can be exacerbated by deficiencies or even excesses of various dietary components [32]. So, a strong reason for periodization is targeting the avoidance of overtraining due to inappropriate training loads, recovery, or nutrition. This can be observed in many sports soliciting combined training even if the specificity of adaptive effects is still insufficiently documented [28] and still discussed. Platonov describes and analyses the following mesocycles: introductory, basic, regenerative-preparatory, regenerative-maintaining. Each of them being specifically oriented [25]. In this context, nutrition has to take a significant place. This implies to adapt macro- and micronutrient intakes to individual requirements not only to maintain robust immunity in athletes but even to cater for requirements. Maintaining physiological functions to allow adaptive effects is crucial to ensure the intended hermetic effects do not fail.

ENERGETIC MACRONUTRIENTS Requirements The energy expenditure—and demand—depends mainly on basic metabolism, thermogenesis, and expenditure related to physical activities [34]. Depending on these factors, energetic macronutrients requirements will be increased whatever the type of activity, and performance level. As the metabolic pathways of the three energetic macronutrients interact, the proportion of each in supplying the energetic requirements is an issue in the general population and its adaptation and a matter of concern for athletes. The more often coaches prescribe periods of prolonged submaximal exercise, moderate periods of training at “threshold,” or shorter high-intensity exercise sessions [35]. Regarding both training and events, the first and most immediate issue to maintain performance is the energy availability. Balanced energy availability is crucial for achieving peak performance and avoiding an OTS. As energy is provided by several macronutrients, the proportion of each is closely dependent on anabolic and more metabolic need. This issue is more and varies regarding acute and chronic exercise. The Principle of Setting Intervals The bases for defining the nutritional requirements are distinct from those underpinning the nutritional strategies oriented toward a nutritional stress to optimize training effects. In the first case, reference values are fixed for general population to satisfy requirements and prevent noncommunicable disease as recently resumed for the French population [36]. In the second, it is the adaptation that is sought by several strategies that are proposed to optimize the training induce adaptions, sometimes by an additional nutritional stress to the internal load. For macronutrient, a reference interval (RI) is set instead of a unique value—differently from vitamins and minerals. It corresponds to the intake interval considered to be healthy for the population. This reference value is specific to energy macronutrients and expressed as a percentage of total energy intakes (TEI). In the European Community, countries

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mostly refer to the European Food and Safety Authority (EFSA) evaluations for the general population. The determination of the distribution values of macronutrient intakes aims at covering the energy requirement by the supply of macronutrients simultaneously to the satisfaction of the needs of nutrients and among the essential ones (proteins, essential amino acids, and essential fatty acids). The RIs for each of the three macronutrients are framed by a low limit below which the intake of a macronutrient is considered insufficient and a high limit beyond which this macronutrient is judged in excess—without being in itself an upper limit (UL). Nutritional reference values are based on the links between each macronutrient and the physiological responses, as well as on the pathophysiological consequences associated with imbalances in macronutrient intake. These requirements are translated in quantity and then expressed as a percentage of the TEI, excepted for proteins which have the particularity of being expressed in absolute value and relative to body weight to meet the needs related to protein turnover. These values are defined for the general population by international (e.g., WHO, EFSA for Europe) or national (e.g., ANSES in France) organizations. The declination of these references requires taking into consideration the specific issues that are introduced at least by both metabolic and anabolic demand. Nutritional requirements are generally and first defined for the general population and for a given energy expenditure averaging 1.6 METs (1 MET51 Metabolic Equivalent of Task) [36]. ANSES has based the RI on the simultaneous consideration of several macronutrients, as previously done by Institute of Medicine [37]. The ULs are determined on the basis of risks of metabolic disorders. The increase energy expenditure due to muscular solicitation requires both quantitative and qualitative changes. Intervals: From General to Exercising Population Energy demand increases in proportion to exercise intensity and energy supply is a critical factor for performance. Physical activity can be categorized according to its intensity, its duration, the type of muscular contraction, and thus the metabolic pathway mainly involved in energy production, in the presence or absence of oxygen: anaerobic or aerobic. In addition to the role of proteins for plasticity, carbohydrates and lipids are crucial in fulfilling the energetic demand; they also contribute to several proper functioning of tissues and organs. Even if amino acids can be converted to glucose or to various intermediates of oxidative metabolism, they generally contribute little to energy production. As protein is continually degraded and synthesized, a constant dietary supply of amino acids is necessary to offset these losses. What differentiates the fixation of the protein requirement is its calculation and initial expression in absolute value and not as a percentage of TEI. As well as for carbohydrates and lipids, the qualitative aspects are to be considered. In the general population, protein intake must be a minimum of 10% TEI for the majority of the general population with, however, 12% of TEI for women over the age of 50 and men over the age of 60 [36]. For proteins, the levels of intakes and the diversity of sources allows the coverage of essential amino acid requirements in the general population. The question of quality only arises in cases where intakes are close to the lower values. In this

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situation, the protein sources have to be chosen to allow a balanced intake of amino acids in the diet. The maximum protein intake is difficult to determine due to a strong apparent tolerance to high protein intakes and the absence of any compelling evidence concerning a metabolic risk 20% of TEI can be reached with no risk [36]. However, very high intakes in manipulated regimens could be at risk of renal failure [38]. In sports subject, investigations on the effects of protein supplementation on endurance performance are fewer than on resistance exercises. It is appropriate for quite high energy expenditure to consider the strict context of covering the needs related to maintaining the muscular mass, in athletes. A daily protein intake of 1.2
1.3 g/kg body weight [39,40] helps to maintain muscle mass. This value is higher than for general population because of the use of some amino acids for energy production, and muscle protein turn-over increased by muscle damage [41]. In resistance exercise, the maintenance or development of muscle mass is taken into account to define the requirements. According to the International Society of Sports Nutrition, daily protein intake in the range of 1.4
2.0 g protein/kg body weight/day (g/kg/day) is sufficient for most exercising individuals [42]. A high biological quality (casein, whey protein, soybean, etc.) in reasonable quantities (20
25 g/day) can play an important role in recovery processes [43,44,45], to 40 g/day as more recently proposed [42]. Indeed, the quality of the proteins has to be considered as extensively recently reviewed by Philips [46]. Amino acid requirements with physical exercise are developed in details in another section of the book. In general population, the threshold of carbohydrate intake below which the risk of metabolic disorders may be increased is set at 40% of TEI [36]. The maximum carbohydrate intake is 55% of TEI, the value above which the risks of insulin resistance, diabetes, cardiovascular disease, and certain cancers are increased for little active population. This value is very specific to nonsport population. Among the three macronutrients, carbohydrates (CHO) metabolism is probably the most altered by physical exercise. For sports, the state of muscle and liver reserves in glycogen is determining in maintaining health, decreasing fatigue, and improving performance [47]. Recommendations for carbohydrate intake range from 6 to 10 g/kg/day [39] depending on disciplines, level of energy expenditure, and environmental conditions. It should correspond to more than 60% of TEI. The distribution of CHO intake over time is crucial in terms of glycogenic repletion, muscular and glycogen savings during exercise. The glycemic index is chosen according to the objective and the distance to the exercise and will be developed in paragraph dedicated to nutritional strategies. Carbohydrates are important substrates for oxidative phosphorylation and energy production in skeletal muscle but fat are too. Their proportion in the energetic supply is strongly dependent on exercise intensity and training period. It is still a controversial subject because of the variability of their metabolic utilization, but mostly because of the believed interest of stressing organism by CHO deprivation seeking to promote the metabolic utilization of fats by β-oxidation stimulation which will be developed later. Fatty acids play a role in the constitution of cell membranes, intracellular signaling, also and especially as potential energy substrates. Fat intake must be a minimum of 35% of TEI, first, to ensure the intake of essential fatty acids and, second, in view of primary prevention of chronic noncommunicable diseases [36]. As for CHO, the quality of their food

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vectors has to ensure the coverage of the requirements for essential fatty acids 1 for linoleic acid set at 4% of TEI for adults arises from a concern to reach a total for polyunsaturated fatty acids (PUFAs) favorable to cardiovascular prevention and to limit intakes to ensure a linoleic acid/ALA ratio below 5, for docosahexaenoic acid 250 mg/day due to its very low rate of conversion from ALA (AI value), for eicosapentaenoic acid 250 mg/day, on the basis of data on prevention, in particular of CVDs (AI value).), vitamins, and minerals, on the one hand, and to guarantee the recommended intakes in monounsaturated fatty acids (MUFAs), PUFAs, and fiber, on the other. The maximum fat intake is 40% of TEI, the value above which the risk of an energy imbalance and its possible consequences is increased. For sports, 30% of TEI in lipids allows covering the essential fatty acid requirement comprising one-third saturated fatty acids, one-third MUFAs, and one-third of PUFAs. As a maximum, outside of any nutritional manipulation intended to stress the metabolism, lipid intakes do not have to exceed 35% of TEI [34]. This is because higher intakes of fat will mechanically reduce the proportion of carbohydrates in the TEI. The fraction of macronutrient intakes is of high dependence on individual and training periods and result from many factors including volume of exercise, age, body composition, TEI, and training status [42]. The relevance of strategies of nutritional stress to optimize lipid utilization as a main fuel and the compatibility with the needs for exogenous CHO availability and metabolism during exercise in both training bouts and competitions is still debated.

Nutrition Strategies: CHO vs Fat, the “Train-Low, Compete-High” Approach The interaction between carbohydrate and fatty acid oxidation is dependent on the intracellular and extracellular metabolic environments that are affected by substrate availability (both from inside and outside of the muscle), exercise intensity, and duration [48]. The evolving model is that athletes should follow an individualized approach, whereby carbohydrate intake is periodized throughout the training cycle according to the fuel needs of each workout, the importance of performing well in the session, and/or the potential to amplify the adaptive response to exercise via exposure to low carbohydrate availability [49]. Fat and carbohydrate are important fuels for aerobic exercise. The availability of exogenous carbohydrate before and during dynamic exercise increases carbohydrate oxidation and reciprocally decreases fat oxidation. Exercise intensity and metabolic demand or changes in CHO availability mobilize the reciprocal shifts in the proportions of carbohydrate and fat that are oxidized. In well-trained athlete, this is achieved by overloading: addition of highintensity interval training to the already high training increases carbohydrate [27]. Targeting this metabolic flexibility, the metabolic stress achieved by exercise-induced overload is maximized by nutritional strategies that use recent scientific data. The “train-low, compete-high” concept is training with low carbohydrate availability to promote adaptations such as enhanced activation of cell-signaling pathways, increased mitochondrial enzyme content and 1

Recommended values are as follows: for alpha-linolenic acid (ALA) was set at 1% of TEI for adult men and women, with the aim of preventing cardiovascular diseases (CVDs) (AI value).

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activity, enhanced lipid oxidation rates, and hence aims at improving exercise capacity. Aiming to increase exercise-induced signaling associated with improved oxidative capacity and glycogen saving during events, there is renewed popular interest in high-fat, low-CHO diets for endurance sports especially in ultraendurance athletes The following is specifically related to the scientific basis of nutritional strategies currently used in some disciplines and dedicated to increase the level of adaptation by adding a metabolic stress to optimize muscle substrates utilization. A key question is to what extent train low has an effect on training-induced adaptive response, and whether or not this effect translates in performance improvement. Train Low Carbohydrate ingestion before dynamic exercise has a potential effect in decreasing free fatty acid (FFA) concentrations and downregulating fat metabolism in skeletal muscle. The reduction in fat metabolism following glucose ingestion appears to be due to the combined effects of decreased adipose tissue FFA release and provision to muscle and to direct inhibitory effects on fatty acid oxidation in the muscle [48]. Ingested glucose increases insulin release which inhibits adipose tissue lipolysis and reduces the plasma (FFA) as elevation in plasma insulin before exercise suppress lipolysis during exercise to the point at which it limits fat oxidation [50]. Increased insulin may also inhibit FFA transport across the plasma and mitochondrial membranes and decrease intramuscular triacylglycerol (IMTG) breakdown in skeletal muscle. Carbohydrate oxidation, from plasma glucose and/or muscle glycogen, is increased [48]. As a consequence, decreasing the fat oxidation during exercise, CHO supply is considered to hinder the exercise-induced adaptive effect on promoting fat use. Training is well known to reduce the reliance on CHO as an energy source; thereby increasing fat oxidation during submaximal exercise. The regulation of fat metabolism and oxidation in skeletal muscle involves multiple regulatory sites, including FFA transport across the muscle membrane with protein carriers, binding and transport of FFAs in the cytoplasm, IMTG synthesis and degradation, FFA transport across the plasma, and mitochondrial membranes facilitated by proteins and potential regulation within the β-oxidation pathway [48]. The ability of proteins to translocate to the membranes during exercise and the new roles of adipose triglyceride lipase and hormone-sensitive lipase in regulating skeletal muscle lipolysis are examples of recent discoveries. At the opposite, the lack of availability of CHO (exogenous and endogenous) stresses the metabolism and upregulates skeletal capacity to oxidize fat—determined by the mitochondrial volume [48]. The exercise-induced increase in p53 phosphorylation and expression of genes associated with regulation of mitochondrial biogenesis and substrate utilization in human skeletal muscle is enhanced in conditions of reduced CHO availability [51]. One of the objectives pursued during these manipulations is to increase the level of oxidation of the fatty acids during exercise, according to its intensity, so as to spare the elective substrate, glucose, in quantity limited in the body. As CHO is the first fuel for the working muscles—what is particularly true as exercise intensity is increasing—availability of CHO has to be maximized: being synthesized and stocked (glycogen) and spared. However, fat-rich diets do not “spare” CHO (i.e., muscle

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glycogen) or improve training capacity/performance but, instead, directly impair rates of muscle glycogenolysis and energy flux. So, there is no clear evidence that enhanced metabolic adaptations translates to improvements in high-intensity exercise performance [52,53,54] albeit greater increases in citrate synthase activity and resting muscle glycogen concentration of the vastus lateralis muscle of recreationally active and elite subjects, respectively [27]. Burke et al. [55] have shown that despite achieving substantial increases in the capacity for fat oxidation during intense exercise, chronic adaptation to a ketogenic low-CHO, high fat diet impaired exercise economy and negated the transfer of traininginduced increases in aerobic capacity into improved performance of a real-life endurance event in elite athletes. The downregulation of CHO metabolism after high-fat feeding that underpins the capacity impairment for high-intensity exercise [49] in both competitive and training bouts [35,49] probably explains partly the lack of proof of a performance advantage. In addition, effects of high-CHO diets and elevated muscle glycogen have positive effects on performance [35,6] and even more when considering timing intakes [56]. Training with a diet rich in carbohydrate and which provides either high or periodized carbohydrate availability around training sessions is associated with improved race outcomes [49]. This underlines the role of CHO provision to maintain the capacity to train at high intensities to achieve an optimal adaptive level. When the exercise is prolonged (2 hours or more), exogenous carbohydrate becomes a fuel which is essential to be ingested and to metabolize [54]. Noteworthy, exogenous carbohydrate oxidation is limited by the intestinal absorption of carbohydrates. It is believed that glucose uses a sodium-dependent transporter SGLT1 for absorption that becomes saturated at a carbohydrate intake around 60 g/hour [19]. Many studies have demonstrated that increases in the hourly rate of CHO and overall energy intake are correlated with faster race times in ultraendurance events [48]. It is likely that the absorptive capacity of the intestines is modified by carbohydrate content of the diet, as intestinal transporters can be upregulated with increased carbohydrate intake [19]. Indeed, the ability to ingest and metabolize CHO seems to be trainable. So, considering that the ability to ingest and metabolize carbohydrates during exercise can be determinant of performance [56], it should be kept in mind that “training low” does not allow for optimized carbohydrate metabolism during the competition. Even if adaptation to a low carb high fat (LCHF) diet increases the muscle’s capacity to utilize fat as substrate of the forced production of succinyl-CoA as is the case in situations of lack of carbohydrate substrates and forced oxidation of branched amino acids, production of ROS by the mitochondria is exaggerated [57] and could increase oxidative stress and damages. Moreover, this stress would also decrease immune function and therefore if used too often may increase infections [6]. The philosophy of undertaking quality training and competition with high CHO availability, to both promote training adaptations and the use of CHO as a substrate for the brain and central nervous system while performing, seems to be preserved [58,35]. The question of whether training bouts with limited CHO intakes are the best option is questionable as on the one side it increases fat oxidation but on the other decreases adaptive effects to CHO intakes, in some cases limits the highest intensity that can be achieved and is likely to increase ROS production at a level that has not been identified to overwhelm or not antioxidant systems. Further research specifying the modalities for which train low would benefit to performance is warranted.

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Compete High Fat oxidation rates increase from low to moderate intensities and then decrease when the intensity becomes high. Maximal rates of fat oxidation have been shown to be reached at intensities between 59% and 64% of maximum oxygen consumption in trained individuals and between 47% and 52% of maximum oxygen consumption in general population [59]. Anyway, CHO intake during the race provides a substantial fuel source and is associated with enhanced performance [60,61], even for endurance events lasting up to 3 hours (in highly trained athletes) where CHO-, not fat-based fuels are the predominant fuel for the working muscles. Indeed, CHO, not fat [61] availability becomes rate limiting for performance [58]. Until the last decade, advices for CHO intake during exercise were essentially based on the supply of glucose in the form of monosaccharide, or polymers. Considering the maximum oxidation flow of the CHO, the maximum intake recommendations have generally been admitted to be set at 60 g/hours, the maximum intestinal absorption capacity being estimated at 1 g/minutes (by saturation of the SGLT1 specific transporter). However, when glucose is combined with another sugar which uses different carriers as glucose and fructose coingestion may increase total carbohydrate intestinal absorption [62], the maximum oxidation flow of the CHOs reaches the value of 1.3 g/minutes and a fructose combination improves performance [39]. Nevertheless, these effects are only observed for exercises longer than 2.5 hours in trained subjects, or for carbohydrate requirements exceeding 1 g/minutes. Glucose and fructose are also metabolized through different pathways after being absorbed from the gut and the ingestion of glucose combined to fructose may thereby profoundly alter hepatic function ultimately raising both glucose and lactate fluxes. This particularly may induce a spectrum of effects on muscle metabolism possibly resulting in an improved performance, and compared to glucose alone, ingestion could also induce several nonmetabolic effects which are so far largely unexplored [62]. The practices aiming to adopt nutritional strategies to ensure that muscle glycogen stores are well stocked prior to competition as it helps delay fatigue are consensual. Indeed, it is unfounded to avoid carbohydrate feeding in the hour before exercise [19]. Preexercise (training or competition) meals should contain high glycemic index (HGI) carbohydrate foods rather than low glycemic index CHO foods because of the more rapid delivery of glucose to liver and muscle via the systemic circulation [18]. During exercise lasting approximately 30
60 minutes, it is not necessary to ingest large amounts of carbohydrate and a mouth rinse with carbohydrate may suffice to a performance benefit [19]. But during participation in multiple-sprint sports, ingesting a well-formulated CHO solution or gel improves endurance capacity and may prevent a significant decay in sprint speeds [18]. As exercise accelerates the rate of glycogen resynthesis, HGI carbohydrates intakes immediately after is essential for training or competing on successive days [18] to replete optimally glycogen stores—at the same time save essential amino acids oxidation—regarding the next event. In both genders, strict weight categories, esthetic quest, or even high loads can lead some athletes to energy restriction and thus unsatisfactory nutritional status. Elite synchronized swimmers have shown to fail to compensate for the rise in energy needs due to high energy expenditure with increased training loads [63]. Female athletes in esthetic events

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(e.g., gymnastics, ballet)—but not only—have often reported low nutrient intakes [64]. Deficiencies are often related. The insufficient energy availability is clinically and more easily evidenced by physiological signs of menstrual dysfunction as oligo- or amenorrhea [65]. As undoubtedly CHO remains the “king” [61] substrate for acute exercise, it can be considered—according to the period and the discipline—that athlete could not only be aware to “compete high” but even to consider the “training high” principle. As requirements vary according to the stressor, and cycle, nutritional strategies have to be thought being closely related to the training period regarding the objectives including whether they are major or intermediate. Consequences on Nutrition Periodization The remaining controversies encourage seeking for to answer to the question of whether this approach is of interest or not, at least considering periodization [47]. There seems to be a few scenarios where LCHF diets are of benefit, or at least are not detrimental, for sports performance [49]. It can be assumed that optimal nutrition results in a decreased risk of energy depletion, and quicker full-recovery [66]. Most of the experiments did not reveal the beneficial effect of fat-rich diets on performance. According to Burke et al. [58], it could make sense to focus on good carbohydrate availability for sessions requiring high intensity or high levels of technique and skill. Regarding nutritional periodization, undoubtedly CHO provision is less important during lower intensity exercise or training sessions at the beginning of the season. The same can be considered regarding fasting, as the most adequate advice is to train while fasting specifically at low intensities and during the preseason period for not disturbing a suitable recovery during a program of training load [67]. However nothing is reported about the stability of the expected effect later in the season and a fortiori in the competitive period. Obviously, individualized nutritional strategy has to be mainly developed targeting carbohydrate delivery to the working muscle at a rate depending on the absolute exercise intensity and the duration of the event. Aiming to individualize nutrition periodization, interesting guidelines have been proposed by Jeukendrup [19] aiming to promote individualization rather than adopting undifferentiated principles, indeed that is confirmed today. Its recent review is particularly complete and to date on this topic [54]. As the nutritional strategy for immediate performance differs from nutrition aiming for optimal training adaptive effect induction, “competition nutrition” and “training nutrition” are now considered as separate entities [61]. Thus, to support both performance and optimize training effects, defining appropriate nutrition implies to take into consideration the constraints and the principles of training and its periodization. The transfer from science to the field is still uneasy. The determinant high CHO availability achieved by the traditional supercompensation protocol and the effect of maximizing the use of fats as substrates remains a challenge. Even if further research is needed to establish optimal dietary periodization plans [53], the very recent analyses of the available data (see for review [54]) confirm the relevancy of dietary periodization, as training has to be. A part of the answer is maybe in modeling the nutritional periodization. Awaiting next studies, mostly clinical, the strategies can only combine the field observations and the knowledge available. A part of it remains quite empirical. Training between high carbohydrate (HCH) and low carbohydrate (LCH) is like walking a tightrope as

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nutritional strategies optimizing fat oxidation could lead to both decreases in CHO ingestion capability and lowest training intensities, and consequently training-induced adaptations and performance.

DOES EXERCISE-RELATED REQUIREMENT JUSTIFY MICRONUTRIENT SUPPLEMENT USE? The needs in vitamins and minerals are subject to variations depending on factors including the period of training or the season, climatic or environmental conditions (temperatures, hypoxia, etc.). Whatever the level of performance, some beliefs are likely for athletes to justify the supplements intakes. The raised question is whether requirements are altered and whether these requirements justify supplement use. Here will be addressed the issue of the micronutrient requirements for health and performance including the place of certain phytochemicals which is still debated, mostly with regard to potential ergogenic effects. The complexity of setting reference values for vitamins and minerals, including safety limits, is primarily addressed to enlighten the issue of the expected supplementation benefits—supported or not by claims—and related health risks. The case of antioxidants will be discussed separately referring to the specific role of the redox signal in adaptation which has been developed earlier.

Setting Reference Values for Micronutrients Micronutrients as a part of the molecules necessary for the fundamental reactions of cellular metabolism are essential to the fulfillment of a particular function. They include mineral and organic compounds, the synthesis of which man is unable of performing. Due to their participation in metabolic, enzymatic activities, inadequate intake in vitamins and mineral and consecutive inadequate micronutrient status leads to an alteration of biological functions incompatible with health and a fortiori the achievement of higher level of performance. The so called reference values include both requirements and upper safety levels. Dietary Reference Values for the General Population The nutritional requirement of a nutrient can be defined as the level of intake that satisfies an adequacy criterion. In the general population, the average nutritional requirement is estimated from the mean of individual input data in relation to a nutritional adequacy criterion of measurements made on an experimental group in a small number of individuals. Experimental studies (depletion and repletion) use criteria such as the one or more nutrient balance, modification of reserve markers, or associated functions. Requirements for substances other than vitamins and minerals that would be the subject of recommendations have not yet been established. A harmonization of the terms used in nutrition has been based on a better characterization of the scientific background (type and quality of data) for the selected values [36]. These concepts are presented in a didactic, up-to-date, and comprehensive manner by Verkaik-Kloosterman et al. [68]. The need can also be calculated by the factorial method II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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taking into account additional components related to particular physiological situations (growth, pregnancy, lactation, exercise, etc.). The terms relating to dietary reference values (DRVs), i.e., the average requirement (AR) is the average daily need within the population, as estimated from individual intake data in relation to a criterion of nutritional adequacy in experimental studies. The population reference intakes (PRI) are calculated from an estimate of the parameters of distribution of the requirement. Most often the PRI is estimated from the AR, to which are added two standard deviations, in order to determine the intake that covers the requirement of almost the entire population (97.5%). As the standard deviation is most often estimated at 15% of the AR, the PRI is therefore 1.3 times the AR. However, to the extent that the average need is not known for all nutrients, population references are based on other data (e.g., epidemiological data) and are considered as AI. AI is the average daily intake of a population or subgroup whose nutritional status is considered adequate. Regarding the risk of adverse health effects due to excess, tolerable upper intake levels (ULs) have been set for vitamins or minerals by EFSA [69] for the European Community. Adverse Effects: Is the UL the Right Limit? The UL values are set to define the tolerable UL of vitamins and minerals intake. As athletes are numerous to consume both supplements and fortified foods, they are potentially highly exposed to health adverse effects. In Europe a reference value based on the hazard identification and its characterization has been set for each vitamin or mineral [69]. Beyond this value, the risk can be considered and is likely to occur with excess consumption of fortified foods or food supplements. In few cases, the hazard cannot be identified and some nutrients may be devoid of UL. In theory, for micronutrients, the UL of safety is the maximum chronic daily intake of a vitamin or mineral considered unlikely to pose a risk of adverse health effects. Actually, the margin of safety between the recommended intake—the “free space” for fortification or supplement [68]—and the intake that would produce adverse effects varies greatly among the nutrients. The UL is probably not the sole reference to be taken into account as a maximum for some nutrients in supplementation alone or in association, as seen for antioxidants hindering some expected effects of training being less “adverse effects” than “side effects.” It can add some reports of increased mortality with chronic intakes of β-carotene and vitamin E even in small proportions, as do high doses of vitamin A. So health risks can be associated with high consumption of antioxidant micronutrients beyond the food vectors as shown by a metaanalysis [70] leading the authors to conclude in a way that antioxidant supplements should be considered as medicinal products and should be subjected to a sufficient premarketing evaluation [71]. The sought effects of supplementation have to be discussed regarding the effects of a naturally rich diet antioxidants (several hundred molecules) vs supplementation (by dietary supplements or fortified foods). A joint model for excess and deficiency has been proposed, so that the dose and origin of toxicity (excess or deficiency) predict the probability of an adverse health effect [72]. If adapted to each micronutrient, the dose could differ from UL. The author propose a closed-form expression for the point at which the exposure
response curves for excess and deficiency cross, corresponding to the exposure level at which the risk of an adverse outcome due to excess is equal to that for deficiency, more than being a fixed and quite high dose regarding biological responses to increased intakes. II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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Exercise-Induced Change in Micronutrients Requirements and References Nutritional requirements vary according to the physiological status of the subjects. Reference values are set for subgroups of the population depending on the physiological situation taking into account their specificities (growth, aging, pregnancy, lactation, etc.). However, national and international institutions rarely take into account the energy expenditure for activities over two METs. It can be recalled that the main item of expenditure is that related to the level of physical activity. The question is in what extent physical exercise changes requirements. It can be raised by two approaches at least. The one—only informative—considers observational studies regarding the nutritional status of athletes, the other aims to calculate the needs regarding biochemical, physiological, and metabolic data, as the calculation of a PRI based on AR. Some athletes are more likely than others to be deficient in micronutrients, e.g., through the application of high workloads [73], during multiple stage races as cyclist tours [74], or the consumption of food products with a low nutrient density, considered as being unavoidable [75], mostly over 4500 kcal [76]. Quite differently, an energy restriction is likely to induce deficits in some trace elements (zinc, copper, selenium) [77
79] and also other micronutrients. A decrease or lack of absorption despite satisfactory dietary intakes is likely to have the same effects. Despite higher total micronutrient intakes, most trained athletes would show biochemical evidence of vitamin and mineral status similar to that of untrained subjects [80]. Nevertheless, the limits of reliability of the biological balance sheets necessitate a complementary nutritional assessment. The assumed increase in micronutrient requirements refers to several exercise-induced changes. Have to be taken into account: the losses at least partially attributed to the sweat losses (vitamin C, iodine, etc.), although in some cases at partially compensated by less urinary excretion, but also the increase in oxygen transport (referring to the increase of erythropoiesis involving in particular iron and vitamin B12), the increase in energy metabolism whose use of substrates involves B-complex vitamins, increase in the production of ROS which potentially exposes to oxidative damage. Nevertheless, a large number of compensatory phenomena—as sweat losses that can be partially compensated by a lesser urinary excretion—explain at least in part the absence of significant changes in need—in the sense that they cannot be covered by food—coupled with the fact that few nutritional references are established by considering physical activity [76,81]. Some values proposed in countries or at international level are consensual, but the current knowledge does not allow establishing recommendations all nutrients as for molybdenum, fluorine, iodine, vanadium, or silicon in athletes. Methods for simultaneously fitting excess and deficiency data in the form of a single U-shaped exposure
response curve have been proposed [72] allowing to set the minimum of which occurs at the exposure level that minimizes the probability of an adverse outcome due to either excess or deficiency. Both inter- and intraindividual variability regarding training periods being more pronounced with physical exercise encourage indeed considering a reasonable range of micronutrient intake, rather than focusing on a single optimal intake level for athletes. The effects of physical exercise on the vitamin status remain controversial, probably because it is difficult to generalize. Considering moderate intensities or durations, physical exercise do not necessarily affect daily micronutrient intake [80]. The exercise-induced

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increase in energy expenditure is likely to increase energy and by this way—if not provided by empty calorie foods—micronutrient intakes. It has long been held that supplementation of diet with either single or multivitamin preparations do not improve physical performance in athletes with a normal biochemical vitamin balance resulting from a wellbalanced diet [76], are unnecessary for the athlete receiving a balanced diet, and are ineffective as ergogenic aids. This is not disproved by recent date. However, to be precise, DRVs setting for sports and high energy expenditure are needed. DRVs for sports have been scarcely set by national or international nutrient recommendation setting bodies, and when done, alignment of methodologies for the derivation of micronutrient dietary reference in sports values remain poor.

Side and Adverse Effects of Supplementation Induced stresses, training loads, performance targets, and the highest exposure to various pathologies, especially during precompetitive periods as observed during the pre-Olympic period [82], constitute a favorable ground for supplementation. As a consequence, selfadministration of products for their presumed benefits and without any medical supervision is a current behavior and represents a public health concern [83] although athletes have higher micronutrient intakes than untrained subjects [80]. Observational studies show that 40%
90% of athletes would be regular users or have recently consumed nutritional supplements [84]. A recent study [85] including Dutch athletes showed that 97% of them had used nutritional supplements during their career, 85% indicating having used supplements during the last 4 weeks. The frequency of this practice questions its nutritional value, the expected effects on performance and ultimately on health. Acute Adverse Effects Looking for immediate functional benefits claimed or not, passed on by social media, a large number of athletes consume high dose of supplements. Adverse side effects of massive doses or misuses have been widely reported in Europe and in France to ANSES. In USA, FDA website contains frequent reports of problems with supplements [86]. The major pitfall is that of exposure to disproportionate health risks, as shown by several health risk assessments carried out following reports of adverse effects related to fortified foods or drinks [87] or supplements [88,89], through declarations within the framework of the nutrivigilance device operated by the ANSES 2 in France. 3 Since the introduction of the national nutrition monitoring system in 2009 and until February 2016, 49 reports of adverse reactions likely to be linked to the consumption of sports supplements have been 2

French Agency for Food, Environmental and Occupational Health Safety.

3

Decree 2010-688 of June 23, 2010 on vigilance on certain foodstuffs. The Hospital Patient Health and Territory law has entrusted the ANSES with the implementation of the system of food vigilance (nutrivigilance). The aim of this device, unique in Europe, is to improve consumer safety by quickly identifying possible adverse effects related to the consumption of certain foods. This vigilance concerns novel foods, foods which are added to nutritional or physiological substances, food supplements, and foodstuffs intended for particular nutritional uses.

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brought to the attention of ANSES [90]. The adverse effects reported are mostly cardiovascular (tachycardia, arrhythmia, stroke) and psychiatric (anxiety disorders, nervousness). The consumption of dietary supplements intended for the development of muscle mass is quite extended and exposes the consumer to health risks [88]. There is, however, a great diversity in the composition of dietary supplements for athletes regarding the substances, combinations, and doses. Plant extracts used by athletes have been little studied and bibliographic data are lacking on the metabolism of their constituents and their possible longterm toxicity. The use of supplements with adrenergic stimulants, agonists of adipocyte receptors to catecholamine, claimed as a means of reducing fat mass, as supplements containing guarana or caffeine [88], presents undeniable risks, mainly cardiovascular, but not only (see for review [124]), as was evaluated for extracts of Citrus aurantium containing psynephrine [89] which has been otherwise proposed as being ergogenic [91] with no mention of risk by the authors. Plant extracts are often metabolized by the same P450 cytochromes as those metabolizing the drugs, exposing them to a risk of pharmacokinetic interaction. Some substances present in sports supplements have the same mechanisms of action as medicines. If these two types of products are consumed concomitantly, a drug interaction cannot be excluded [87] and has to be considered in case of medication. Some plants used in the formulation of food supplements are absent from the lists of authorized plants and the quality of the products is not well informed. Restrictions on use and side effects are often absent from product labels. There is still a large number who believes that the “racers edge” may be found in a tablet [75], but in this context, their safe use cannot be guaranteed. Hindrance of Adaptive Effects as a Side Effect Oxidants are formed as a normal product of aerobic metabolism and continuously produced at rest as a natural consequence of the use of molecular oxygen by oxidative metabolism. During exercise, the acceleration of metabolic reactions to cover the muscle demand in energy results in an increased production of ROS. The imbalance between proxidant and antioxidant overwhelm the defense systems reactions and thus initiate oxidative stress which can provoke damage in various cellular components such as lipids [92
94], proteins [95], and nucleic acids [96,9]. Mainly of mitochondrial source in the active muscles, the ROS produced during the exercise are also secondary to muscle damage per se and related to a redox-dependent regulation of immune responses to skeletal muscle damage induced by intense eccentric exercise [97]. Cell targets are protected by antioxidant systems. An antioxidant is state by Haliwell and Gutteridge [98] as “any substance that when present at low concentrations compared with that of an oxidizable substrate significantly delays or inhibits oxidation of that substrate”; antioxidants are likely to protect against oxidative damages. Antioxidant defense involves both enzymatic and nonenzymatic strategies that include a set of enzymes [glutathione (GSH) peroxidase , catalase, and superoxide dismutase] specialized in the reduction of ROS into stable chemical species. Endogenous antioxidant systems depend on the availability of cofactors (Cu, Se, Zn, etc.) and exogenous chemical compounds capable of neutralizing ROS, such as vitamins E diet (tocopherols and tocotrienols), and C (ascorbic acid), β-carotene, and GSH, flavonoids, provided coenzyme q10, carotenoids provided by

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diet. Some convert the free radicals into less reactive substances, some minimize availability by protein binding to, and others are free radical scavengers. In the lipid phase, tocopherols and carotenes as well as oxy-carotenoids are of interest. In the aqueous phase, there are ascorbate, GSH, and other compounds. In addition to the cytosol, the nuclear and mitochondrial matrices and extracellular fluids are protected. In the athlete, studies on the trace element status are numerous for zinc, selenium, copper, and manganese with antioxidant function—notably enzymatic cofactors of peroxidases or dismutases. These antioxidants add significantly to the defense provided by the enzymes superoxide dismutase, catalase, and GSH peroxidases of the endogenous antioxidant system. Strengthening the endogenous antioxidant system with training reduces the risk of cellular damage during exercise [99,92,100]. Since the beginning of the research in this area in the end of 1980s, the most studied are selenium, vitamins C and E, sometimes completed by several compounds as chosen phytochemicals. Although several points of discussion still existed, the question of whether antioxidant vitamins and antioxidant enzymes play a protective role in exercise-induced muscle damage was answered affirmatively in the past [101]. We observed in one of our studies, a reinforcement of antioxidant system and reduced muscle damages with supplementation. Actually, there were overloaded and in nutrtionnal deficit at the time of inclusion in the study [102], in this case tapering is of high benefit. Moreover, the suggestion that repetition of training sessions induces chronic oxidative stress as shown by high lipid peroxidation in athletes [93] is far from being widely shared. During the late 1980s and after, the literature widely suggested that dietary supplementation with antioxidants have favorable effects by detoxifying the peroxides produced during exercise and are capable of scavenging peroxyl radicals and therefore prevent muscle damage [101]. As a consequence, and quite consensually, antioxidant vitamin supplementation was recommended to individuals performing regular heavy exercise. These results should be reconsidered in the light of the evolution of the methods and epidemiological data regarding chronic effects of high antioxidants intakes. Since the past decade, experimental studies take systematically into account confusing factors as dietary intake, training status, exercise protocol, and type of muscle-damage marker used. They have become less conclusive skating away from a systematic recommendation to use supplements or event fortified foods. It is becoming increasingly evident that the production of ROS is indispensable to the process of adaptation [6,103]. Thus the well-being of an antioxidant supply raises the question of these protective effects with regard to the cell, or on the contrary, hindrance of the inductive signals of the targeted adaptations. Today a growing body of evidence indicates detrimental effects of antioxidant supplementation on the health as doses—even above UL—that can also result in prooxidative effects [104], causing risks of decreased recovery, health [99], and performance benefits of training. Although ROS are associated with harmful biological events, they are also essential to the development and optimal function of every cell [105]. Last decades have provided body of evidence on the pivotal role of ROS production on adaptive effects, and it is noteworthy that Davies et al. in 1982 [8], while discovering in vivo the ROS production with exercise, already hypothesized a role of ROS in mitogenis. Well-conducted training results in increased activity of several major antioxidant enzymes and overall antioxidant status as a small surplus of ROS appears to promote cellular antioxidant defense capacity

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due to increased gene expression of antioxidant enzymes [12], upregulation of uncoupling proteins (UCPs), and suppression of inflammatory and proteolytic pathways [106]. Supplementation with high levels of antioxidants can blunt the normal increase in mitochondria with endurance training [105] and slow down the endogenous antioxidant synthesis [107]. Emerging evidence strongly suggests that additional contribution antioxidants may, by lowering the physiological concentration of ROS, hamper positive adaptations to training [14,61] as the molecular regulation of endurance training response can be altered. The intervention with the antioxidant supplementations has a suppressive role on the exercise-associated upregulation of Hsp70 transcription. By limiting the signaling effect of ROS needed to strengthen endogenous systems [107], exogenous antioxidants may exert a detrimental hindrance of training effects. The issue is probable to be thought taking into account the matrix and the specific U-shaped dose
response relationship of each nutrient. A part of the answer could be in the time of supplementation regarding the hormesis U-shape. If the antioxidants are supplemented before the ROS reach levels for maximum adaptive response, the antioxidants would depress the physiological response. If the supplementation arises when the concentration of exercise-generated ROS is associated with a declining physiological response, the supplementation would result in enhanced performance and delayed fatigue [103]. Indeed, if done, exercise-induced responses and have to be taken into account in combination with antioxidants pharmacodynamics. The risks of exceeding the capacities of the endogenous antioxidant systems are not observable for a moderate physical activity. However, some claim that ultraendurance athletes may use supplements—as far as is necessary, and in keeping with advice from healthcare providers—to support training and events performance and aid in recovery [18], nothing proves that requirements cannot by met by food. As the effectiveness of these systems is physiologically increased in response to the level of physical activity, it seems that adaptation takes place without requiring additional supplements of antioxidants other than those brought spontaneously by a diet rich in fruits and vegetables. In a balanced diet, there is indeed no benefit from antioxidant supplementation [108]. A body of evidence advocates for avoiding systematic antioxidant supplementation in athletes as in recreational sports, as a contribution in the form of fruits and vegetables allows to benefit from the synergistic effect of antioxidant nutrients (in vitamin C, but also in phytonutrients, etc.) and phytochemicals present in the diet in a more general way. In nonenvironmental-stressing situations, the majority of the scientific evidence is against recommending the indiscriminate use of antioxidant supplementation. This can be considered differently as the additional hypoxia-induced stress is likely to increase ROS production at level which is likely to overwhelm the endogenous antioxidant system and induce hormetic effect failure of training or performance achievement in middle or longterm exposure (mountaineering). Additionally, in high altitudes, exercise-stimulated hunger is alleviated, appetite energy intakes decreased, and the decrease in vectors of antioxidants argues in not excluding fortified foods or supplementations. The question is whether the athlete has to cope with a high environmental and acute stress or whether the hypoxia is used to provoke adaptive effects (training vs punctual event in altitude). In the first case, additional intakes of micronutrient have to be thought in accordance with the adaptions sought, avoiding the hindrance of ROS positive effects, in the second, nutrition aims to protect from ROS-induced damages which are likely to be increased with hypoxia. The added undernutrition justifies in some cases supplementation or fortified food intakes. II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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Anyway, over the specificity or the uncertainty of beneficial effects of supplementations, adverse effects have been widely reported with high chronic or acute doses. Seeking for functional effects and in the belief of “more is best,” the cumulative effects of supplements and fortified food are likely to expose the athlete to high doses. Both health risks and adaptive effects hindrance associated with high intakes of micronutrients are becoming now better documented. The increased and systematic use of fortified products, even being in the framework of the application of Regulation 1925/2006 [109], exposes the consumer—including sport—to hazards depending on the use. These foods—whose maximum doses of vitamins and minerals to be incorporated in fortified foods are still not fixed by the European regulation—are, moreover, potentially carriers of health claims. Indeed, one issue is how to deal with the health risk regarding the claimed beneficial effects. van der Beek [76] to cite Williams in 1976: “Only through further controlled research may we eliminate false advertising claims for food supplements in athletics and the myths associated with nutrition and athletic performance [110].” The issue is not so recent. The European regulation on nutrition and health claims [111] entered in force in 2006, aiming at assess health claims to not mislead the consumer before the product being on the market replaced the a posteriori assessments—as done by the majority of Member States—by an a priori one. The target population being is an issue for assessments [112]. In the specific case of physical activity, what matters is that claims are appropriately assessed, so that athletes could actually not be misled.

HEALTH CLAIMS AND THE “BENEFICIAL” EXPECTED EFFECTS Physical activity and especially strenuous athletic training is believed to be harmful for nutritional status. Consequently, the need for micronutrient supplements to ensure adequate nutritional status is a widespread belief in physically active people.

Health Claims and Applications to Sport Nutrition Functional foods for high energy expenditure fall within the scope of the regulation on nutrition and health claims [111], but these are authorized in very small numbers. Many applications for claiming specific effects on targeting sport have been rejected and to date sole five of them have been positively evaluated by EFSA for creatine [113] as the existing evidence suggests that creatine have a positive impact on athletic performance [114], as well as proteins [115] electrolytes, CHO solutions [116], and ascorbic acid [117]. They have been authorized by the European Commission. Actually, the active subject is potentially targeted by other claims than those specifically related to physical exercise. Some subjects tempted to use fortified foods or dietary supplements specifically targeting the active subject are increasing the intakes of vitamins or minerals involved in various physiological functions in a more general way (“bone capital,” “lean mass gain,” “immunity,” etc.) with no deficit. In this case, these claims are misleading [112]. The use of nutrition and health claims made on foods refers to Regulation (EC) No. 1924/2006 of the European Parliament and of the Council [111]. A health claim is thus

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defined as any message or representation “that states, suggests or implies that a relationship exists between on the one hand, a food category, a food or one of its components, and second, the health.” To enter the authorization procedure by the European Commission, health claims must be assessed on their validity by EFSA on the basis of available scientific data. Scientific expertise includes steps specified in the guidelines of EFSA [118]: (1) the food or ingredient is defined and characterized; (2) the claimed effect is defined and has a physiological benefit; (3) a cause and effect relationship is established between consumption of the food/constituent and the claimed effect. The evaluation focuses on the nutritional composition of the product; the justification of the claim; and the functional effects or health attributed to a given nutrient, the coherence with public health policies, and the formulation of the claim. The accuracy of target populations and conditions of use has to be subject to a special attention. The establishment of a sufficiently high standard of evidence to substantiate the claim remains a critical issue (see for review [112]) as an isolated result of research cannot give rise to a transposable message to the consumer. The most complete body of knowledge available has to be drawn in the wide spectrum of molecular biology to epidemiology supporting the validation of the claim. So, the availability or the production of quality clinical studies is a critical point of the outcome of the evaluation. The highly dynamic nature of the physiological state of the athlete removes it even more from the potential target of these allegations. The assessment of antioxidant claims raises the question of how far a molecule whose role has proven antioxidant biochemical behaves like an “antioxidant” systemically. EFSA questioned the validity of the claim “antioxidant” for a lot of nutrients and rejected systematically the claims excepted in two cases [119]. However, several opinions validated claims’ effects on the cell. The evaluation focuses on the available studies, mostly on cells or animals, as the highlight of the antioxidant effect is a complex exercise for methodological reasons including those related to circulating markers in humans. An issue is that regulatory provisions do not guarantee the simultaneous evaluation of risks and benefits. Entering in force, regulation should encourage the coherence of research investments needed to substantiate the beneficial effects as well as to prove the lack of health hazards (intrinsic or loss of chance). The risk assessment should not to be devoted to the sole public authorities whose assessment highly depends on the scientific corpus available at a t-time of the expertise. An additional issue is that the search for benefits can paradoxically be accompanied by effects opposite to those expected, as can be the case of antioxidants [107] whose effects are certainly “antioxidants” on the cell but for which no effect on health can validly be claimed [119] and has not been evaluated favorably by EFSA other than cellular effects [120]. Beyond that, the search for some “effects”—legally claimed or not—is likely to expose to unexpected effects or health risks. So-Called or So-Understood Ergogenic Aids and Risks Observational data show that almost all athletes had used dietary supplements at some time during their athletic career and supplements intended for sports are of high diversity [88]. Some nonnutritive substances are believed to have ergogenic effects which are among those expected. On a representative sample of 816 Greek athletes, 39.4% (n 5 253) of the total sample reported they used Nutritional Supplement (NS) for performance

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enhancement purposes. Of them, 19.6% reported they rarely used NS, 15.7% said they consumed supplements sometimes/often, and 4.0% reported quite systematic use of NS [121]. Proteins, amino acids, creatine, multivitamins, and wide range of herbal products (e.g., Tribulus terrestris, maca) are used as “legal” performance enhancers in elite and recreational sports [122]. Sahlin [123] outlined in a review that Cr, bicarbonate, and β-alanine could have ergogenic effects during high-intensity exercise. Among the several extracts or/and phytochemicals thought to be ergogenic, the most popular stimulant and documented is caffeine [124] which after being banned is mentioned on the monitoring list by the World Anti-Doping Agency (WADA) since 2004. Over the question of caffeine alone appears the specific issue of the marketing position of energy drinks and the voluntary confusion conveyed by marketers with sport drinks. This confusion has to be prevented, as energy drinks are not formulated for sports, and moreover because effects of caffeine intake before and during exercise is of concern as several health issues regarding cardiovascular risk, hyperthermia, are now documented [87], and need to be further properly documented. Moreover, bibliographic data are lacking on the metabolism of plant extracts used by athletes have been poorly studied and constituents and their possible long-term toxicity [88]. Athletes turn to the use of dietary products with presumed ergogenic properties as an alternative to prohibited drugs [125]. Man has to keep in mind that many commercial products are contaminated with steroid-like chemicals [126,127]. About 20% of nonhormonal nutritional supplements contain anabolic androgenic steroids. The WADA established in 1999 to fight doping in sports updates annually its list of prohibited performance enhancement drugs. Although a substance is not classified as illegal substances according to the WADA prohibited substances list, there is evidence that athletes using these supplements risk inadvertent doping infractions due to contaminated products [122,126] as food supplements intended for the active or sporting subject taming circuits are sometimes insufficiently controlled. Although that some companies take great care to supply highquality products, problems with the integrity of commercially available supplements are well documented [86]. Only additional controlled research may help answer some of the questions that still remain relative to nutrition and athletic performance.

DISCUSSION Together with a positive balance between stress and adaptive response targeting hormesis, the nutritional strategy is a part of athlete methods to improve performances. Low intakes can alter performance as AIs play a protective role. It is difficult to recommend strictly defined intakes of macro- and micronutrient, as variables such age, gender, environmental conditions, training cycle, and, most of all, adaptive state underpin the requirements. It appears that many controversies remain not only in particular to the share to be attributed to the CHO intake according to the period and the strategies of optimization of lipid use but also in terms of meeting micronutrient needs. In general, some constants appear that can be followed in a range of situations. Overall, a basic principle to consider, whatever the activity, is that training aims at the optimization of adaptations, whereas

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event is aimed at a punctual highest level of performance. Requirements vary according to the period of training program and nutrition is strained by the strong exigencies of training periodization. Regarding micronutrient requirements, examples of chronic deficiencies that actually require micronutrient supplementation are few. Diet is a dynamic balance which can be broken down taking supplement—whereas a well-balanced diet full of natural antioxidants can minimize the level of oxidative stress produced during overloaded training. Only particular situations may lead occasionally a health professional to propose fortified foods or supplements with the goal of reaching a normal nutritional status and to restore biochemical and physiological functions in targets biomarkers. This is in the cases where the hormesis principle can no longer be applied due to a too high load (functional or environmental stress) to induce adaptation, as ultraendurance events, hypoxic stresses, as altitude training, or mountaineering, loss of appetite. This can also occur in cases with energy restriction intake, severe weight-loss practices, or unbalanced diets with low micronutrient density—what is ethically questionable. It should not be avoided at all costs, but reasoned. The biological and nutritional assessment is of great utility, even necessary. In this context, for athletes and athlete support personnel, in particular in their initial training, education and training programs should aim to provide updated and accurate information on nutritional supplements [UNESCO 2005 4] More generally speaking, there cannot be expected an ergogenic effect from vitamins and mineral. Changes in energy, macronutrients, vitamins and minerals requirements do not require the use of functional foods as a prerequisite for meeting nutritional needs. In general, vitamins and minerals requirements can be covered by a balanced diet unless there is an individual biological demonstration to the contrary. To date there is little scientific evidence to substantiate claims regarding performance increase in well-balanced diets. Thus, only a compensatory effect can be expected when correcting a deficiency. As it is usual to consider the U- or J-shaped curve of the dose
response relationship considering a given nutrient [72], risks as well as benefits have both to be considered to prevent inappropriate micronutrient intake-induced adverse or side effects. Risk/benefit analysis emerge on evidence for an unknown risk of supranutritional intakes, a supposed impairment of adaptive effects, and a still unknown long-term risk. In addition, because products are likely to be faked [126], and because specific regulations to nutritional ergogenic aids are poorly enforced, they should be used with caution and only after careful product evaluation for safety, efficacy, potency, and legality. Among factors associated with a decreased level of performance due to illness in athletes preparing for Olympic Games competition, including hygiene practices, low energy availability has been shown to be the highest attributable fraction in the population [82]. Some deficiencies are even observed with inadequate intakes in nonenergetic nutrients, underlining the key role of energy supply, and generally speaking of nutrition. This highlights for coaches and support staff the crucial role of nutrition—among other factors including recovery, travel, sleep, rest, active rest, relaxation strategies, and emotional support as recommended by International Olympic Committee [29,128]—in maintaining health as prerequisite to the optimization of adaptive responses to training and avoiding 4

International Convention against Doping in Sport. Art. 19-2(d).

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OTS. As poorly conducted physical training is an aggravating factor in food-related disorders, a poor diet quality is an aggravating factor in overtraining related disorders. Whatever the strategies, training is now usually periodized and nutrition has to be—if not already done—incorporated in the periodization to cope with increased needs and allow positive training-induced adaptations. The time-factor has even to be taken into account in the very short term, before, during, and after exercise to support performance and recovery. Regarding “time-nutrient,” the most recent position stand of the International Society of Sports Nutrition [56] is the demonstration of a processing and promising opening field. Whatever the timescale, individuals respond differently to any given stimulus, even from training or diet, beyond certain broad general principles, the need for an individualized approach is beyond question. Any shortage of a vitamin is therefore linked to suboptimal metabolism which in the long term will result in decreased performance or even illness. But most of the studies showing effects of supplementation intake have been performed in deficient subjects and does not seem to produce any effect when the diet is adequate. Vitamin supplementation in quantities exceeding those needed to obtain optimal blood levels has never been shown to improve performance. In case of no compensatory need, supplementation has to be avoided as excessive intakes of micronutrients pose a health risk. Only specific situations (deficiency in diets, unbalanced diet, metabolic disorders, etc.) identified by a complete nutritional or biological balance may lead a health professional to propose fortified foods or supplements to compensate existing deficits. Examples of chronic deficiencies that actually require supplementation in are few and each supplementation has to be based on biological or nutritional appraisal. These situations are not necessarily specific to the increase in energy expenditure. Perceived as conferring health advantages, the use of supplements is likely to create an illusory sense of invulnerability. The athlete may feel itself dispensed from having appropriate eating habits and behaviors and endanger his health [129]. Observational data have shown that dietary counseling results in better informed choices with respect to the use of nutritional supplements related to performance, recovery, and health [85]. Coaches appear to be the main influence and source of information for athletes. However, observational studies [130] show science-based information is still insufficient. As a consequence, whatever the requirements, athletes have to benefit from a science-based information and accompaniment from professionals involved in their practice. Practicing has to remain a part of a healthy lifestyle to achieve both health and performance. Hence, if nutrition is a health issue, before being a performance issue, nutritional strategies have to be thought once this prerequisite has been met. Comprehensive and source providing harmonized guidelines and recommendations to both protect the health of and promote performance is to date not available and remain to be produced. Precise, adapted, and consensual recommendations to articulate training and nutrition require further, well-controlled research and collegial expertise. As the problem to solve integrates multiple variables, a mathematic method approach could be developed, such as a linear program using an iterative method which by repeated use gives the solution to any number of variables, as it has been done recently in France for the general population [36] using the simplex algorithm to achieve the optimal solution.

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CONCLUSION AND KEY MESSAGES The nutritional strategies to improve adaptive responses differ according to the duration and configuration of the sport, depending not only on the training cycle (fundamental preparation, basic, taper, competitive, etc.) but also on environmental stress (heat, cold, altitude exposure for training and/or competition), weight control (wrestlers, ballerinas, jockeys, judokas, etc.), or body composition. Physical exercise is a potent hormetic conditioner. ROS act as messenger in exercise-induced adaptive response. Antioxidants improve the biological status from the point of view of damage but exogenous are likely to inhibit endogenous responses. Low CHO training modifies the metabolism favorably without the expected effects on performance. Micronutrient supplementation—whose biochemical roles are confirmed or consolidated by scientific assessments of claims—is not a direct performance enhancer, and not without health risk. Some are solely likely to be required in particular environmental conditions or states of deficits. Far from the systematic use or rejection of any approach, good practices are integrative and probably located between the two. Physicians and dieticians need to be aware among uncertainties and data gaps through research on acute and long-term effects of some substances. Diet education has to be enhanced and athletes need to be aware of the potential health risks of any practice. A coordinated risk
benefit analysis based on available information can be conducted before choosing any option. Healthy state has to be maintained as a prerequisite by appropriate loads and requirements coverage. On the basis of established scientific knowledge, and in the making, at a reasonable distance from generalities, the individualization of nutrition and the periodization of training are clearly the keys to performance and imperative maintenance of health. The evolving knowledge may allow the integration of relevant and precise parameters combining training and nutrition. Modeling the training and nutrition aiming to target hormesis effects is most probably the next step to promote control of performance optimization.

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Proteins and Amino Acids and Physical Exercise Stephen D. Patterson1, Mark Waldron1,2 and Owen Jeffries1

1

St Mary’s University, Twickenham, London, United Kingdom 2University of New England, Armidale, NSW, Australia

INTRODUCTION Proteins are among the most abundant organic molecules in living organisms and are essential for all key functions within the human body. Like carbohydrates and fats, they contain carbon, hydrogen, and oxygen molecules; however, in addition, they also contain nitrogen. They are made from chains of amino acids (the monomers that make up a protein), which link together to create a polypeptide. There are 20 total amino acids. Nine of which are called essential amino acids (EAAs) because the body cannot manufacture them, namely, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, and must be consumed in the diet. The remaining 11 are the nonEAAs, which can be produced by the body, namely alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. When consumed from the diet, amino acids are used to synthesize proteins and other biomolecules or can be oxidized as a source of energy. Three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine have demonstrated unique characteristics for their roles in protein metabolism [1], neural function [2], and blood glucose and insulin regulation [3]. Protein sources with a higher concentration of BCAAs and the other essential amino acids (EEAs) are suggested to be of higher protein quality and more effective at promoting protein synthesis [4]. Throughout the day, proteins are broken down and remade. This turnover allows for damaged proteins to be replaced by new proteins, formed in response to exercise training. This chapter provides an overview of protein and amino acid supplementation before, during, and in recovery from exercise. This review will cover both classical and contemporary literature and discuss the optimal protein sources, required amounts needed by athletes, and the ergogenic effect for exercise performance and recovery.

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SOURCES OF PROTEIN INTAKE Milk proteins have been widely investigated, relating to their role in augmenting adaptations and recovery from exercise training [5]. Milk protein has a high biological value, making it a good source of EEAs and BCAAs [6]. Two of the main proteins in milk are casein and whey, making up 80% and 20% of milk proteins, respectively. Casein is considered a “slow” protein, as it slowly empties from the stomach leading to a slow and prolonged appearance of amino acids in the blood [7]. Whey is considered a “fast” protein due to its rapid digestion that provides higher concentrations of amino acids in the blood. However, this response is short-lived [7]. Both proteins, ingested after resistance exercise, lead to net muscle protein synthesis [7]. Milk ingestion has been shown to have potential benefits for hydration [8], endurance capacity following glycogen depleting exercise [9], accelerate recovery from muscle damaging exercise [10], and improve the gains in skeletal muscle hypertrophy and strength following resistance training [5]. Whey protein results in greater muscle protein synthesis postexercise compared with both casein and soy protein [11]. It is believed that whey protein increases amino acid concentrations at faster rate in the blood in comparison to casein and soy. Casein protein has also been demonstrated to be superior to soya in stimulating muscle protein synthesis [11]. In contrast, the soya protein resulted in a more rapid increase in total amino acids in the blood, whereas the casein appeared at a much slower rate. It may be beneficial to consume this protein prior to sleep as to provide a sustained delivery of amino acids during the overnight fast [12].

PROTEIN REQUIREMENTS The protein requirement for athletes is still a controversial topic. Athletes consume dietary protein to repair and rebuild skeletal muscle and connective tissues following intense training bouts or athletic events. It has been suggested that minimum dose of 20 g of whey protein is required following resistance exercise to increase protein synthesis rates in young individuals [13]. In contrast, this dose should be increased to 40 g to maximize rates of protein synthesis in older individuals [14]. Some believe that athletes do not require additional protein intake, whilst others suggest that the intakes are higher than the 0.8 g/kg recommended to sedentary individuals [15]. One of the reasons for confusion is due to the different sporting activities carried out. For example endurance trained athletes require approximately 1.2 1.4 g/kg, whilst strength and power athletes require 1.8 2.0 g/kg. Furthermore, protein intakes as high as 3.0 g/kg aren’t harmful and may even be of some benefit to the athlete [16]. It is suggested that these requirements can be achieved through food intake alone; however, for convenience, supplemental protein may be used.

ENDURANCE EXERCISE PERFORMANCE Acute Supplementation of Protein and Amino Acids A common misnomer among athletes is that carbohydrates and fats provide the only important fuel for endurance exercise performance. Indeed, the effects of endurance II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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exercise training on the regulation of protein metabolism is less understood and underappreciated. However, protein accounts for 5% 10% of total substrate oxidation, which can be increased as a function of exercise intensity [17] or in the glycogen depleted state [18]. To fully acknowledge the role of protein and amino acid supplementation on endurance performance, research from both continuous endurance exercise (i.e., running and cycling) and multiple sprint sports (i.e., football and rugby) should be considered. Both of these prolonged types of activity rely predominantly on aerobic metabolism [19]. One approach has been to add a small amount of whole protein (typically 7 20 g) to a preestablished ergogenic carbohydrate solution. For example, Ivy et al. [20] was one of the first to show that ingestion of a 200 mL bolus of 7.75% liquid carbohydrate plus 1.94% protein supplement (12 g/kg) was sufficient to increase endurance capacity by 36% compared to carbohydrate alone. These findings have been mirrored by Saunders et al. [21], where a 29% increase in cycling time to exhaustion following ingestion of 9 g/kg of whey protein plus carbohydrate vs carbohydrate alone. Owing to the nonisocaloric supplements provided in these studies, it is unclear whether these findings are the result of additional caloric value or the protein supplement alone. In contrast to those studies describing an ergogenic effect of protein supplements with suboptimal carbohydrate delivery, when delivery of carbohydrate has been at least 60 g/h, the addition of protein has provided no ergogenic advantage during either time-to-exhaustion [22] or time-trial performance tests [23]. Thus, there is essentially no evidence to support the use of protein supplements to enhance endurance exercise performance when carbohydrate delivery has been optimal and no evidence to support a direct effect of protein supplements on muscle glycogen utilization or muscle metabolism during exercise. Notwithstanding these limitations, the evidence shows that a coingested protein supplement is capable of providing energy to facilitate energy demanding processes, such as prolonged muscle contractions during endurance exercise, but the magnitude of this response depends upon certain factors, such as the fed state of the athlete and willingness to feed high amounts of carbohydrate. As well as possessing a glycogen sparing role, amino acids are known to be potent stimulators of insulin. Indeed, greater postexercise insulin responses have been infrequently observed with carbohydrate and protein intake [24 26]. If protein supplementation increased the insulin response, this might augment glucose uptake into the muscle during transient rest periods, such as those observed during intermittent sprint sport activity [27]. Therefore, ingestion of protein could facilitate the delivery of high-energy substrates (carbohydrate), via its insulinotropic mechanisms, to fuel high-intensity exercise and, potentially, increase glycogen resynthesis rates. However, others have reported no change in the insulin response after carbohydrate and protein ingestion [20,28 30]. Similarly, insulin responses are not acutely increased during multiple sprint sport exercise [31]. These inconsistent findings question the role of insulin after protein supplementation but might be explained by the dose of protein provided or by the type of amino acid composition. For example, van Loon et al. [32] demonstrated that the composition of amino acids can alter the insulin response, with the addition of leucine, phenylalanine, and tyrosine eliciting the most notable responses. Research is ongoing in this area in an attempt to elucidate the ergogenic role of protein-related insulin responses. A separate strategy that has received some attention to improve performance is the ingestion of BCAAs during exercise. This is in contrast to the typical approach of using whole protein sources. Historically, it has been thought that exogenous BCAA supply of II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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7.5 12.0 g during exercise might have a sparing effect on muscle glycogen [33], thus preventing or attenuating the net rate of protein degradation [34]. Furthermore, this has led to the postulation that the BCAA supply during prolonged exercise may reduce central fatigue [35]. The so-called central fatigue hypothesis resides on the commonly observed increase in free fatty acid (FFA) mobilization from adipose tissue during exercise. As the mobilization of FFA in the blood is greater than its rate of utilization, blood FFA concentration increases during exercise [36]. The newly available FFA are understood to compete with free tryptophan (f-TRP) for the binding site on albumin, which normally transports them in the blood, causing parallel increases in circulating f-TRP and alteration of the f-TRP:BCAA ratio. This is problematic because such an imbalance increases the f-TRP transport across the blood brain barrier, which is subsequently converted to serotonin in the brain—a neurotransmitter that has been associated with feelings of tiredness and lethargy and may be a possible mediator of fatigue. One caveat to this mechanism, as suggested by Meeusen et al. [37], is that brain function is not solely determined by a single neurotransmitter. Rather, it was suggested that an increase in the central ratio of serotonin to dopamine is associated with feelings of tiredness and lethargy. It is, therefore, possible that the abovementioned role of serotonin in the development of central fatigue is overestimated. Nevertheless, these processes suggest that BCAA supplements taken before or during prolonged exercise may have beneficial effects on some of the metabolic causes of fatigue, such as glycogen depletion and/or central fatigue.

Adaptations to Endurance Training With Protein and Amino Acid Supplementation Whilst there is limited evidence for acute protein supplementation and its impact on endurance performance, there is more information available for the use of chronic supplementation and its impact in augmenting adaptations to endurance training. There is evidence in support of a preferential stimulus for mitochondrial protein synthesis during the early stages of aerobic training for untrained individuals [38], which appears consistent with faster gains in VO2max or treadmill running time to exhaustion that have been attributed to protein supplementation [39,40]. In addition, mean increases of 20% 50% in mitochondrial enzyme activity, such as cytochrome oxidase, have been reported in as little as 6 10 training sessions [41,42], supporting the potential for rapid changes in protein synthesis to be augmented with supplementation during early phases of training. Despite this, the only study that compared changes in mitochondrial enzyme activity did not demonstrate faster gains during the initial weeks of training with protein supplementation [39]. Moreover, preferential effects of protein supplements on rates of mitochondrial protein synthesis decrease as participants become more adapted to the training stimulus [23]. Nevertheless, greater changes in metrics of aerobic performance such as VO2max, treadmill run time, or rowing time to exhaustion have been reported following protein supplementation for several weeks for both untrained [40] and trained participants [43]. Clearly, additional research is warranted to elucidate the mechanism(s) responsible for improvements in VO2max and other metrics of aerobic performance observed following only 4 6 weeks of protein supplementation.

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Key Points • Acute supplementation with BCAAs may offset fatigue during prolonged endurance exercise. • When adequate carbohydrate is delivered, adding protein to carbohydrate does not appear to improve acute endurance performance. • There are relatively few investigations on the effects of protein supplementation on endurance performance.

RESISTANCE EXERCISE PERFORMANCE Protein turnover is defined as the constant cellular processes of protein synthesis and protein breakdown, controlling the quantity and quality of protein in a biological system. An inequality between muscle protein synthesis and muscle protein breakdown can lead to muscle protein accrual/hypertrophy (e.g., exercise training and nutrition) or muscle loss/atrophy (e.g., sarcopenia, inactivity, malnutrition, and muscle wasting).

Acute Responses to Resistance Exercise With Protein Supplementation To assess the acute muscle protein anabolism (growth) response, researchers have used Amino acids (AAs) as tracers (stable and isotopically labeled), alongside muscle biopsies to measure muscle protein synthesis and muscle protein breakdown in humans in vivo. Both resistance exercise [44] and protein ingestion [45] are known to stimulate muscle protein synthesis, which is necessary for the accretion of skeletal muscle mass. However, following resistance exercise, both muscle protein synthesis and muscle protein breakdown are increased compared with rest, but net balance is less negative [44,46]. Moreover, combining protein or amino acid ingestion with an acute bout of resistance exercise has been shown to further augment muscle protein synthesis, whilst fractional breakdown rate is thought to slightly decrease, presumably due to insulin- and/or amino acid mediated effects, and net protein balance becomes positive [44]. The majority of the literature examining protein metabolism with resistance exercise and protein/amino acid supplementation has been studied in the early postexercise recovery (1 6 hours) period. Less is known regarding the muscle protein synthesis response in the later period (6 24 hours), although it has been shown that a single bout of resistance exercise improves the muscle protein synthesis response in the morning 24 hours postexercise [47,48]. Regardless of when muscle protein synthesis is elevated following exercise, there is very little understanding of the translational relevance of these changes in protein turnover in relation to the chronic adaptations observed following exercise training. The acute physiologic responses to exercise and protein supplementation have been heavily researched, mainly to help gain a mechanistic insight into the drivers of adaptations to training. However, recent findings question the relevance of these types of studies in relation to chronic adaptations [49,50].

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Chronic Adaptations to Resistance Exercise With Protein Supplementation Resistance exercise, when repeated over a period of time, can stimulate a range of health benefits, such as improving body composition and neuroendocrine and cardiovascular function and increasing muscle size and strength [51]. A prodigious amount of investigation has been directed toward understanding these adaptations and determining if an enhancement effect occurs with protein and/or amino acid supplementation. It’s common practice for many strength athletes and bodybuilders to consume protein supplements around and even during training—but is this warranted? The early adaptations observed following resistance exercise are mainly neural, which raises some doubts about the role of protein supplementation with untrained individuals. Moreover, when the confounding influence of neural adaptations during the early training period are removed, no effect of protein supplementation on measures of muscle strength is observed [52].It has been suggested that a training stimulus of at least 6 8 weeks in duration with appropriate progressions in intensity, frequency, and duration is necessary before measurable changes in phenotype and muscle function occur and reflect altered rates of protein signaling and expression; however, these effects are often marginal [53]. Progressive, periodized resistance training, consisting of exercises for all major muscle groups performed for 6 weeks, in combination with protein supplementation resulted in greater gains in muscle strength and lean muscle mass compared to placebo, in men and women [54]. Furthermore, in trained participants, changes in muscle cross sectional area and muscle contractile protein that were related to gains in muscle strength were observed after 8 11 weeks of supplementation that involved resistance training 3 4 times per week [55,56]. Also observed are greater increases in muscle fiber area [5,57] and myofibrillar protein content [58] following resistance training with protein supplementation rather than carbohydrate. In contrast, other studies in young men have shown no effect of protein on gains in muscle size [59,60] or strength [52,57,60]. This may be due to the measurement of strength not necessarily reflecting the functional gains in lean mass. Furthermore, the wide range of protein supplements used (whey, casein, BCAAs) may impact the adaptations observed. A recent metaanalysis [61] of 49 studies, including 1863 participants, demonstrated that dietary protein supplementation increased changes in strength (1RM), fat-free mass (FFM), and muscle cross sectional area during periods of prolonged ( . 6 weeks) resistance exercise. The impact of protein supplementation on gains in FFM was reduced with increasing age and was more effective in resistance-trained individuals. However, protein supplementation beyond total protein intakes of 1.62 g/kg day resulted in no further resistance training induced gains in FFM. In agreement with this finding, protein supplementation during resistance training was found to not enhance resistance exercise-induced increases in myofiber hypertrophy, satellite cell content, or myonuclear addition in young healthy men, when protein intake is adequate during muscle overload [62,63].

Key Points • Protein supplementation alongside resistance training may not augment adaptations in untrained individuals due to focus on neural adaptations.

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• A training stimulus of 6 1 weeks may be necessary to observe adaptations to skeletal muscle hypertrophy and muscle strength following protein supplementation with resistance training. • Resistance exercise-induced adaptations may not be augmented when protein intake is adequate during muscle overload

RECOVERY FROM EXERCISE Interventions that help to attenuate the effects of muscle damage are beneficial to the athlete by reducing the decline in physical function and permitting greater engagement with training in the days following exercise [64 66]. Furthermore, interventions that lower the perception of fatigue and delayed onset muscle soreness (DOMS) or maintain the intracellular anabolic environment would also support the training or adaptation process. Whole protein supplementation has long been considered as a potential candidate recovery agent, despite the collective equivocal findings. Milk or milk derivatives, such as whey isolate or hydrolysate have typically been chosen to augment recovery from different forms of exercise, but it is not clear if these forms of protein offer sufficient nutritional content to for full recovery or whether they can be ingested at suitable quantities throughout the day. For example, Baty et al. [67] administered 13.5 g of a milk-based protein before, during, and after performing three sets of eight repetitions at eight repetition max. They reported no benefit of supplementation on recovery of strength, but they did find reductions in plasma creatine kinase (CK) and myoglobin (Mb). Gilson et al. [68] supplemented subjects with a similar dose (0.3 g/kg/h) of chocolate milk over 4 days of intensified training and found no change in muscle function but did report lower CK and Mb. Indeed, to the best of our knowledge, only Cockburn et al. [69,70] or Rankin et al. [71] have reported improvements in muscle function and reduced indirect muscle damage markers (CK and Mb flux) after providing milk products at similar rates (500 mL milk) to recover form muscle damaging exercise. These findings question the efficacy of some whole proteins to facilitate full recovery from exercise but demonstrate the capacity to partly block cellular damage. BCAAs, which include leucine, isoleucine, and valine, are commonly used in isolation, or in combination, as prophylactic interventions to attenuate symptoms of muscle damage [72]. Supplementation of leucine, one of the most researched BCAAs, has also been suggested to suppress muscle proteolysis [73] and reduce protein oxidation [74] after muscle-damaging exercise, thus helping to maintain the integrity of the muscle cell membrane. This is relevant because skeletal muscle proteins, such as CK, lactate dehydrogenase, or Mb, are known to exit the cell and indirectly infer cellular damage and act as surrogate markers of muscle damage. The appearance of indirect markers of muscle damage, such as CK, has been reduced by both short- [75 77] and long-term amino acid supplementation (loading) periods [78 80]. For example, Kirby et al. [77] reported significant reductions in serum Mb and CK concentration 24 hours after eccentrically biased exercise with 250 mg/kg body mass of leucine 30 minutes before, during, and immediately postexercise and the morning of each recovery day. Similarly, Howatson et al. [79] provided participants with 10 g of

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BCAA (2:1:1 leucine, isoleucine, and valine) twice per day after eccentric muscle damage and reported a significant reduction CK efflux at 24-hour postexercise. However, not all studies have reported this [81,82], which might relate to the well-established intersubject variability of CK activity postexercise [83]. It is also possible that leucine alone is more effective than mixed BCAA solutions for cellular recovery, owing to the reported competition between leucine, isoleucine, and valine for cellular transport and subsequent metabolism [84]. Therefore, it is possible that valine and isoleucine can inhibit the effects of leucine on muscle protein synthesis, and that the combination of all three BCAAs is unnecessary or detrimental to muscle recovery [85]. The attenuation in CK efflux after BCAA supplementation has been linked to a reduction in secondary damage, caused by the inflammatory response [65]. That is, the derangement of intracellular Ca21 homeostasis, caused by the insult of heavy resistance exercise, initiates a series of intracellular events that lead to the activation of proteolytic and lipolytic pathways, thus damaging the cell membrane [86]. The dampening of acute inflammatory responses might also explain the commonly reported reductions in DOMS following mixed amino acid [76], isolated leucine [77], or mixed BCAA supplementation [75,79,82]. Reductions in DOMS and cell damage might, in turn, explain the accelerated recovery of muscle function after leucine or mixed BCAA supplementation [77,79]. Administration of leucine-rich amino acids has also been shown to reduce the appearance of inflammatory cytokines, whilst increasing muscle protein synthesis after eccentric exercise in rodents [87] and after endurance exercise in athletes [88]. Rowlands et al. [88] provided 15 g of leucine to athletes as part of a balanced macronutrient recovery meal. The authors demonstrated decreased leukocyte migration and connective tissue development, indicating the acute antiinflammatory and proteinogenic properties of leucine rich supplementation. Muscle soreness is partly related to local inflammation, whereby local swelling acts to sensitize nociceptors (pain receptors) located in the muscle [66]. Inflammation is a necessary part of the recovery process that follows acute mechanical damage of the myofibers [65]. The proposed antiinflammatory effects of leucine postexercise might explain its capacity to lower muscle soreness. However, there are putative roles for all BCAAs during the acute inflammatory phase of muscle damage. This is because of the known transamination of all BCAA into glutamate and, thus, contribution to the glutamate-glutamine pool, which is a known substrate for inflammatory cells [89]. Indeed, given the role of glutamine as an inhibitor of leucine oxidation [90], it is possible that optimal combinations of leucine and glutamine would ameliorate recovery through antiinflammatory processes. It is, therefore, important that future research is designed to examine the effects of BCAAs, most notably leucine, in combination with other antiinflammatory agents on the prolonged (i.e., days) recovery from exercise. The accelerated return to baseline physical function among those supplementing BCAAs or isolated leucine can be explained by the combined reduction in muscle soreness and protection and/or regeneration of the passive structural and contractile elements in the muscle. In addition, powerful multijoint activities, such as a counter-movement jump, have been shown to improve after supplementation of BCAAs at 0.087 g/kg body mass (2:1:1 leucine, isoleucine, and valine) twice daily [82] (see Fig. 13.1). It is known that movements involving the stretch-shortening cycle (SSC) can be reduced by muscle damage, partly owing to structural changes in noncontractile elements and an associated loss of II. PATHOPHYSIOLOGY OF SKELETAL MUSCLE: THE IMPORTANT ROLE OF DIET AND NUTRIENTS

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FIGURE 13.1

Changes in isometric strength, perceived soreness (DOMS), countermovement jump (CMJ) and creatine kinase (CK) from % baseline, after muscle damage for placebo (black lines) and BCAA (Blue lines) groups. Adapted from Waldron et al. [82].

100 90 80 600 300 100

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250 175 100 Baseline

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muscle-tendon stiffness [91]. In vitro studies have shown that leucine administration can promote the restoration of damaged connective tissue in rat skeletal muscle [92], which is responsible for the transfer of energy between the muscle and tendon structures [93]. Therefore, it is feasible that the repair of damaged connective tissue can be facilitated by BCAA supplementation, thus supporting energy transfer during SSC movements. There are other mechanisms, such as impairment of reflex sensitivity [91] that might explain the poorer countermovement jump (CMJ) performance postmuscle damage. The lowered DOMS after BCAA supplementation is likely to cause less neural inhibition, thus enabling improved reflex sensitivity and performance [94].

Key Points • Whole proteins, such as milk-based beverages, can help to attenuate symptoms of muscle damage but their effects are inconsistent. • Supplementing either mixed BCAA solutions or isolated leucine in doses ranging between 5 and 20 g/day, typically multiplied across 2 3 feedings, appear to accelerate recovery from muscle damaging exercise. • These improvements are, in most cases, characterized by improved perception of muscle soreness, increased muscle function, and decreased leakage of intracellular proteins into the blood. • There have been no reported detrimental effects of BCAA ingestion in any of the relevant studies.

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[48] Fry CS, Drummond MJ, Glynn EL, Dickinson JM, Gundermann DM, Timmerman KL, et al. Skeletal muscle autophagy and protein breakdown following resistance exercise are similar in younger and older adults. J Gerontol A Biol Sci Med Sci 2013;68(5):599 607. [49] Mitchell CJ, Churchward-Venne TA, Parise G, Bellamy L, Baker SK, Smith K, et al. Acute post-exercise myofibrillar protein synthesis is not correlated with resistance training-induced muscle hypertrophy in young men. PLoS ONE 2014;9(2):e89431. [50] Mitchell CJ, Churchward-Venne TA, Cameron-Smith D, Phillips SM. Last word on viewpoint: what is the relationship between the acute muscle protein synthetic response and changes in muscle mass? J Appl Physiol 1985;118(4):503 2015 Feb 15. [51] Kraemer WJ, Deschenes MR, Fleck SJ. Physiological adaptations to resistance exercise. Implications for athletic conditioning. Sports Med 1988;6(4):246 56. [52] Erskine RM, Fletcher G, Hanson B, Folland JP. Whey protein does not enhance the adaptations to elbow flexor resistance training. Med Sci Sports Exerc 2012;44(9):1791 800. [53] Hulmi JJ, Kovanen V, Lisko I, Selanne H, Mero AA. The effects of whey protein on myostatin and cell cyclerelated gene expression responses to a single heavy resistance exercise bout in trained older men. Eur J Appl Physiol 2008;102(2):205 13. [54] Candow DG, Burke NC, Smith-Palmer T, Burke DG. Effect of whey and soy protein supplementation combined with resistance training in young adults. Int J Sport Nutr Exerc Metab 2006;16(3):233 44. [55] Cribb PJ, Williams AD, Stathis CG, Carey MF, Hayes A. Effects of whey isolate, creatine, and resistance training on muscle hypertrophy. Med Sci Sports Exerc 2007;39(2):298 307. [56] Joy JM, Lowery RP, Wilson JM, Purpura M, De Souza EO, Wilson SM, et al. The effects of 8 weeks of whey or rice protein supplementation on body composition and exercise performance. Nutr J 2013;12:86 2891-12-86. [57] Andersen LL, Tufekovic G, Zebis MK, Crameri RM, Verlaan G, Kjaer M, et al. The effect of resistance training combined with timed ingestion of protein on muscle fiber size and muscle strength. Metabolism 2005;54 (2):151 6. [58] Willoughby DS, Stout JR, Wilborn CD. Effects of resistance training and protein plus amino acid supplementation on muscle anabolism, mass, and strength. Amino Acids 2007;32(4):467 77. [59] Coburn JW, Housh DJ, Housh TJ, Malek MH, Beck TW, Cramer JT, et al. Effects of leucine and whey protein supplementation during eight weeks of unilateral resistance training. J Strength Cond Res 2006;20(2):284 91. [60] Lemon PW, Tarnopolsky MA, MacDougall JD, Atkinson SA. Protein requirements and muscle mass/strength changes during intensive training in novice bodybuilders. J Appl Physiol 1985;73(2):767 75 1992 Aug. [61] Morton RW, Murphy KT, McKellar SR, Schoenfeld BJ, Henselmans M, Helms E, et al. A systematic review, meta-analysis and meta-regression of the effect of protein supplementation on resistance training-induced gains in muscle mass and strength in healthy adults. Br J Sports Med 2017;52(6):376 84. [62] Reidy PT, Fry CS, Igbinigie S, Deer RR, Jennings K, Cope MB, et al. Protein supplementation does not affect myogenic adaptations to resistance training. Med Sci Sports Exerc 2017;49(6):1197 208. [63] Reidy PT, Borack MS, Markofski MM, Dickinson JM, Deer RR, Husaini SH, et al. Protein supplementation has minimal effects on muscle adaptations during resistance exercise training in young men: a double-blind randomized clinical trial. J Nutr 2016;146(9):1660 9. [64] Cheung K, Hume P, Maxwell L. Delayed onset muscle soreness: treatment strategies and performance factors. Sports Med 2003;33(2):145 64. [65] Howatson G, van Someren KA. The prevention and treatment of exercise-induced muscle damage. Sports Med 2008;38(6):483 503. [66] Proske U, Morgan DL. Muscle damage from eccentric exercise: mechanism, mechanical signs, adaptation and clinical applications. J Physiol 2001;537(Pt 2):333 45. [67] Baty JJ, Hwang H, Ding Z, Bernard JR, Wang B, Kwon B, et al. The effect of a carbohydrate and protein supplement on resistance exercise performance, hormonal response, and muscle damage. J Strength Cond Res 2007;21(2):321 9. [68] Gilson SF, Saunders MJ, Moran CW, Moore RW, Womack CJ, Todd MK. Effects of chocolate milk consumption on markers of muscle recovery following soccer training: a randomized cross-over study. J Int Soc Sports Nutr 2010;7:19 2783-7-19. [69] Cockburn E, Hayes PR, French DN, Stevenson E, St Clair Gibson A. Acute milk-based protein-CHO supplementation attenuates exercise-induced muscle damage. Appl Physiol Nutr Metab 2008;33(4):775 83.

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[70] Cockburn E, Robson-Ansley P, Hayes PR, Stevenson E. Effect of volume of milk consumed on the attenuation of exercise-induced muscle damage. Eur J Appl Physiol 2012;112(9):3187 94. [71] Rankin P, Stevenson E, Cockburn E. The effect of milk on the attenuation of exercise-induced muscle damage in males and females. Eur J Appl Physiol 2015;115(6):1245 61. [72] da Luz CR, Nicastro H, Zanchi NE, Chaves DF, Lancha Jr. AH. Potential therapeutic effects of branchedchain amino acids supplementation on resistance exercise-based muscle damage in humans. J Int Soc Sports Nutr 2011;8:23 2783-8-23. [73] Zanchi NE, Nicastro H, Lancha Jr. AH. Potential antiproteolytic effects of L-leucine: observations of in vitro and in vivo studies. Nutr Metab (Lond) 2008;5:20 7075-5-20. [74] Shimomura Y, Kobayashi H, Mawatari K, Akita K, Inaguma A, Watanabe S, et al. Effects of squat exercise and branched-chain amino acid supplementation on plasma free amino acid concentrations in young women. J Nutr Sci Vitaminol (Tokyo) 2009;55(3):288 91. [75] Shimomura Y, Inaguma A, Watanabe S, Yamamoto Y, Muramatsu Y, Bajotto G, et al. Branched-chain amino acid supplementation before squat exercise and delayed-onset muscle soreness. Int J Sport Nutr Exerc Metab 2010;20(3):236 44. [76] Nosaka K, Sacco P, Mawatari K. Effects of amino acid supplementation on muscle soreness and damage. Int J Sport Nutr Exerc Metab 2006;16(6):620 35. [77] Kirby TJ, Triplett NT, Haines TL, Skinner JW, Fairbrother KR, McBride JM. Effect of leucine supplementation on indices of muscle damage following drop jumps and resistance exercise. Amino Acids 2012;42 (5):1987 96. [78] Coombes JS, McNaughton LR. Effects of branched-chain amino acid supplementation on serum creatine kinase and lactate dehydrogenase after prolonged exercise. J Sports Med Phys Fitness 2000;40(3):240 6. [79] Howatson G, Hoad M, Goodall S, Tallent J, Bell PG, French DN. Exercise-induced muscle damage is reduced in resistance-trained males by branched chain amino acids: a randomized, double-blind, placebo controlled study. J Int Soc Sports Nutr 2012;9:20 -2783-9-20. eCollection 2012. [80] Sharp CP, Pearson DR. Amino acid supplements and recovery from high-intensity resistance training. J Strength Cond Res 2010;24(4):1125 30. [81] Jackman SR, Witard OC, Jeukendrup AE, Tipton KD. Branched-chain amino acid ingestion can ameliorate soreness from eccentric exercise. Med Sci Sports Exerc 2010;42(5):962 70. [82] Waldron M, Whelan K, Jeffries O, Burt D, Howe L, Patterson SD. The effects of acute branched-chain amino acid supplementation on recovery from a single bout of hypertrophy exercise in resistance-trained athletes. Appl Physiol Nutr Metab 2017;42(6):630 6. [83] Clarkson PM, Ebbeling C. Investigation of serum creatine kinase variability after muscle-damaging exercise. Clin Sci (Lond) 1988;75(3):257 61. [84] Cynober LA. Plasma amino acid levels with a note on membrane transport: characteristics, regulation, and metabolic significance. Nutrition 2002;18(9):761 6. [85] De Bandt JP, Cynober L. Therapeutic use of branched-chain amino acids in burn, trauma, and sepsis. J Nutr 2006;136(1 Suppl):308S 313SS. [86] Gissel H, Clausen T. Excitation-induced Ca21 influx and skeletal muscle cell damage. Acta Physiol Scand 2001;171(3):327 34. [87] Kato H, Miura K, Nakano S, Suzuki K, Bannai M, Inoue Y. Leucine-enriched essential amino acids attenuate inflammation in rat muscle and enhance muscle repair after eccentric contraction. Amino Acids 2016;48 (9):2145 55. [88] Rowlands DS, Nelson AR, Raymond F, Metairon S, Mansourian R, Clarke J, et al. Protein-leucine ingestion activates a regenerative inflammo-myogenic transcriptome in skeletal muscle following intense endurance exercise. Physiol Genomics 2016;48(1):21 32. [89] Nicastro H, da Luz CR, Chaves DF, Bechara LR, Voltarelli VA, Rogero MM, et al. Does branched-chain amino acids supplementation modulate skeletal muscle remodeling through inflammation modulation? Possible mechanisms of action. J Nutr Metab 2012;136937 2012. [90] Holecek M, Sprongl L, Tilser I, Tichy M. Leucine and protein metabolism after bilateral nephrectomy in rats: the role of hepatic tissue. Res Exp Med (Berl) 2000;200(1):53 65. [91] Komi PV. Stretch-shortening cycle: a powerful model to study normal and fatigued muscle. J Biomech 2000;33(10):1197 206.

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[92] Pereira MG, Silva MT, Carlassara EO, Goncalves DA, Abrahamsohn PA, Kettelhut IC, et al. Leucine supplementation accelerates connective tissue repair of injured tibialis anterior muscle. Nutrients 2014;6 (10):3981 4001. [93] Turrina A, Martinez-Gonzalez MA, Stecco C. The muscular force transmission system: role of the intramuscular connective tissue. J Bodyw Mov Ther 2013;17(1):95 102. [94] Nicol C, Kuitunen S, Kyrolainen H, Avela J, Komi PV. Effects of long- and short-term fatiguing stretchshortening cycle exercises on reflex EMG and force of the tendon-muscle complex. Eur J Appl Physiol 2003;90(5-6):470 9.

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Physical Exercise in Chronic Diseases Bente K. Pedersen The Centre of Inflammation and Metabolism and The Centre for Physical Activity Research, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

INTRODUCTION This chapter presents an up-date of previously published reviews “Evidence for prescribing exercise as therapy in chronic disease” from 2006 [1] and “Exercise as medicine
evidence for prescribing exercise as therapy in 26 different chronic diseases” [2]. Physical activity represents a cornerstone in the primary prevention of at least 35 chronic conditions [3]. However, over the past two decades considerable knowledge has accumulated concerning the significance of exercise as the first line treatment of several chronic diseases. It is interesting that regular muscle work has impact on many chronic diseases with very different phenotypical presentation. However, the identification of skeletal muscle as a secretory organ has created a new paradigm: muscles produce and release myokines, which work in a hormone-like fashion and exert specific endocrine effects on distant organs [4,5]. Some myokines are thought to induce direct antiinflammatory responses with each bout of exercise and mediate long-term exercise-induced improvements in metabolic and cardiovascular risk factors, having an indirect antiinflammatory effect [6
8]. Therefore, contrary to fears that physical activity might aggravate inflammatory pathways, exercise has now been accepted as a potential treatment for patients with chronic diseases and disorders, which are linked with low-grade chronic inflammation. Here, I provide the reader with the evidence-based basis for prescribing exercise as medicine for adiposity, hyperlipidemia, metabolic syndrome, type 2 diabetes, type 1 diabetes, hypertension, coronary heart disease (CHD), heart failure, cerebral apoplexy, intermittent claudication, chronic obstructive pulmonary disease (COPD), asthma, and cancer.

METHODS A comprehensive literature search was carried out for each diagnosis in the Cochrane Library and MEDLINE databases (search terms: exercise therapy, training, physical fitness,

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physical activity, rehabilitation, and aerobic). I have primarily identified systematic reviews and metaanalyses and thereafter identified additional controlled trials.

METABOLIC DISEASES Obesity Background Several studies show a U-shaped association between BMI and mortality, which means that both low and high BMI are associated with increased risk of premature death. The risk associated with low BMI is associated with decreased lean body mass and not reduced fat mass [9]. A review from 2014 [10] attempted to quantify the joint association of cardiorespiratory fitness (CRF) and weight status on mortality from all causes using metaanalytical methodology. Ten articles were included in the final analysis, and pooled hazard ratios were assessed for each comparison group (i.e., normal weight-unfit, overweight-unfit and -fit, and obese-unfit and -fit) using a random-effects model. Compared to normal weight-fit individuals, unfit individuals had twice the risk of mortality regardless of BMI. It appears that overweight and obese-fit individuals have similar mortality risks as normal weight-fit individuals. Evidence-Based Physical Training The importance of physical activity for weight loss assessed by body weight or BMI is controversial, but physical training leads to a reduction in fat mass and abdominal obesity, in addition to counteracting loss of muscle mass during dieting. Strong evidence exists that physical activity is important for preventing weight gain in general, as well as for maintaining body weight after weight loss. A Cochrane Review from 2006 [11] comprising 3476 overweight or obese individuals studied 41 randomized controlled trials and concluded that physical activity alone induced significant weight loss, while physical activity combined with a restricted diet and dietary counseling was more effective. High-intensity physical activity was more effective than moderate activity. The authors defined physical training as “any form of physical exercise that is repeated regularly for a certain period of time.” A prerequisite was that the physical training had to be quantifiable. The physical training intervention mainly consisted of walking, using an exercise bike, jogging, and weight training. In most of the studies, the intensity of the training was greater than 60% of the maximum oxygen uptake/heart rate. The participants exercised most frequently for 40
50 minutes per session, three to five times a week. All of the studies showed that physical exercise induced a slight reduction in body weight and BMI. The combination of exercise and diet resulted in an average greater weight loss [difference: 1.0 kg, 95% confidence interval (CI) 0.7
1.3 kg, n 5 2157] and a greater decrease in BMI (difference: 0.4 kg/m2, 95% CI 0.1
0.7 kg/m2, n 5 452) than diet alone. Without diet, high-intensity physical training (B60% of the maximum oxygen uptake/pulse) led to greater weight loss (difference: 1.5 kg, 95% CI 0.7
2.3 kg, n 5 317) than low-intensity physical training.

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The Cochrane Review showed that physical training for overweight and obese adults had positive effects on both body weight and risk factors for cardiovascular disease. Physical training combined with a restricted diet/dietary counseling reduces body weight slightly but significantly more than a restricted diet/dietary counseling only. Studies with physical training without dietary change showed that high-intensity physical training reduced body weight more than low-intensity physical training. These results are consistent with other metaanalyses [12,13]. A literature review of 26 articles [14] assessed the independent effects of normal weight versus obesity: fit versus unfit and physically active versus physically inactive. The risk of all-cause mortality and cardiovascular death was lower in individuals with high BMI who were physically fit compared to individuals with normal BMI and a lower level of physical fitness. The literature review, however, could not confirm results from other studies that showed that a high level of physical activity gave the same protection as being physically fit. Individuals with a high BMI and a high level of physical activity had a greater risk of developing type 2 diabetes and cardiovascular disease than those with a normal BMI and low level of physical activity. There are many possible explanations as to why physical fitness and not a high level of physical activity protect against the serious health consequences of overweight and obesity. Information on physical activity in most studies is based on self-reported information, which is subject to considerable inaccuracy, while fitness is an objective measure. Another possible explanation is that primarily physical activity of high intensity leads to improved fitness, and thereby protection against diseases is associated with obesity. Obesity is often associated with hypertension, hypercholesterolemia, hypertriglyceridemia, and insulin resistance. The effect of physical training on these risk markers is described below. Obesity is also frequently associated with erectile dysfunction, which physical training can contribute to prevent [15,16]. Possible Mechanisms Physical training increases energy expenditure and induces lipolysis, whereupon the fat mass is reduced, if the energy expended is not compensated for with an increase in caloric intake. Type of Training For the purpose of weight loss, a large volume of moderately intense aerobic exercise is recommended, preferably in combination with strength training. Because physical fitness has an independent impact on preventing diseases associated with obesity, it is recommended that moderate physical activity be combined with activities that build fitness in the form of high-intensity physical activity. The goal is at least 60 minutes of moderately intense physical activity daily. Many overweight and obese patients have, however, concomitant hypertension or symptomatic ischemic cardiovascular disease. As a result, recommendations must be individualized. There are no general contraindications; however, training should take into account any competing diseases. With ischemic heart disease, brief rigorously intensive workouts should be refrained from. With hypertension, strength training should be performed with light weights and low contraction velocity.

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Hyperlipidemia Background Hyperlipidemia is a group of disorders of lipoprotein metabolism entailing elevated blood levels of certain forms of cholesterol and triglyceride. Primary hyperlipidemia caused by environmental and genetic factors are by far the most frequent, accounting for 98% of all cases. Isolated hypercholesterolemia and combined dyslipidemia are the most frequent types of dyslipidemia and are due to excessive intake of fat in most people. These types of dyslipidemia entail an elevated risk of atherosclerosis. There is consensus that physical activity protects against the development of cardiovascular diseases [17,18], and it has been suggested that one of many mechanisms could be a positive effect of exercise on the lipid profile of the blood [19,20]. Epidemiological studies indicate that physical activity prevents hyperlipidemia [21,22]. Evidence-Based Physical Training Today, evidence shows that a large volume of physical training, independent of weight loss, has a beneficial effect on the lipid profile of the blood. A number of review articles summarize this knowledge [20
36]. A 2007 metaanalysis studied the effect of training on high-density lipoprotein (HDL) cholesterol. The analysis included 25 randomized controlled trials. The training comprised walking, cycling, or swimming [37]. Training had a significant but moderate effect on HDL cholesterol. The minimum amount of physical activity needed to cause an effect was 120 minutes of physical activity weekly or an energy expenditure equivalent to 3780 kJ. The duration of the physical activity was more important than its intensity. Each time the duration of the physical activity was increased by 10 minutes, the HDL cholesterol level increased on average by 1.4 mg/dL (0.036 mmol/L). The average effect of physical activity on HDL is clinically relevant, albeit somewhat smaller than the effect achieved when using drugs that lower lipid levels [38]. It is estimated that each time HDL increases 0.025 mmol/L, the cardiovascular risk goes down by 2% for men and by at least 3% for women [39,40]. Training induced a mean increase of 0.036 mmol/L in the level of HDL. For the subgroup of individuals with a BMI of less than 28 and a total cholesterol level over 5.7 mmol/L, it was found that exercise induced an increase of 0.054 mmol/L in the level of HDL [41]. For the latter group, physical training was thus able to reduce the cardiovascular risk by about 4% for men and by 6% for women. A review article from 2014 [36] includes 13 published investigations and two review articles and conclude that both aerobic, resistance exercise and the combination of aerobic and resistance training have impact on cholesterol levels and blood lipids. A randomized clinical controlled trial evaluated the effect of training volume and intensity in a study comprising 111 physically inactive overweight men with mild-to-moderate hyperlipidemia [41]. The subjects were randomized to a control group or 8 months of high volume/high-intensity physical training [32 km/week at 65%
80% maximum oxygen uptake (VO2max)]; low volume/high intensity [19 km/week at 65%
80% of VO2max], or low volume/low intensity (19 km/week at 40%
55% of VO2max). This study distinguishes

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itself by evaluating an extensive lipid profile in which the size of the lipid-protein particles is also included. Subjects were asked to maintain their weight, and individuals with excessive weight loss were excluded. Despite this, there was a small but significant amount of weight loss in the training groups. All of the training groups achieved a positive effect on their lipid profile compared to the control group, but there was no significant difference in the effect of training in the two groups with a low volume of exercise, despite the fact that the high intensity group achieved a greater improvement in fitness. There is a significantly better effect from a high volume of physical training on virtually all lipid parameters, despite the fact that the two groups with high intensity training achieved the same improvement in fitness level. There was no effect on the total cholesterol level. High volume/high intensity training reduced the level of low-density lipoprotein (LDL), intermediate-density lipoproteins (IDL), and small LDL particles and increased the size of the LDL particles and the level of HDL. All of the groups had a positive effect on the level of triglyceride, Very-low-density lipoprotein (VLDL) triglyceride, and the size of VLDL. Thus, the volume of training had clear effects, but the intensity of the training had less impact. A 2010 metaanalysis compared 13 randomized controlled trials that examined the effect of resistance training on parameters related to metabolic syndrome. Resistance training showed a significant effect on obesity, hemoglobin A1c (HbA1c), and systolic blood pressure (BP) (SBP), but no effect on total cholesterol, HDL cholesterol, or LDL cholesterol [42]. A 2012 systematic review [43] assessed the effect of supervised exercise interventions on lipid profiles and BP control in patients with type 2 diabetes. Forty-two randomized controlled study (RCTs) (2808 subjects) met inclusion criteria and were included in the metaanalysis. It was concluded that supervised exercise is effective in improving BP control, lowering LDL-C, and elevating HDL-C levels in people with diabetes. Possible Mechanisms Training increases the ability of muscles to better burn fat instead of glycogen. This is achieved by activation of a number of enzymes in the skeletal muscle necessary for lipid turnover [44]. Type of Training There is solid evidence that physical training should be of a large volume, assessed as the distance covered or energy expended. There is evidence for an effect of both aerobic training and resistance training. If light to moderately intense physical activity is preferred, then it is necessary to train twice as long compared to doing high-intensity physical activity. Many patients with hyperlipidemia have hypertension or symptomatic ischemic heart disease. Recommendations should thus be largely tailored to the individual. Treatment should follow the general recommendations for physical activity for adults, but it is recommended that the volume be increased, for example, to 60 minutes of moderately intense physical activity daily most days of the week. Alternatively, it is possible to increase the intensity and halve the time or to alternate. According to the previously

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mentioned dose
response study [41], it is advantageous to walk or run at least 20 km a week, preferably 30, in order to control one’s cholesterol level with physical activity. There are no general contraindications.

Metabolic Syndrome Background Metabolic syndrome is also known as insulin resistance syndrome, since one of the traits of the disorder is reduced insulin activity. There are several definitions for metabolic syndrome, but it encompasses abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. The International Diabetes Federation [45] defines metabolic syndrome as follows: Abdominal obesity, i.e., waist circumference . 94 cm for men and . 80 cm for women, plus at least two of the following four risk factors: Plasma concentration of triglycerides

$ 1.7 mmol/L

Plasma concentration of HDL cholesterol

,1.0 mmol/L for men and ,1.2 mmol/L for women

Blood pressure

Systolic blood pressure $ 130 mmHg or diastolic blood pressure $ 85 mmHg or receiving antihypersensitive therapy

Plasma concentration of glucose (fasting)

$ 5.6 mmol/L or type 2 diabetes

Metabolic syndrome rarely occurs in people with normal weight but it can occur, and there is a higher incidence among members of the Pakistani and Turkish ethnic minorities than members of the general population with the same BMI. Metabolic syndrome is a precursor of type 2 diabetes, and large-scale epidemiological studies show that physical activity can prevent the onset of metabolic syndrome [46,47]. Evidence-Based Physical Exercise PHYSICAL EXERCISE AND THE METABOLIC SYNDROME

A metaanalysis from 2013 [48] examined the effect of physical training on individual parameters of the metabolic syndrome. The analysis included seven randomized controlled studies. Exercise induced significant decreases on waist and systolic/diastolic blood and as well as an increase in HDL-cholesterol. Moreover, there was a significant improvement in fitness level. PHYSICAL EXERCISE AND INSULIN RESISTANCE/PREVENTION OF TYPE 2 DIABETES

En metaanalysis from 2015 [49] finds strong evidence for an effect of physical training on insulin resistance and glucose control. High intense interval training (INT) is more efficient than continuous training. A 2008 Cochrane Review [50] assessed the effect of a combination of diet and physical exercise as prophylaxis against type 2 diabetes. Physical exercise varied from a recommended increase in daily physical activity to supervised

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physical training of varying intensity and up to several times a week. Most programs included walking, running, or cycling at different intensities. The diets were low calorie with reduced fat and high fiber. The participants in the analysis had a pathological glucose tolerance and/or metabolic syndrome. The analysis included eight trials with 2241 participants in one group, who were prescribed physical activity and placed on a diet as described above, and 2509 control persons. The studies ran over a period of 1 and 6 years. Exercise and diet significantly lowered the risk of type 2 diabetes [risk ratio (RR) 0.63, 95% CI 0.49
0.79]. A significant impact on body weight, BMI, waist-to-hip ratio, and waist circumference was also identified, as was a moderate impact on blood lipids. The intervention had a marked effect on both SBP and diastolic BP (DBP) [50]. The isolated effect of exercise alone as prevention against diabetes in patients with pathological glucose tolerance is sparsely documented, but there is solid evidence pointing to the effect of combined physical exercise and diet. A Chinese study divided 577 people with pathological glucose tolerance into four groups, diet, exercise, diet 1 exercise, and control, and monitored them over 6 years [51]. The risk of diabetes fell by 31% (P , 0.03) in the diet group, by 46% (P , 0.0005) in the exercise group, and by 42% (P , 0.005) in the diet 1 exercise group. In a Swedish study, 6956 men aged 48 were given a health check-up. Those with pathological glucose tolerance were divided into two groups: (1) exercise 1 diet (n 5 288) and (2) no intervention (n 5 135) [52] and were monitored over 12 years. The mortality rate was the same in the intervention group as among the healthy control group (6.5 versus 6.2%) and lower than in the group with pathological glucose tolerance, which did not exercise (6.5 versus 14%). Thus, among all the participants with pathological glucose tolerance, there was a predictive effect of intervention but not a predictive effect of BMI, BP, smoking, cholesterol, or glucose level. Two randomized controlled trials included people with pathological glucose tolerance and found that changes in lifestyle protected against development of type 2 diabetes. A Finnish study randomized 522 overweight middle-aged men and women with pathological glucose tolerance to physical exercise and diet or control [53] and monitored them over 3.2 years. The lifestyle intervention consisted of individual counseling on reduction of calorie intake, reduction of fat intake, and an increase in fiber-rich foods and daily physical activity. The risk of type 2 diabetes fell by 58% in the intervention group. The greatest effect was recorded with the patients who underwent the most extensive lifestyle changes [54,55]. An American study randomized 3234 people with pathological glucose tolerance to either treatment with metformin or a lifestyle program involving moderate physical activity in the form of at least 150 minutes of brisk walking a week and a reduced-calorie diet or no intervention. The subjects were monitored over 2.8 years [56]. The lifestyle intervention group had a 58% lower risk of contracting type 2 diabetes. Thus, the reduction matched the findings in the Finnish study [53], while the metformin treatment only reduced the risk of diabetes by 31%. As can be seen, it is not formally possible to assess the isolated effect of exercise with respect to diet in three of the studies mentioned [52,53,56], but the intervention group experienced only a moderate weight loss. In the Finnish study, weight loss after 2 years was 3.5 kg in the intervention group versus 0.8 kg in the control group [53]. The intervention group thus experienced a drop in

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BMI from around 31 to around 30 in the Finnish study [53] and from 34 to 33 in the American study [56]. PHYSICAL EXERCISE AND ABDOMINAL OBESITY

Visceral fat constitutes an independent risk factor for developing heart disease [57]. A cross-sectional study showed that overweight men with a high level of fitness have a significantly lower visceral fat than overweight men with a poor level of fitness [58]. A metaanalysis from 2017 [59] examined the effect of physical training on ectopic adiposity in patients with type 2 diabetes. The analysis included 24 studies (n 5 1383) and found that aerobic but not strength training had significant impact on abdominal adiposity. A group of young, healthy, normal-weight men who normally walked 10,000 paces every day reduced their paces to 1500 per day over a period of two weeks. They experienced a significant rise in volume of visceral fat (7%) despite a total average weight loss of 1.2 kg [60]. Irrespective of other fat deposits, abdominal obesity is a major risk factor for hyperlipidemia [61,62], lower glucose tolerance [63], insulin resistance [64], systemic inflammation [65], hypertension [66], type 2 diabetes [67], and all-cause mortality [68]. There is a link between regular physical activity, with or without weight loss, and reduction in visceral fat volume [69
72]. Increasing physical activity to 60 minutes/day over 3 months has been found to reduce visceral fat volume by about 30% [73,74]. It should be emphasized, however, that changes in visceral fat volume as a response to physical exercise vary considerably and that it is not possible to identify a clear correlation between amount of physical exercise and reduction in visceral fat [74
76]. In relation to reduction of visceral fat tissue deposits, however, no specific method exists (surgery, diet, physical activity, etc.) for achieving this. Intervention-induced reduction of visceral fat tissue deposits relates to reduction of the total volume of fat tissue and the initial ratio of volume of visceral fat tissue to volume of total fat, regardless of how the reduction in fat tissue is achieved [77]. Studies have shown that an increase in daily physical activity leads to a significant reduction in quantity of visceral fat and/or abdominal circumference, despite no or minimal alteration in total body weight. Thus studies on people with type 2 diabetes show that 2
3 months of regular moderate-intensity aerobic training leads to a significant reduction in quantity of visceral fat (227% to 245%) [78
80]. There is a corresponding finding for healthy, normal-weight premenopausal women [81]; healthy, middle-aged men [82]; and HIV-positive men with lipodystrophy [83]. Middle-aged, normal-weight, or overweight men and overweight women can expect to see a reduction in visceral fat volume (210 to 219%) after 3 months of regular physical activity. These results also apply to older, overweight individuals (60
80 years) [84]. As a result of exercise, either strength or stamina training for 80 minutes a week, test subjects did not accumulate visceral fat after dieting and losing weight, while the control group that did not exercise increased their volume of visceral fact by 38% [85].

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Possible Mechanisms The mechanisms behind the effect of physical exercise on blood lipids, hypertension, and insulin resistance (type 2 diabetes) are described in other sections. Type of Training Resistance and aerobic exercises can both be recommended as effective treatments for people with the metabolic syndrome. A metaanalysis included 12 trials (n 5 626) and concluded that although differences in some diabetic control and physical fitness measures between resistance exercise and aerobic exercise groups reached statistical significance, there is no evidence that they are of clinical importance [86]. There is also no evidence that resistance exercise differs from aerobic exercise in impact on cardiovascular risk markers or safety. Using one or the other type of exercise for fighting type 2 diabetes may be less important than doing some form of physical activity. Future long-term studies focusing on patient-relevant outcomes are warranted. There is solid evidence that physical exercise should ideally be in large amounts. If light to moderately intense physical activity is preferred, then it is necessary to train twice as long compared to doing high-intensity physical activity. Many patients with metabolic syndrome have hypertension or symptomatic CHD. Recommendations should thus be largely tailored to the individual. Treatment should follow the general recommendations for physical activity for adults, but it is recommended that the volume be increased, for example, to 60 minutes of moderately intense physical activity per day. Alternatively, it is possible to increase the intensity and halve the time or to alternate. There has been an increase in technology-based interventions, and a recent review provides a systematic and descriptive assessment of the effectiveness of technology to promote physical activity in people with type 2 diabetes, which could also be applied for people with the metabolic syndrome [87]. For the latter review, technology included mobile phones and text messages, websites and computer-learning-based technology, and excluded telephone calls. In total, 15 articles were eligible for review: web-based (9), mobile phone (3), CD-ROM (2), and computer based (1). All studies found an increase in physical activity, but only nine were significant. Thus, in general, technology-based interventions to promote physical activity were found to be effective. There are no general contraindications; however, training should take into account any competing diseases. People with CHD, should refrain from high intensity work outs. People with hypertension should perform strength training only with light weights and low contraction velocity.

Type 2 Diabetes Background The global prevalence of diabetes is predicted to increase from 171 million individuals (2.8%) in 2000 to 336 million (4.4%) in 2030 [88]. Type 2 diabetes is a metabolic disease characterized by hyperglycemia and abnormalities in glucose, fat, and protein metabolism [89,90]. The disease is due to insulin resistance in the striated muscle tissue and a beta cell

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defect that inhibits the increase in insulin secretion to compensate for insulin resistance. In almost all cases, type 2 diabetes is present for several years before being diagnosed, and over half of all newly diagnosed diabetes patients exhibit signs of late diabetic complications, in particular diabetic macroangiopathy in the form of ischemic heart disease, stroke, and lower limb ischemia, but microvascular complications, such as nephropathy, retinopathy, and especially diabetic maculopathy, are also common. In patients with newly diagnosed type 2 diabetes, the prevalence of peripheral arteriosclerosis is 15%, CHD 15%, stroke 5%, retinopathy 5%
15%, and microalbuminuria 30%. Furthermore there is a high incidence of other risk factors: for example, 80% of patients are overweight, 60%
80% have hypertension, and 40%
50% have hyperlipidemia [91
93]. The excess mortality rate in patients with type 2 diabetes is 60% [91
93]. Multifactorial intensive intervention can prevent late diabetic complications [94]. Evidence-Based Physical Exercise IMPACT ON METABOLIC CONTROL

The positive gains from physical exercise for patients with type 2 diabetes are very well documented, and there is an international consensus that physical exercise is one of the three cornerstones in the treatment of diabetes, along with diet and medication [95
97]. Several reviews [98,99] and metaanalyses [100
103] report that increased physical exercise produce a significant improvement in glucose control in people with type 2 diabetes, yielding an average improvement in HbA1c of between 20.4% and 20.6%. We have recently shown that a high volume of supervised exercise training (U-TURN) for 1 year accompanied by a healthy diet can replace glucose-lowering medicine in patients with type 2 diabetes [104]. Reduction in medication occurred in 74% of the patients while 56% had discontinued their glucose-lowering medicine after 1 year of intervention. A 2011 metaanalysis concluded that structured exercise training that consists of aerobic exercise, resistance training, or both combined is associated with HbA1c reduction in patients with type 2 diabetes. Structured exercise training of more than 150 minutes/week is associated with greater HbA1c declines than that of 150 minutes or less per week. Physical activity advice is associated with lower HbA1c, but only when combined with dietary advice [103]. A systematic review and metaanalysis from 2014 compared resistance exercise with aerobic exercise and concluded that there was no evidence that resistance exercise differs from aerobic exercise in impact on glucose control, cardiovascular risk markers, or safety. Using one or the other type of exercise for type 2 diabetes may be less important than doing some form of physical activity [86]. Measures of fasting glucose and HbA1c do not accurately represent glycemic control because they do not reflect what occurs after meals and throughout the day in the freeliving condition [105]. An accumulating body of evidence now suggests that postprandial glucose fluctuations are more tightly correlated with microvascular and macrovascular morbidities and cardiovascular mortality than HbA1c or fasting glucose, stagnant measure of glycemia. It is therefore important that unlike medications, which generally have a poor effect at improving postprandial glucose, exercise has been proven effective in reducing postprandial glycemic excursions in as little as a few days [105,106].

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Possible Mechanisms There is extensive literature on the effect of physical training on type 2 diabetes; however, the mechanisms are only briefly outlined here. Physical training increases insulin sensitivity in the trained muscle and muscle-contraction-induced glucose uptake in the muscle. Mechanisms include increased postreceptor insulin signaling [107], increased glucose transporter (GLUT4) mRNA and protein [108], increased glucose synthesis activity [109] and heksokinase [110], lower release and higher clearance of free fatty acids [111], and increased transport of glucose to the muscles due to an enlarged muscle capillary network and blood flow [110,112,113]. Resistance training increased insulin-mediated glucose uptake, GLUT4 content, and insulin signaling in skeletal muscles in patients with type 2 diabetes [114]. Physical activity increases blood flow and thus so-called sheer stress on the vessel wall, which is assumed to be a stimulus for endothelial nitrogen oxide, which induces smooth muscle cell relaxation and vasodilation [115]. The antihypertensive effect is assumed to be mediated via a less sympathy-induced vasoconstriction in a fit condition [116]. Type of Training Aerobic training and resistance training are both beneficial; however, a combination of the two is perhaps the optimal form of exercise for people with type 2 diabetes [117]. Evidence also suggests that high-intensity exercise improves glycemic control more than low-intensity exercise. A 2003 metaanalysis assessed the effect of a minimum of 8 weeks of physical exercise [118] and found a link between relatively high-intensity physical training and a decrease in HbA1c (r 5 20.91, P 5 0.002), while no significant link between quantity of physical activity and a decrease in HbA1c (r 5 20.46, P 5 0.26) was established. These correlations partly contradict an intervention trial, which showed that regular physical training increased insulin sensitivity in physically inactive people who did not have diabetes—an effect which was greater for those who spent a good deal of their time being physically active—but that the intensity of the activity was not significant [119]. Recent studies of interval-training programs have shown remarkable results on glycemic control [120
123]. In this context, it has been shown that interval-walking training more favorably improves glycemic control in T2DM subjects when compared to energy-expenditurematched continuous-walking training [124,125]. Scheduled daily exercise is ideal with regard to insulin therapy and adjustment and regulating diet. Most patients with type 2 diabetes can engage in physical activity without following any particular instructions or rules. It is important, however, for patients being treated with sulphonylurea, postprandial regulators, or insulin to receive guidance in order to avoid hypoglycemia. Precautions include monitoring blood sugar, adjusting diet, and adjusting medication. Overall, avoiding physical activity carries greater risks than engaging in physical activity; however, special precautions are necessary. Physical activity should be postponed in the case of a blood sugar level .17 until it has been corrected. The same applies to low blood sugar ,7 mmol/L if the patient is receiving insulin therapy. In the case of hypertension and active proliferative retinopathy, it is recommended that high intensity training or training involving Valsalva maneuvers be avoided. Strength training should be done with light weights and at low contraction velocity.

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In the case of neuropathy and the risk of foot ulcers, body-bearing activities should be avoided. Repeated strain on neuropathic feet can lead to ulcers and fracture. Treadmill exercises, long walks/runs, and step exercises are not recommended, while nonbodybearing exercise is recommendable, such as cycling, swimming, and rowing. One should be aware that patients with autonomic neuropathy may have severe ischemia without symptoms (silent ischemia). These patients typically suffer from resting tachycardia, orthostatism, and poor thermoregulation. There is a danger of sudden cardiac death. It may be advisable to consult a cardiologist and carry out an exercise ECG or a myocardial scintigraphy. Patients should be instructed to avoid physical activity in cold or hot temperatures and to ensure adequate hydration during physical activity.

Type 1 Diabetes Background Type 1 diabetes is an autoimmune disease that occurs in children or adults. The disease is caused by the destruction of beta cells in the pancreas, which stops production of insulin. The etiology is still unknown, but environmental factors (e.g., viruses and chemicals), genetic disposition, and autoimmune reactions all play a part. Evidence-Based Physical Exercise Patients with type 1 diabetes have a high risk of developing cardiovascular disease [126], and physical activity offers good prevention [127]. It is therefore important for patients with type 1 diabetes to be physically active on a regular basis. Insulin requirement decreases during physical activity, which is why patients must reduce their insulin dose if they plan to do physical training [128] and/or ingest carbohydrates in connection with training [129]. Patients with type 1 diabetes thus need guidance on how to avoid hypoglycemia so that they, like others, can benefit from the positive effects of physical activity against other diseases. A systematic review from 2014 analyzed physical activity interventions in children and young people with type 1 diabetes mellitus. A total of 26 articles (10 randomized and 16 nonrandomized studies), published in the period 1964
2012, were reviewed. Metaanalyses showed potential benefits of physical activity on HbA1c, BMI, triglycerides, and cholesterol [130]. There are relatively few studies that shed light on the specific impact of training in patients with type 1 diabetes, but in general little or no difference in glycemic control can be identified in patients with type 1 diabetes who are physically active compared to those who are inactive [131,132]. Some studies find no improvement in HbA1c with physical training [133
137], whereas other find that the most physically active patients have the lowest HbA1c [138
140]. A large study included 4655 patients and found an inverse dose
response association was found between physical activity level and HbA1c [140]. Another study showed that intense physical activity was associated with better metabolic control in patients with type 1 diabetes. An observational, cross-sectional study included 130 adult patients with type 1 diabetes. The study found no differences in HbA1c levels in relation to time dedicated to moderate

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physical activities. However, patients who dedicated more than 150 minutes/week to intense physical activity had lower levels of HbA1c (HbA1c: 7.261.0% versus 7.861.1% versus 8.061.0% in more than 149 minutes, between 0 and 149 minutes or 0 minutes of intense physical activity per week, respectively) [138]. On the other hand, patients with type 1 diabetes—like nondiabetics—improve insulin sensitivity [133], which is associated with a lower (ca. 5%) reduction in the exogenous insulin requirements [134]. Endothelial dysfunction is a trait of some [141
144], though not all [145
149], patients with type 1 diabetes, and the effect of physical training on this parameter is only sparsely illuminated. Endothelial function has been found to be both improved [150,151] and unchanged [132] after physical training. Physical training possibly has a positive impact on the lipid profile, also in patients with type 1 diabetes. Controlled studies show that training reduces the level of LDL cholesterol and triglycerides in the blood [136] and increases the level of HDL cholesterol [136] and HDL cholesterol/total cholesterol ratio [133,136]. The ratio, however, has not been thoroughly investigated and there might also be a difference between the sexes [135]. In uncontrolled or cross-sectional trials, a link has been found between training and an increase in HDL2 cholesterol and a decrease in serum triglyceride and LDL cholesterol [152,153]. A randomized controlled trial examined the effect of 30
60 minutes of running at a moderate intensity three to five times a week over 12
16 weeks. The study included young men with type 1 diabetes (n 5 28 and the control group n 5 28). Aerobic training increased fitness, exercise capacity and improved lipid profile [136]. A controlled study showed that 4 months of aerobic training increased fitness by 27% (P 5 0.04), reduced insulin requirement (P , 0.05) [154], and improved endothelial function [150] in patients with type 1 diabetes. Possible Mechanisms Physical training increases glucose uptake in the muscle, which is induced by muscle contraction. The lipoproteins in the blood appear to be significant in the development of atherosclerosis, also in patients with type 1 diabetes [155]. Physical training affects the lipid composition of the blood in a desirable way [41]. Type of Training Most experience has been drawn from aerobic training, but in principle, patients with type 1 diabetes can take part in all forms of sport, if contraindications/precautions are observed. There are some indications that high intense exercise has a more profound effect on glycemic control compared to moderate exercise. Training needs to be regular and planned in line with insulin treatment and adjustment and dietary regulation. The risk of hypoglycemia is lower with INT than with moderate intensity continuous training, since training at high intensity stimulates glucose production in the liver more than moderate intensity training [156]. It is very important for patients to be carefully informed and educated. They must be instructed on steps for avoiding hypoglycemia, which include monitoring blood sugar, adjusting diet, and adjusting insulin [157,158]. When starting on a specific training program, patients should frequently measure their blood sugar level during and after training and thus learn what their individual response

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is to a given strain over a given duration. Patients must be instructed on how insulin and consumption of carbohydrates are adjusted according to the physical activity. Ideally, training should always be at the same time of day and of more or less the same intensity. It is important to drink before and during the physical activity, especially when the training is over a long period and in hot weather. Patients should be particularly aware of their feet 1 / 2 neuropathy and footwear. The recommendations have to be tailored to the individual and take into account late diabetic complications, but both aerobic fitness and strength training are advisable, either a combination of the two or on their own. The goal should be at least 30 minutes of moderate intensity exercise daily. Overall, avoiding physical activity carries greater risks than being active; however, special precautions are necessary. Physical activity should be postponed in the case of a blood sugar level .14 mmol/L and ketonuria, and blood sugar level .17 mmol/L without ketonuria, until this has been corrected. The same applies to low blood sugar ,7 mmol/L. In the case of hypertension and active proliferative retinopathy, it is recommended that high intensity training or training involving Valsalva maneuvers be avoided. Resistance training should be done with light weights and in short series. In the case of neuropathy and the risk of foot ulcers, body-bearing exercise should be avoided. Repeated strain on neuropathic feet can lead to ulcers and fracture. Jogging/ walking treadmill, long walks/runs, and step exercises are not recommended, while nonbody-bearing physical exercise is recommendable, such as cycling, swimming, and rowing. One should be aware that patients with autonomic neuropathy may have severe ischemia without symptoms (silent ischemia). These patients typically suffer from resting tachycardia, orthostatism, and poor thermoregulation. There is a danger of sudden cardiac death. It may be advisable to consult a cardiologist and carry out an exercise ECG or a myocardial scintigraphy. Patients should be instructed to avoid physical activity in cold or hot temperatures and to ensure adequate hydration during physical activity.

CARDIOVASCULAR AND PULMONARY DISEASES Cerebral Apoplexy Background Cerebral apoplexy (stroke, cerebrovascular accident, apoplexy) is defined by WHO as a rapid onset disorder of brain function with symptoms lasting more than 24 hours or leading to death, and where the probable cause is vascular. The reasons are infarction due to cardiac embolism, intracerebral hemorrhage, or subarachnoid hemorrhage after ruptured aneurysm. The average age is 75 years, but 20% of the patients are less than 65 years of age. Depending on localization of the brain damage, different parts of the brain functions are affected, but the majority of stroke patients have unilateral paresis of the upper and lower extremities, while about one-third also have aphasia. Moreover, most of the patients need hospitalization and will require rehabilitation [159].

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Most stroke patients are affected cognitively and emotionally after their attack. Approximately one-third of them experience poststroke depression [160]. These effects coupled with low physical function make it difficult to comply with recommendations for physical activity. Patients with stroke generally have low levels of physical activity [161]. Physical inactivity is a risk factor for atherosclerosis and hypertension, which explains why physical inactivity in epidemiological studies is a prognostic factor for apoplexy [162
171]. Stroke patients who have a relatively high level of physical activity have been found to have comparatively fewer severe stroke episodes and better recovery of function after 2 years [170]. Evidence-Based Physical Training A Cochrane Review from 2016 [172] includes 58 controlled studies, n 5 2797. The authors conclude that cardiorespiratory training and, to a lesser extent, mixed training reduce disability during or after usual stroke care; this could be mediated by improved mobility and balance. There is sufficient evidence to incorporate cardiorespiratory and mixed training, involving walking, within poststroke rehabilitation programs to improve the speed and tolerance of walking; some improvement in balance could also occur. There is, however, insufficient evidence to support the use of resistance training. A Cochrane study from 2017 [173] concludes that people who receive electromechanical-assisted gait training in combination with physiotherapy after stroke are more likely to achieve independent walking than people who receive gait training without these devices. A systematic review from 2016 [174] concludes that aerobic exercise may have a positive effect on improving global cognitive ability and a potential benefit on memory, attention, and the visuospatial domain of cognition in stroke survivors. Thus, there is evidence that aerobic exercise in patients with stroke has a positive effect on walking speed and function. Furthermore, there is support for an effect on mortality. A metaanalysis from 2014 included 38 randomized controlled trials. There was high evidence that in the subacute stage of stroke, specific walking training resulted in improved walking speed and distance compared with traditional walking training of the same intensity. In the chronic stage, walking training resulted in increased walking speed and walking distance compared with no/placebo treatment, and increased walking speed compared with overall physiotherapy. On average, 24 training sessions for 7 weeks were needed [175]. A metaanalysis from 2013 of randomized trials included nine studies of treadmill training comprising 977 participants and found evidence that for people with stroke who can walk, treadmill training without body weight support results in faster walking speed and greater distance than no intervention/nonwalking intervention and the benefit is maintained beyond the training period [176]. Another study from 2013 included metaanalyses of randomized controlled trials with mortality outcomes comparing the effectiveness of exercise and drug interventions with each other or with control (placebo or usual care). In total, they included 16 (4 exercise and 12 drug) metaanalyses. Three trials concerned exercise interventions among patients with stroke (n 5 227) compared with 10 trials of anticoagulants (n 5 22 786), 14 of antiplatelets (n 5 43 041), and three directly comparing anticoagulants with antiplatelets

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(n 5 11 567). Physical activity interventions were more effective than drug treatment among patients with stroke (odds ratios, exercise versus anticoagulants 0.09, 95% credible intervals 0.01
0.70, and exercise versus antiplatelets 0.10, 0.01
0.62) [177]. Since physical activity helps to prevent risk factors for stroke, i.e., hypertension, atherosclerosis, and type 2 diabetes, it is likely that physical training of stroke patients can prevent new episodes of stroke, but there is no evidence to support this. A Cochrane Review from 2013 included 45 trials, involving 2188 participants, which comprised cardiorespiratory (22 trials, 995 participants), resistance (8 trials, 275 participants), and mixed training interventions (15 trials, 918 participants). It was concluded that there is sufficient evidence to incorporate cardiorespiratory and mixed training, involving walking, within poststroke rehabilitation programs to improve the speed and tolerance of walking; improvement in balance may also occur. Presently, there is, however, insufficient evidence to support the use of resistance training [178]. However, individuals with hemiparesis are often older and have a low level of activity, and it must be assumed that this group can achieve the same advantages from strength training as individuals without neurological deficits. In other words, physically active people with hemiparesis will have lower mortality, more active years, reduced risk of metabolic syndrome, and increased bone density [179,180]. Possible Mechanisms Patients with apoplexy have poor physical function and a low level of (age-adjusted) fitness [181,182], which means that they have less energy to carry out rehabilitation. Their poor level of fitness is presumably also related to the smaller number of motor units that can be recruited during dynamic muscle contractions [183], to the altered composition of fibers resulting from prolonged physical inactivity and to reduced oxidative capacity in the paretic muscle [184]. The absolute energy expenditure per submaximum workload in the hemiplegic patient is greater than what is seen in normal individuals of the same age and weight [185]. The increased energy expenditure is related to inefficient patterns of movement and spasticity [186]. Aerobic exercise breaks this vicious cycle by improving aerobic capacity and reducing energy exertion. This increases the patient’s overall physical capabilities and the ability to carry out rehabilitation. Type of Training Physical therapy methods will not be reviewed here [187,188]. The training program should be individualized but focus on walking and cardiorespiratory training. There are no contraindications, but specific measures incorporating partial body weight support may be necessary.

Hypertension Background Hypertension is a significant risk factor for stroke, acute myocardial infarction, heart failure, and sudden death. The borderline between low and normal BP is fuzzy, as the incidence of these cardiovascular diseases already rises from a relatively low BP level. A

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metaanalysis involving 61 prospective studies (1 million people) showed a linear relationship between decrease in the risk of cardiovascular mortality and decrease in BP to a SBP of below 115 mmHg and a DBP of below 75 mmHg [189]. A decrease of 20 mmHg in SBP or 10 mmHg in DBP halves the risk of cardiovascular mortality. Thus, for example, a person with SBP of 120 mmHg has half the risk of cardiovascular mortality as a person with SBP of 140 mmHg [189]. Hypertension is defined as SBP . 140 and DBP . 90 mmHg. According to this definition, about 20% of the population have high BP or require BP lowering medication [190]. However, the borderlines between optimal and normal BP and between mild, moderate, and severe hypertension are arbitrary. [190]. Large-scale epidemiological studies indicate that regular physical exercise and/or fitness prevents hypertension or lowers BP [191,192]. Evidence-Based Physical Training EFFECT ON RESTING BLOOD PRESSURE (NORMOTENSIVE AND HYPERTENSIVE)

Several metaanalyses have concluded that physical exercise has a positive effect on BP in both normotensive and hypertensive cases [193
203]. A metaanalysis included randomized controlled trials lasting $ 4 weeks investigating the effects of exercise on BP in healthy adults (age $ 18 years) [199]. The study included 93 trials, involving 105 endurance, 29 dynamic resistance, 14 combined, and 5 isometric resistance groups, totaling 5223 participants (3401 exercise and 1822 control). SBP was reduced after endurance [ 2 3.5 mmHg (confidence limits 24.6 to 22.3)], dynamic resistance [ 2 1.8 mmHg (23.7 to 20.011)], and isometric resistance [ 2 10.9 mmHg (214.5 to 27.4)] but not after combined training. Reductions in DBP were observed after endurance [ 2 2.5 mmHg (23.2 to 21.7)], dynamic resistance [ 2 3.2 mmHg (24.5 to 22.0)], isometric resistance [ 2 6.2 mmHg (210.3 to 22.0)], and combined [ 2 2.2 mmHg (23.9 to 20.48)] training. BP reductions after endurance training were greater (P , 0.0001) in 26 study groups of hypertensive subjects [ 2 8.3 (210.7 to 26.0)/ 2 5.2 (26.8 to 23.4) mmHg] than in 50 groups of prehypertensive subjects [ 2 2.1 (23.3 to 20.83)/ 2 1.7 (22.7 to 20.68)] and 29 groups of subjects with normal BP levels [ 2 0.75 (22.2 to 10.69)/ 21.1 (22.2 to 20.068)]. BP reductions after dynamic resistance training were largest for prehypertensive participants [ 2 4.0 (27.4 to 20.5)/ 2 3.8 (25.7 to 21.9) mmHg] compared with patients with hypertension or normal BP. It was concluded that endurance, dynamic resistance, and isometric resistance training lower SBP and DBP, whereas combined training lowers only DBP. Data from a small number of isometric resistance training studies suggest this form of training has the potential for the largest reductions in SBP. A metaanalysis from 2010 focused specifically on the effect of isometric exercise, which has not traditionally been recommended as an alternative to dynamic exercise [204]. Five trials were identified including a total of 122 subjects. Isometric exercise for ,1 hours/week reduced SBP by 10.4 mmHg and DBP by 6.7 mmHg. Also this study found that isometric exercise induces changes in BP that are similar to that of endurance or dynamic resistance training and similar to those achieved with a single pharmacological agent. Interestingly, a smaller study suggested that even handgrip exercise had effect on BP lowering effects [205].

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Another metaanalysis from 2013 included aerobic exercise training studies among previously sedentary older adults [198].Twenty-three studies, representing a total of 1226 older subjects, were included in the final analysis. Robust statistically significant effects were found when older exercisers were compared with the control group, representing a 3.9% reduction in SBP and a 4.5% reduction in DBP. A metaanalysis was carried out in 2007 [201] involving randomized controlled trials in which the training consisted of either endurance or resistance training. The metaanalysis was based on 72 trials and 105 study groups. Physical training was found to induce a significant reduction in resting BP and SBP/DBP, measured during outpatient visits, of 3.0/2.4 (P , 0.001) and 3.3/3.5 mmHg (P , 0.01), respectively. The reduction in BP was more pronounced for the 30 hypersensitive trial groups, in which an effect of 26.9/ 2 4.9 was achieved, while the normotensive group achieved an effect of 21.9/ 2 1.6 (P , 0.001). Training had a positive effect on a number of clinical and paraclinical variables, namely, systemic vascular resistance, plasma noradrenalin, plasma renin activity, body weight, abdominal girth, fat percentage, homeostatic model assessment (HOMA), and HDL cholesterol. An expert panel of the American College of Sports Medicine (ACSM) [193] extrapolated data from a total of 16 studies involving patients with hypertension (SBP . 140 mmHg; DBP . 90 mmHg) and found the effect of physical training in people with hypertension to be a decrease in BP of 7.4 (systolic) and 5.8 mmHg (diastolic). A general finding was that the BP lowering effect of physical training was most pronounced in the patients with the highest BP. A metaanalysis from 2011 identified studies that had examined the effect of strength training on BP and other cardiovascular risk factors in adults [206]. The study included 28 randomized, controlled trials, involving 33 study groups and 1012 participants. Overall, resistance training induced a significant BP reduction in 28 normotensive or prehypertensive study groups [ 2 3.9 (26.4; 21.2)/ 2 3.9 (25.6; 22.2) mmHg], whereas the reduction [ 2 4.1 (20.63; 11.4)/ 21.5 (23.4; 10.40) mmHg] was not significant for the 5 hypertensive study groups. When study groups were divided according to the mode of training, isometric handgrip training in 3 groups resulted in a larger decrease in BP [ 2 13.5 (216.5; 210.5)/ 26.1(28.3; 23.9) mmHg] than dynamic resistance training in 30 groups [ 2 2.8 (24.3; 21.3)/ 22.7 (23.8; 21.7) mmHg]. This metaanalysis supports the BP-lowering potential of dynamic resistance training and point at an interesting effect of isometric handgrip training. The latter study adds to previous metaanalysis [207,208]. BP was measured daily (24 hours) in 11 studies [193] and showed the same effect from training as the studies mentioned above. ACUTE EFFECT OF PHYSICAL ACTIVITY

Physical activity induced a decrease in BP after it was carried out. This decrease in BP typically lasted for 4
10 hours but was measured up to 22 hours later. The average decrease was 15 and 4 mmHg for SBP and DBP, respectively [193]. This means that people with hypertension can achieve normotensive values many hours of the day, which should be seen as having considerable clinical significance [193]. Overall, it is well documented that training for hypersensitive people induces a clinically relevant lowering of BP. Conventional treatment using BP lowering medication typically brings about a decrease in DBP of the same level [209
212], which in the long run lowers

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the risk of strokes by an estimated 30% and the risk of ischemic cardiac death by 30%. A metaanalysis involving 1 million people calculates that a reduction in SBP of just 2 mmHg reduces stroke mortality by 10% and ischemic cardiac death mortality by 7% among middleaged people [189]. These calculations coincide with findings from earlier analyses [209,213]. Possible Mechanisms The BP lowering effect of physical training is assumed to be multifactorial but appears to be independent of weight loss. The mechanisms include neurohormonal, vascular, and structural adaptations. The antihypersensitive effect includes decreased sympathetically induced vasoconstriction in a fit condition [214] and a decrease in catecholamine levels. Hypertension often occurs in conjunction with insulin resistance and hyperinsulinemia [215,216]. Physical training increases insulin sensitivity in the trained muscle and thus reduces hyperinsulinemia. The mechanisms include increased postreceptor-insulin signaling [107], increased glucose transporter (GLUT4) mRNA and protein [108], increased glycogen synthase activity [109] and hexokinase [110], lower release and increased clearance of free fatty acids [111], and increased transport of glucose to the muscles due to a larger muscle capillary network and blood flow [110,112,113]. Prolonged hypertension leads to hypertrophy and in the long term also to systolic dysfunction [217
220]. Many patients are characterized by chronic low-grade inflammation with increased levels of, for example, C-reactive protein [221]. The latter has a negative prognostic value [222,223]. Physical training augments diastolic filling in the left ventricle [224,225], increases endothelial vasodilator function [226,227], and induces antiinflammatory effects [228]. Type of Training All patients with hypertension (both those receiving medical treatment as well as those not receiving treatment) benefit from physical training, which should either be in the form of endurance training, dynamic strength training or isometric training. In accordance with guidelines from ACSM, people with BP . 180/105 should not begin regular physical activity until after pharmacological treatment has been initiated (relative contraindication) [229]. An increase in risk of sudden death or stroke in physically active people with hypertension has not been ascertained [229,230]. ACSM advises caution in the case of highintensity dynamic training or strength training with very heavy weights. Heavy strength training can lead to very high pressure in the left ventricle ( . 300 mmHg), which can be potentially dangerous. In particular, patients with congestive hypertrophy should refrain from heavy strength training. Other precautions depend on comorbidity.

Coronary Heart Disease Background The term CHD refers to a pathophysiological condition where a decrease in blood flow to the heart muscle causes ischemia, i.e., reduces oxygen supply. The most common cause is atherosclerotic constriction in the coronary arteries, but myocardial ischemia can also occur in patients with heart valve disease, hypertrophic cardiomyopathy, severe

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hypertension, and abnormal tendency toward coronary spasm. Level of physical activity and CRF with cardiovascular endpoints in healthy people and in patients with CHD [231]. Evidence-Based Physical Training There is solid evidence demonstrating the effect of physical training on patients with CHD. Physical training improves survival rates and is assumed to have a direct effect on the pathogenesis of the disease. A Cochrane Review from 2016 [232] included 63 trials which randomized 14,486 people with CHD. The population included predominantly postmyocardial infarction (MI) and postrevascularization patients and the mean age of patients within the trials ranged from 47.5 to 71.0 years. Women accounted for fewer than 15% of the patients recruited. Exercise-based cardiac rehabilitation (CR) reduced cardiovascular mortality compared with no exercise control. The overall risk of hospital admissions was reduced. The latter review is in agreement with previous metaanalyses [233
235]. Physical training of patients with CHD has been found to reduce total cholesterol and triglyceride levels and SBP. Many of the subjects in the training groups had stopped smoking [OR 5 0.64 (95% CI 0.50
0.83)] [234]. Possible Mechanisms The mechanism behind the prognostic gain from physical training is undoubtedly multifactorial and includes training-induced increased fibrinolysis, decreased platelet aggregation, improved BP regulation, optimized lipid profile, improved endothelium-mediated coronary vasodilation, increased heart rate variability and autonomic tone, a beneficial effect on a number of psychosocial factors, and a generally higher level of supervision of patients. Physical training is believed to have a beneficial effect by enhancing CRF, reducing myocardial oxygen demand at a certain exercise level, having a beneficial effect on autonomic and coronary endothelial function, and improving cardiovascular risk profile, including BP, HDL/LDL ratio, weight, glycemic control, and psychological well-being [236,237]. Type of Training The recommendation is to offer CR that includes physical training as a key component to all patients with CHD. Before training is commenced, there should be an evaluation of exercise capacity in order to create an individual training program. The recommended method for evaluating exercise capacity is a symptom-limited exercise test. Trained personnel (physiotherapist, nurse, laboratory assistant) under a doctor’s supervision can carry this out. It is difficult to make precise recommendations about the duration, frequency, and intensity of the training due to a lack of comparable studies. From 1995
2007, the American Heart Association and the American College of Cardiology guidelines [238] recommended aerobic training lasting 20
60 minutes three to five times a week at an intensity of 50%
80% of the patient’s maximum level (defined as VO2max, maximum heart frequency or maximum symptom-limited exercise capacity). The training can be continual or interval, e.g., walking, running, step machine, cycling, rowing, or stair walking. The aerobic training presumably should be backed up by strength training and training intensity should be increased as the patient’s exercise capacity increases.

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Physical training is also advisable for patients with angina pectoris who are not candidates for revascularization [239]. It is not clear how long the supervised training program should be, but the bulk of the studies that were part of the abovementioned metaanalysis involved a duration of 6
24 weeks with a weighted average of approximately 11 weeks. The effect was not determined by duration but by the overall “training dose” and no difference was found in mortality after training programs involving a generally large dose as opposed to a low dose [234]. Lengthier training programs are aimed at ensuring that the patient achieves a training effect and partly at helping to incorporate new exercise habits. The working group assessed that training should extend over 12 weeks, with a shorter program or longer program for selected patient groups after assessment. In areas with scant provision of facilities by the local authorities, it is recommended that a training program be put together in conjunction with the hospital. Based on the above, the following is recommended: • Physical exercise is advisable for all patients with stable CHD. In the case of acute coronary disease (ACD), training can be embarked on 1 week after revascularized STElevation Myocardial Infarction (STEMI)/NSTEMI and 4
6 weeks after coronary artery bypass grafting (CABG). • All patients who have been hospitalized with ACD and/or are not fully revascularized should be examined by a cardiologist before any training program is initiated. • In order to organize an individual training program, the training should be preceded by an assessment of exercise capacity. The recommended method for this is a symptomlimited exercise test, which can be carried out by trained personnel (physiotherapist, nurse, laboratory assistant) under a doctor’s supervision. • Supervised training with individually organized training programs after an initial exercise test: two to five 30
60-minute sessions a week at an intensity of 50%
80% of maximum exercise capacity. • Twelve weeks of organized aerobic training and possibly INT combined with resistance training, especially for the elderly and patients with muscle weakness. • Daily low-intensity training (walking) over 30 minutes, increasing the level under the supervision of the rehabilitation team. The following contraindications are in agreement with a European working group [240]: • Acute myocardial infarction (AMI) or unstable angina, until the condition has been stable for at least 5 days • Dyspnea at rest • Pericarditis, myocarditis, endocarditis • Symptomatic aortic stenosis • Severe hypertension. There is no established, documented borderline BP value deemed to be the cutoff point for increased risk. Generally it is recommended that demanding physical exercise be avoided in the case of SBP . 180 or DBP . 105 mmHg • Fever • Serious noncardiac disease

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Heart Failure Background Heart failure is a condition where the heart is unable to pump sufficiently to maintain blood flow to meet the metabolic needs of the peripheral tissue [241]. Heart failure is a clinical syndrome with symptoms such as fluid retention, breathlessness or excessive tiredness when resting or exercising, and with objective symptoms of reduced systolic function of the left ventricle at rest. Asymptomatic left ventricular dysfunction is often the precursor of this syndrome. Symptoms vary from very light restriction of function to seriously disabling symptoms. Heart failure is often divided into left-sided (the most frequent and best researched type) and right-sided heart failure and into acute (pulmonary edema, cardiogenic shock) and chronic heart failure. Heart failure is often caused by ischemic disease but can also be caused by, for example, hypertension or valvular heart disease [241]. Maximum oxygen uptake (VO2max) is lower in patients with heart failure [240,242,243]. This is caused, among other things, by the reduced pumping function of the heart and by peripheral conditions in the muscles [240,244,245]. A common symptom with heart failure patients is muscle atrophy, tiredness, and reduced muscle strength [246
248]. Heart failure patients are characterized by defects in the renin
angiotensin system, increased levels of cytokines, also tumor necrosis factor (TNF) [249], increased noradrenalin [250], and insulin resistance [251]. These metabolic conditions can all be significant factors in the development of muscle atrophy with heart failure [248], although no direct link between VO2max and noradrenalin [252] has been found. Thus, heart failure patients tend to have poor fitness, poor muscle strength, and muscle atrophy. The characteristic symptom of tiredness is presumably related to diminishing physical ability. While there was a consensus in the 1970s that patients with all stages of heart failure should be advised to refrain from physical activity and be prescribed bed rest [253], the consensus now is the opposite [240]. Evidence-Based Physical Training International guidelines recommend physical training for patients with congestive heart failure (CHF) after a large number of studies have demonstrated the beneficial effect on central and peripheral factors and on function level, New York Heart Association (NYHA) class and quality of life, without presenting any significant risks [254,255]. The effect of physical training on patients with heart failure has been assessed in numerous metaanalyses [256
262]. There is consistent evidence of the beneficial effect of training patients with heart failure. A definite impact on heart-failure-related hospitalization, physical function, and quality of life has been identified. The studies were carried out on stable patients in NYHA class II and NYHA class III, and most of the studies excluded patients with competing diseases, such as diabetes or COPD. A 2010 Cochrane Review assessed the effect of physical training on patients with congestive heart failure. The analysis identified 19 randomized controlled trials that compared training over a minimum of 6 months with a nonexercise control group. The 19 studies included 3647 patients, the majority of them men in NYHA class II-III with a left ventricular ejection fraction of less than 40%. Unlike several other earlier analyses, which were based on

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fewer studies, no significant difference in mortality was found between the training group and the control group. There was an insignificant effect of the same magnitude as in earlier studies. Heart-failure-related hospitalizations were significantly lower in the exercise group (RR: 0.72, 95% CI: 0.52
0.99). Furthermore, a clear enhancement of quality of life was ascertained (standardized mean difference (SMD): 20.63, 95% CI: 20.80 to 0.37) in the exercise group [257]. The randomized trials generally included CHF patients with systolic failure (EF , 40%), documentation for the effect of training with isolated diastolic failure is sparse. The latest randomized clinical trials on physical training were carried out with heart failure patients who were presumably following a more optimal program of medical treatment compared to the initial studies. For example, in a 2009 study, 94% of the patients were being treated with beta blockers and angiotensin receptor blockers and 45% had an implanted defibrillator or pacemaker [263]. A Cochrane Review from 2014 [264] updated the Cochrane Review previously published in 2010 [257] and had focus on mortality, hospitalization admissions, morbidity, and health-related quality of life for people with heart failure. Randomized controlled trials of exercise-based interventions with 6 months’ follow-up or longer compared with a no exercise control that could include usual medical care. The study population comprised adults over 18 years and were broadened to include individuals with HFPEF in addition to Heart failure with reduced ejection fraction (HFREF). The authors included 33 trials with 4740 people with heart failure predominantly with HFREF and NYHA classes II and III. This latest update identified a further 14 trials. There was no difference in pooled mortality between exercise-based rehabilitation versus no exercise control in trials with up to 1-year follow-up (25 trials, 1871 participants: RR 0.93; 95% CI 0.69
1.27, fixed-effect analysis). However, there was trend toward a reduction in mortality with exercise in trials with more than 1 year of follow-up (6 trials, 2845 participants: RR 0.88; 95% CI 0.75
1.02, fixedeffect analysis). Compared with control, exercise training reduced the rate of overall (15 trials, 1328 participants: RR 0.75; 95% CI 0.62
0.92, fixed-effect analysis) and heart failure specific hospitalization (12 trials, 1036 participants: RR 0.61; 95% CI 0.46
0.80, fixed-effect analysis). Exercise also resulted in a clinically important improvement in disease specific health-related quality of life measure. Two studies indicated exercise-based rehabilitation to be a potentially cost-effective use of resources in terms of gain in quality-adjusted life years and life-years saved. Regarding mode of exercise, a metaanalysis showed that in clinically stable patients with heart failure with reduced ejection fraction vigorous to maximal aerobic INT is more effective than traditionally prescribed moderate-intensity continuous aerobic training for improving peak oxygen uptake (VO2) [265]. The latter conclusion was supported by yet another metaanalysis [266]. Possible Mechanisms Training increases myocardial function assessed at maximum minute volume [240,267
272], increases systemic arterial compliance [271,272], increases stroke volume [272], counteracts cardiomegaly [272], induces positive changes in the exercising muscle [240,267,273,274], and increases the anaerobic threshold [240,267,273,275
277]. Exercise reduces the sympathetic nervous system and the renin
angiotensin system

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[240,268,278,279]. Exercise further induces muscle cytochrome C oxidase activity, which leads to reduced local expression of proinflammatory cytokines and inducible nitric oxide synthase and increases insulin-like growth factor-1 [280]. Thus, physical training is able to inhibit the catabolic processes in the heart failure patient and counteract muscle atrophy. Physical training lowers the concentration of circulating TNF receptors 1 and 2 [281], TNF and FAS-ligand (FAS-L) [282], and the quantity of circulating adhesion molecules [283] in patients with heart failure. Physical training lowers the expression of cytokines in the skeletal muscle [284] and in the blood stream [285]. Type of Training Many studies have demonstrated a beneficial effect from INT, which is possibly more effective than moderate continual aerobic training [240,286]. Patients can start on INT, beginning on a low exercise capacity, and gradually increase duration, frequency, and intensity [240]. Practitioners used to be reluctant to recommend resistance training out of concern that increased vascular resistance would increase cardiac load more than aerobic training. There is no evidence that a combination of aerobic and resistance training produces better or worse results than aerobic training alone [260]. Based on the above, the following is recommended: • Training is recommended for all heart failure patients in NYHA function class II
III on a fully titrated medication regimen and well compensated for 3 weeks. • All patients should be assessed by a cardiologist before embarking on a training program. • For the sake of caution and in order to determine individual exercise capacity, the training should be preceded by a symptom-limited exercise test. • A supervised, tailored training program is recommended after an initial exercise test. Training programs for heart failure patients with very low exercise capacity must be structured with short daily sessions of low intensity exercise, gradually increasing duration as the program progresses. When the patient is able to train for 30 consecutive minutes, training frequency should be cut down to two to three sessions a week and training intensity gradually stepped up. As a rule, training is not recommended for patients in NYHA IV, although there are studies in which selected patients trained without presenting any hazards. The following contraindications are in agreement with a European Working Group [240]: Relative: • • • • •

. 1.8 kg weight increase over 1
3 days Decrease in SBP during exertion (exercise test) NYHA IV Complex ventricular arrhythmia when resting or during exertion (exercise test) Heart frequency at rest .100 Absolute contraindications:

• Worsening of functional dyspnea or newly occurring dyspnea at rest over 3
5 days

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Significant ischemia at low exertion (,2 METS or 50 W) Acute illness or fever Recent thromboembolism Active pericarditis or myocarditis Moderate/difficult aortic stenosis Valve insufficiency requiring surgery AMI within the preceding 3 weeks Newly occurring atrial fibrillation

Intermittent Claudication Background Arterial insufficiency in the lower limbs (lower limb ischemia, leg ischemia) is a chronic obstructive disease in the aorta below the outlet of the renal arteries, iliac artery, and the arteries in the lower limbs probably caused by atherosclerosis. It is estimated that at least 4% of all people above the age of 65 have peripheral arteriosclerosis, which in 50% of cases causes intermittent pain (intermittent claudication). A minority of patients experience the progression of peripheral arteriosclerosis, which results in pain while at rest and ulcerations. Owing to the realization that medical treatment of the disease has limited efficacy, the international consensus today is that physical training is a key factor in the treatment of patients with intermittent claudication [287]. As the intermittent claudication becomes more severe, function level decreases and quality of life becomes affected. Increasing pain when walking and the consequent fear of moving gradually causes the patient to become static and socially isolated. In the long term, this leads to deterioration of fitness and the progression of arteriosclerosis, reduced muscle strength, and muscle atrophy, trapping the patient in a vicious circle of poor fitness, pain, and social isolation. Physical activity can be used to interrupt this vicious circle and directly affect the pathogenesis of the disease by increasing fitness and muscle strength, changing pain thresholds and the perception of pain, allaying fear, and preventing disease progression. Evidence-Based Physical Training There is solid evidence demonstrating the beneficial effect of physical training on patients with intermittent claudication. A Cochrane Reviews from 2014 [288] included 30 trials, involving a total of 1816 participants with stable leg pain. The follow-up period ranged from 2 weeks to 2 years. The types of exercise varied from strength training to polestriding and upper or lower limb exercises; generally supervised sessions were at least twice a week. Most trials used a treadmill walking test for one of the outcome measures. Twenty trials compared exercise with usual care or placebo, the remainder of the trials compared exercise to medication or pneumatic calf compression. Overall, when taking the first time point reported in each of the studies, exercise significantly improved maximal walking time when compared with usual care or placebo: mean difference (MD) 4.51 minutes (95% CI 3.11
5.92) with an overall improvement in walking ability of approximately 50%
200%. Walking distances were also significantly improved: pain-free walking distance MD 82.29 m (95% CI 71.86
92.72) and maximum walking distance MD 108.99

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meters (95% CI 38.20
179.78). Improvements were seen for up to 2 years. The effect of exercise, when compared with placebo or usual care, was inconclusive on mortality, amputation, and peak exercise calf blood flow due to limited data. At 3 months, physical function, vitality, and role of physical was reported to be improved with exercise in two trials. At 6 months, five trials reported outcomes of a significantly improved physical summary score and mental summary score secondary to exercise. The authors concluded that exercise programs are of significant benefit compared with placebo or usual care in improving walking time and distance in people with leg pain from intermittent claudication who were considered to be fit for exercise intervention. This was obtained without any demonstrable effect on the patients’ ankle blood-pressure measurements. The results were not conclusive for mortality and amputation. In a controlled study, physical training was compared with percutaneous transluminal angioplasty (PTA) and the finding was that there was no significant difference after 6 months [289]. A review article by Chong et al. [290] assessed the results of physical training (9 studies, 294 patients) and PTA (12 studies, 2071 patients). The authors concluded that it was essentially impossible to compare the effect of two treatments in one noncontrolled study design but reported that the effect of PTA was minimally better than training, although PTA involved the risk of serious side effects. A randomized study compared the effect of (1) physical exercise alone, (2) surgery, and (3) physical exercise 1 surgery. All groups achieved the same effect on walking distance, but there were side effects in 18% of the patients who underwent surgery [291]. Another randomized study compared the effect of physical training and antithrombotic therapy [292]. A significantly larger improvement in walking distance was recorded for the group that exercised (86%) compared with the group that received medication (38%). A metaanalysis found that training programs were substantially cheaper and involved fewer risks than either surgery or PTA [293]. Furthermore, a metaanalysis has shown that quality of life increases with walking distance [294]. A recent review analyzed the safety of supervised exercise training in patients with intermittent claudication [295]. There has been concern regarding the safety of performing supervised exercise training because intermittent claudication patients are at risk for cardiovascular events. The review selected 121 articles, of which 74 met the inclusion criteria. Studies represent 82,725 hours of training in 2876 patients with intermittent claudication. Eight adverse events were reported, six of cardiac and two of noncardiac origin, resulting in an only all-cause complication rate of one event per 10,340 patient-hours. Possible Mechanisms Physical training programs for patients with heart failure increase local production of the vascular endothelial growth factor (VEGF) [296], which induces the production of collaterals and thus increases blood flow. The formation of VEGF is stimulated by muscle contractions during ischemia. This is presumably a key mechanism, which also explains the importance of training beyond the pain threshold. However, a clinical effect of exercise that does not affect ankle BP has been demonstrated [297], and there is generally a poor correlation between ankle BP and improvement of walking distance [298]. Physical activity increases endothelial function in the lower limbs [299]. We assume that the effect of the physical training is to a large degree linked to improved fitness and increased muscle

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strength. Furthermore, the patients’ experience of being able to pass the pain threshold probably has a psychological effect and consequently their perception of pain changes. Type of Training The vast majority of studies only assess the effect of walking exercise measured with a treadmill test, while there is little information on other forms of training. One study found beneficial effects from Nordic walking compared to a nonexercising control group [300]. Nordic walking involves walking using poles in both hands, which partly provide support and partly force the patient to move the upper body, increasing overall exertion. There is limited information on the importance of walking speed or intensity but strong evidence to suggest that the effect is increased if training is carried out until the onset of ischemia symptoms. Controlled trials provide some, albeit limited, evidence that supervised training is more effective than unsupervised training [301
304]. The effect of training is reinforced if the patient quits smoking [305] and the amount of training is a determining factor in efficacy [306]. Physical activity should preferably be in the form of walking exercise, which should initially be supervised through regular visits to a therapist. In many cases, training can take place in the home and it should take place at least 3 days a week. The patient should walk until just past the onset of pain and then take a short break until the pain has receded, after which the walking exercise should be resumed. Training sessions should be at least 30 minutes each time and the training program should be lifelong and supervised in the initial 6 months. Feedback is in the form of a patient logbook recording walking distance, distance/time before onset of pain, and training frequency. Walking distance should be tested before and after 3 months, and subsequently once a year. Ischemic pain does not necessarily occur in the case of bike exercise, which is the reason why walking exercise is preferable. If bike exercise is chosen, the patient must be instructed to pedal using the front of the foot and the same training principles apply as for walking. There are no general contraindications. Supervised exercise training can safely be prescribed in patients with intermittent claudication because an exceedingly low all-cause complication rate is found. Routine cardiac screening before commencing exercise training is not required.

Chronic Obstructive Pulmonary Disease Background COPD is characterized by an irreversible decrease in lung function. Advanced-stage COPD is a long and painful process of gradually increasing and ultimately disabling breathlessness as the main symptom. Today the international consensus is that rehabilitation programs are an important part of COPD treatment, which follows from the realization that drug therapy for COPD is inadequate. A vicious cycle of deterioration in physical capacity, shortness of breath, anxiety, and social isolation develops. Rehabilitation can break this cycle by introducing physical training, psychological support, and networking with other COPD patients [307].

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Reduction in muscle strength is a major cause of reduced exercise capacity and physical functional level [308]. A minor study showed that muscle mass in the quadriceps was approximately 15% less and muscle strength about 50% lower in elderly men with COPD than in healthy, physically inactive peers [309]. Evidence-Based Physical Training The positive impact of physical exercise for patients with COPD is well documented. A 2015 Cochrane Review/metaanalysis [310] added to previous metaanalyses [311
314]. The 2015 update includes 65 RCTs involving 3822 participants. A total of 41 of the pulmonary rehabilitation programs were hospital based, 23 were community based, and one study had both a hospital component and a community component. Most programs were of 12 or 8 weeks duration with an overall range of 4
52 weeks. The authors found statistically significant improvement for all included outcomes. In four important domains of quality of life [Chronic Respiratory Questionnaire (CRQ) scores for dyspnea, fatigue, emotional function, and mastery], the effect was larger than the minimal clinically important difference of 0.5 units. Statistically significant improvements were noted in all domains of the St. George’s Respiratory Questionnaire, and improvement in total score was better than 4 units. Both functional exercise and maximal exercise showed statistically significant improvement. Researchers reported an increase in maximal exercise capacity [mean Wmax (W)] in participants allocated to pulmonary rehabilitation compared with usual care. In relation to functional exercise capacity, the 6-minute walk distance mean treatment effect was greater than the threshold of clinical significance. The subgroup analysis, which compared hospital-based programs versus community-based programs, provided evidence of a significant difference in treatment effect between subgroups for all domains of the CRQ, with higher mean values, on average, in the hospital-based pulmonary rehabilitation group than in the community-based group. Subgroup analysis performed to look at the complexity of the pulmonary rehabilitation program provided no evidence of a significant difference in treatment effect between subgroups that received exercise only and those that received exercise combined with more complex interventions. Studies show that rehabilitation programs lead to fewer hospitalizations and thus save resources [315,316]. Most studies use high intensity walking exercise. One study compared the effect of walking or cycling at 80% of VO2max versus working out in the form of Callanetics exercises and found that high intensity training increased fitness while the workout program increased arm muscle stamina. Both programs had a positive effect on the experience of dyspnea [317]. Oxygen treatment in conjunction with intensive training for patients with COPD increased training intensity and thus improved fitness in one study [318], but not in another [319]. It is recommended that oxygenation therapy should be provided at the end of training if the patients are hypoxic or become desaturated during the training [320]. Training to music gave better results than without music [321], presumably because patients who run with music perceive the physical exertion to be less, even though they are doing the same amount of exercise. Specific training for inspiratory muscles increased the stamina of these muscles but did not give the patients a lower perception of dyspnea or improved fitness [322].Thus, strong evidence exists that endurance training as part of pulmonary rehabilitation in patients with COPD improves exercise capacity and health-related quality of life. However, dyspnea limits the exercise intensity.

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Therefore, resistance training, which may cause less dyspnea, could be an alternative. Moreover, low muscle mass is associated with increased risk of mortality [323]. A recent systematic review [324] compared the effect of resistance and endurance training. The authors included eight randomized controlled trials (328 participants) and found that resistance training appeared to induce the same beneficial effects as endurance training. It was therefore recommended that resistance training should be considered according to patient preferences when designing a pulmonary rehabilitation program for patients with COPD. The same authors performed another systematic review [325] in which they assessed the efficiency of combining resistance training with endurance training compared with endurance training alone. For this analysis, they included 11 randomized controlled trials (331 participants) and 2 previous systematic reviews. They found equal improvements in quality of life, walking distance, and exercise capacity. However, they also found moderate evidence of a significant increase in leg muscle strength favoring a combination of resistance and endurance training and recommend that resistance training should be incorporated in rehabilitation of COPD together with endurance training. Possible Mechanisms Physical activity does not improve lung function in patients with COPD but increases CRF via the effect on the muscles and the heart. Patients with COPD have chronic inflammation, which may be a cause of the decrease in muscle strength observed in COPD patients. Patients with COPD have higher TNF levels in blood [326] and muscle tissue [327]. TNF’s biological impact on muscle tissue is manifold. TNF affects myocyte differentiation, induces cachexia and thus a potential decrease in muscle strength [328]. A Danish study showed that smoking inhibited protein synthesis in the muscles, which can potentially also lead to loss of muscle mass [329]. Training can presumably have an impact on this process. Another Danish study showed that physical training counteracted the increase in protein degradation seen in people with COPD [330]. Type of Training All patients with COPD, particularly the more severe cases, benefit from physical training. Initially, the physical training must be supervised, individually tailored, and include a combination of endurance training and strength training. Endurance training can be walking or cycling at 70%
85% of VO2max) [331]. Supervised training over 7 weeks produced better results with regard to a number of respiratory parameters than a 4-week program [332]. There are no general contraindications. The training should take competing diseases into account. For patients with low oxygen saturation (SaO2 , 90%) and dyspnea when at rest, exercising with oxygenation is recommended.

Bronchial Asthma Background Bronchial asthma (asthma) is a chronic inflammatory disorder characterized by episodic reversible impairment of pulmonary function and airway hyperresponsiveness to a variety of stimuli [333]. Allergies are a major cause of asthma symptoms, especially in children,

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while many adults have asthma without an allergic component. Environmental factors, including tobacco smoke, and air pollution, contribute to the development of asthma. Physical exercise poses a particular problem for asthmatics as physical activity can provoke bronchoconstriction in most asthmatics [334]. Regular physical activity is, on the other hand, important in the rehabilitation of patients with asthma [335]. Asthmatics need to be taught how to prevent exercise-induced symptoms in order to benefit, just like other people, from the positive effects of physical activity against other diseases. With children in particular, it is important that they are taught how physical activity can be adapted to asthma due to its importance for their motor and social development. Exercise-induced asthma can be prevented by warming up thoroughly and by using a number of antiasthma drugs, e.g., short or long-acting beta-agonists, leukotriene antagonists, or chromones. Another way to help eliminate some exercise-induced symptoms is to adjust the prophylactic treatment so that the asthma and thus airway responsiveness are under control. Regular intake of antiasthma medicine, especially inhaled steroids, is crucial to enabling physical training. Moreover, it is important to be aware of triggers such as airway infections or triggers in the surroundings where physical activity is carried out, e.g., pollen, mold fungus, cold, air pollution, and tobacco smoke. Some studies [336
338] but not in others [339] have found physical fitness to be poor in asthmatics. Irrespective of how physically fit the patient is, guidance and medicine are important to enabling all asthmatics the opportunity to be physically active without worrying about the symptoms. Evidence-Based Physical Training A Cochrane Review from 2013 [340] included randomized trials of people over 8 years of age with asthma who were randomized to undertake physical training or not. Physical training had to be undertaken for at least 20 minutes, two times a week, over a minimum period of 4 weeks. Twenty-one studies including 772 participants were included. Physical training was well tolerated with no adverse effects reported. None of the studies mentioned worsening of asthma symptoms following physical training. Physical training showed marked improvement in cardiopulmonary fitness as measured by an increase in maximum oxygen uptake; however, no statistically significant effects were observed for forced expiratory volume in 1 second, forced vital capacity, minute ventilation at maximal exercise (VEmax), or peak expiratory flow rate. It was concluded that physical training showed a significant improvement in fitness, though no effects were observed in other measures of pulmonary function. A Cochrane study concluded that swimming training is beneficial for children and adolescents with asthma [341]. A noncontrolled trial showed that it is possible for adult asthmatics to participate in high intensity training [342]. The patients trained in an indoor swimming pool at 80%
90% of maximum oxygen uptake (VO2max) for 45 minutes, initially once a week and then twice a week for 10 weeks. Physical fitness improved and there were fewer cases of exercise-induced asthma attacks, less anxiety in connection with physical exertion, and less of a feeling of dyspnea. At the 3-year follow-up examination, 68% of the patients were still physically active and trained one to two times a week [343]. Physical training has a positive effect on the psychosocial morbidity and quality of life of asthma patients [344,345].

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Possible Mechanisms Physical activity does not improve lung function in patients with asthma, but it does increase cardiorespiratory condition via its effect on the muscles and heart. A common hypothesis [346] is that physical training in asthmatics helps reduce ventilation during exertion, thus reducing the risk of provoking an asthma attack during physical activity. It is also possible that physical training induces an antiinflammatory effect in the lungs [347]. Type of Training The physical training program must be individually tailored and should primarily consist of aerobic training of moderate to high intensity, for example, running, cycling, playing ball, or swimming. Some patients benefit from local treatment with beta-2 agonists or leukotriene antagonists 10
20 minutes prior to training [348]. The treatment must be prescribed by a doctor, and it is important that daily prophylactic treatment is optimal. Warming up at light intensity for about 15 minutes is also beneficial. The recommendation for individuals who are unfit is to start training at low intensity and then gradually increase to moderate intensity, just as the duration of the physical activity should be increased gradually. After 1
2 months, the training should be carried out at least 3 days a week. In cases of acute exacerbation of asthmatic symptoms, a pause in training is recommended. If the patient has an infection, a break in training is recommended until the patient has been asymptomatic for 1 day, after which time training can gradually be resumed.

Cancer Background Cancer and cardiovascular disease are the primary causes of premature death in our part of the world. Cancer is the name given to a group of diseases dominated by uncontrolled cell growth resulting in the compression, invasion, and degradation of surrounding fresh tissue. Malignant cells can be transported through the blood or lymphatic fluid to peripheral organs and cause secondary colonies (metastases). The underlying mechanism common to all cancer diseases is changes in genetic material (mutation), which can be caused by environmental factors, such as tobacco, radiation, pollution, infections, or possibly nutrition. Mutations can cause the cell properties to change and the mechanisms that control the cell’s life span to be disturbed. Thus, cancer cells can live unhindered and uncontrolled. Cancer symptoms are diverse and depend on tumor type and locality. However, many types of cancer cause weight loss, including loss of muscle mass, as well as fatigue and reduced physical capacity as a result of decreased fitness and muscle atrophy. Patients become physically inactive due to general malaise, poor appetite, demanding treatment regimes (surgery, chemotherapy), radiotherapy, and other factors or a combination of factors together with their generally difficult situation. Chemotherapy increases risk of infection and leads to physical inactivity and thus loss of muscle mass and a decrease in fitness. It has been estimated that physical inactivity could account for cancer patients’ poor physical condition by one-third [349]. Fatigue is one symptom that is not only associated with patients with active or advanced cancers but is also found with patients who have undergone radical treatment [350]. This condition affects patients’

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quality of life, and in recent years greater focus has been put on the importance of physical activity to enable cancer patients to function normally and to enhance their quality of life [351
354]. Evidence-Based Training Epidemiological studies demonstrate that physical active lifestyle reduces the risk for at least 13 different types of cancer [355]. In addition, evidence exists that physical activity reduces the risk for recurrence of cancer in breast [356,357], colon [358,359], and prostate [360]. According to these studies, people who are physically active, at least to the extent proposed by general recommendations (www.cdc.gov), almost double their chance of survival [356,358,361
364]. There is ample evidence that physical training for cancer patients has a positive impact on fitness, muscle strength, and physical well-being in the broadest sense [365
367]. Numerous randomized controlled trials have been conducted to determine efficacy of exercise on cancer-related fatigue. A metaanalysis [368] included 72 randomized controlled trials and concluded that exercise had a moderate effect on reducing fatigue compared with control intervention. Exercise also improved depression and sleep disturbance. Exercise effect was larger in the studies published in 2009 or later. Taken together, the results suggest that exercise is effective for the management of cancer-related fatigue. One study [369] examined the effect of physical training in groups as a supplementary measure in addition to conventional therapy (adjuvant therapy or treatment for advanced cancer). The study involved 269 patients with cancer, of which 73 were men aged 20
65 who represented 21 different cancer diagnoses. Patients with metastases in the brain or bones were excluded from the program. The program included a combination of high intensity fitness training, resistance training, relaxation, and massage 9 hours a week over 6 weeks. This intervention was found to reduce fatigue, improve quality of life, improve aerobic capacity, muscle strength, physical and functional activity, and emotional well-being. Physical activity both during and after treatment can increase quality of life and reduce fatigue in women with breast cancer [370
373], and physiotherapy for women with breast cancer after surgery can prevent lymphedema [374]. Possible Mechanisms Physical activity increases fitness and muscle strength, which relieves fatigue and strengthens physical ability. Physical exercise is also thought to boost patients’ selfconfidence and psychological well-being. Exercise may reduce tumor growth via several mechanisms including (1) vascularization and blood perfusion, (2) immune function, (3) tumor metabolism, and (4) muscle-to-cancer cross talk. Insight into these mechanistic effects is emerging and suggest that exercise decreases tumor growth via immune mechanisms [375,376]. Type of Training Cancer patients should aim to exercise according to generally recommended levels of physical activity (www.cdc.gov). Initially the training should be individually tailored and supervised. It should ideally include both aerobic training and resistance training. Cancer

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patients who have completed their therapy typically feel tired as well as physically and, in some cases, mentally weak. Patients benefit from a mixture of moderate and high intensity aerobic training combined with resistance training. The aerobic physical exercise should start at a low intensity and be gradually stepped up to moderate and finally high intensity, gradually increasing the duration of the training at the same time. The aerobic training should be combined with resistance training, which also starts at a low exertion level and short durations. It is recommended that training should be supervised but that relative and absolute contraindications should be observed. Even hospitalized and bedridden patients can profit from physical training [377], but there is sparse information about exercise during chemotherapy or radiotherapy. It is important to emphasize that this patient group is so heterogeneous that standard proposals make no sense and for many, especially elderly cancer patients, the focus ought to be on retaining mobility and physical ability. It is advised that patients undergoing chemotherapy or radiotherapy with a leukocyte count below 0.5 3 10(9)/L, hemoglobin below 6 mmol/L, thrombocyte count below 20 3 10(9)/L, temperature above 38 C should not exercise. Patients with bone metastases should avoid resistance training with heavy weights. In the case of infection, it is recommended that training be interrupted for at least one whole day without symptoms, after which time training should be slowly resumed.

PERSPECTIVE Although there still is a need to define the most optimal type and dose of exercise, it is now time that the health systems create the necessary infrastructure to ensure that supervised exercise can be prescribed as medicine. Moreover, it is important that society in general support a physical active lifestyle, including how infrastructure and architecture may influence the population’s physical activity level.

Acknowledgment This article is based upon a translation and an update of our book “Fysisk aktivitet—ha˚ndbog om forebyggelse og behandling” published by the National Board of Health, Copenhagen in 2003 and 2011 [ISBN 87-91232-78-3 (2003) 978-87-7104-243-6 (2011)]; available at www.sst.dk/publikationer and an upcoming 2018 update. The Centre for Physical Activity Research (CFAS) is supported by a grant from TrygFonden.

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Whey Protein and Muscle Protection Yves Boirie Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CHU ClermontFerrand, Service de Nutrition Clinique, CRNH Auvergne, Clermont-Ferrand, France

SKELETAL MUSCLE LOSS AND PROTEIN NUTRITION Sarcopenia is defined as the progressive loss of muscle mass and function during aging or the consequence of inadequate nutrition, muscle disuse, or chronic disease [1]. Despite its detrimental impacts on physical performance, mobility, bone formation, immunity, metabolism, and global quality of life, it is still largely underestimated [1,2]. Clinically, sarcopenia increases the risks of disability, dysautonomia, falls, fractures, insulin resistance, and frailty in many countries, so that it is now be recognized as a disease by a new ICD code [3,4]. Consequently, muscle loss associated with chronic disease promotes extended hospital stays, but higher risk of infectious and noninfectious complications leading to overall mortality. Therefore to “compress morbidity” with extended life expectancy, preventing age-related or chronic disease-related sarcopenia is of critical importance to maintain mobility at older age in order to preserve autonomy and quality of life as long as possible. There are numerous determinants of sarcopenia, such as environmental, physical, and internal metabolic factors, but inadequate nutritional intakes significantly contribute to muscle loss through the metabolic actions of dietary proteins [5,6]. Even with adequate protein consumption, aging in muscle is characterized by a blunted anabolic response to nutritional stimuli [7 9] which suggests that current protein recommendations in older subjects might not be sufficient for preserving muscle mass and quality on a long-term basis in older age [10], especially considering age-associated clinical states. For these reasons, recommendations for protein intake in elderly subjects have been discussed by the PROT-AGE study group [10] and by ESPEN experts [11] who concluded that older people should consume a daily protein intake of at least 1.0 1.2 g/kg/d in healthy state and 1.2 1.5 g/kg/d in disease state. Of note, the amount of protein consumed during one meal is key for the short-term muscle synthesis activation as meals containing about 30 g of protein with more than 2.5 3 g leucine are associated with greater leg fat-free mass and

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a higher strength [12]. However, beyond the quantity of protein, the qualitative aspects of dietary protein, i.e., its digestibility and its composition in essential amino acids, can modulate protein metabolism and beneficially impact muscle anabolism.

“Fast and Slow” Protein Concept The daily amount of dietary proteins consumed by humans intended to cover nitrogen and amino acid requirements for body and muscle protein preservation particularly in older subjects [13,14]. However in many clinical situations, protein intakes are reduced and an improvement in the quality of dietary proteins is able to compensate for a lower quantitative contribution. The qualitative factors of dietary proteins conventionally based on the content in essential amino acids and their digestibility. To evaluate the quality of food proteins in humans, the protein quality indicator currently used is the digestible indispensable amino acid score which has replaced the protein digestibility corrected for the amino acid score [15] highlighting the fact that dietary amino acids should be identified as individual nutrients together with their bioavailability. However, assessing the nutritional value of proteins using prediction scores is still challenging. Thus, alternative integrative approaches have been developed to better understand the nutritional quality of proteins and their utilization by body tissues. Among these approaches, kinetic analysis of protein assimilation using stable isotopes, especially by using intrinsically labeled dietary proteins such as milk protein [16,17], meat [18], or egg protein [19], have demonstrated that the speed of digestion of dietary proteins is a key regulator of postprandial protein metabolism [20,21]. Indeed, results from studies in animals, in young or older adults, have indicated that absorption kinetics of dietary proteins can influence the metabolic efficiency of food proteins. Thus, the metabolic impact of dairy proteins has been investigated since these proteins are highly consumed, technologically processed for the separation of the different milk components, and they behave in the digestive tract according singular and metabolic properties. Among the milk proteins, whey protein, a soluble protein, has been considered as a fast-digested protein since after its ingestion, digestion, and absorption, the appearance of dietary amino acids derived from its digestion is high, fast but transient. On the contrary, caseins, the main constitutive milk proteins, clot in the stomach which delays gastric emptying and results in a slower, lower but prolonged release of dietary amino acids [20]. More importantly, the pattern of dietary protein digestion/absorption was able to affect postprandial protein anabolism, independently of the amino acid composition of the proteins [22], suggesting that an increased amino acid availability may overcome a low-score quality dietary protein. This idea was recently demonstrated in older adults receiving two doses of soluble proteins or of casein showing that muscle protein synthesis after “fast” protein administration at the lower dose was as efficient as with a large dose of “slow” protein [23]. This stimulation of muscle protein synthesis was closely related to the intensity of dietary amino acids entry, especially plasma leucine peak which was higher with the “fast” proteins than with the slower ones as in another study comparing whey and casein effects [24]. From these data, it is suggested that difference in gastric emptying and digestion and absorption kinetics between casein and whey protein are an important underlying factor. Indeed, different factors related to food structure, physical

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properties, protein matrix, and sources can modulate gastric emptying and the gastrointestinal digestion of its nutritional compounds. The food matrix of proteins may affect the residence time of milk proteins in the stomach : for example, a rennet gel produced with heat-treated milk compared with that of an acid gel, despite identical nutrient compositions, exhibited great differences in their protein digestion kinetics and amino-acid bioavailability [25]. Caloric density of the meal is also able to delay gastric emptying [26]. Inversely, using a low calories leucine-enriched whey protein supplement promoted a higher rise in total, essential, and branched chain amino acids levels compared to casein proteins or to a high caloric products in healthy elderly subjects [27]. The metabolic effects of the “fast” proteins are mediated by the gastrointestinal behavior of these products which modify postprandial amino acid concentrations. Interestingly, changing gastric emptying by acting on the composition of the nutritional components of a nutritional formula could be a novel approach to speed up the assimilation and the tolerance of enteral products. For example, the clotting of enteral nutrition products in the stomach was prevented by using various mixtures of protein from different sources as reflected by the mixing of vegetable proteins, such as pea and soy, with casein proteins which then behave in a similar manner to a “fast” protein [28]. Considering other protein sources, meat provides an important source of daily protein. Meat proteins have a good balance of essential amino acids and a high digestibility. Regarding the speed of protein digestion absorption, meat proteins digestion and absorption are close to “fast” proteins since the postprandial variations in amino acids and leucine concentration appear to be close to what is observed with whey proteins. It is noticeable that chewing capacity improved amino acids availability in older subjects suggesting that protein texture is a determinant of protein digestion rate [29]. Also, when different technological processes were applied to meat preparation, protein digestion rate was affected. Recent reports on the macrostructure and microstructure of the meat have shown that either cooking temperature [30] or meat texture like minced beef [31] affected the speed and the efficiency of protein digestion: high cooking temperature slow down digestion rate whereas minced beef was more rapidly absorbed than beef steak. Recently, intrinsically labeled eggs and poultry meat have been produced with stable isotope through noninvasive oral tracer administration and given to volunteers, to assess in vivo dietary protein digestion and absorption kinetics together with whole-body and muscle protein response to protein intake in humans [19]. Altogether, these works clearly show that labeling milk, meat, or egg proteins with stable isotopes have largely contributed to improve our understanding on dietary protein distribution and assimilation in relation to the quantity and the quality of protein foods in humans.

INTEREST OF “FAST” PROTEINS FOR MUSCLE ANABOLIC RESPONSE Muscle “anabolic resistance” may occur with aging, acute or chronic disease, or during immobilization, meaning that muscle protein synthesis response to meal is blunted. However, skeletal muscle keeps its ability to respond to high and sustained hyperaminoacidemia-induced experimentally by infused or orally administered amino

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acids [32]. This observation suggests that a higher nutritional signals activating protein synthesis pathway, i.e., high plasma amino acids (AA) or insulin, may overcome this metabolic resistance. It also means that the threshold for muscle protein synthesis may be lowered by nutritional, physical, or pharmacological compounds [33,34]. Physiologically, muscle protein synthesis is particularly stimulated by EAAs [35] and leucine [36,37] in older people. Therefore, to counteract sarcopenia, nutritional interventions including different dietary sources designed to increase the availability of amino acids have been proposed to maintain the postprandial stimulation of protein synthesis in older subjects [38] alone or in combination with physical exercise [39]. As described above, the magnitude of changes in amino acids availability can be controlled by amino acids content and composition of dietary proteins [34], but the administration of proteins with different digestion rates can be another nutritional strategy [20]. Whey proteins are interesting dietary proteins for improving muscle anabolism as they have high leucine content and are considered as “fast” proteins [34]. The choice among the various sources of dietary proteins, defined by their amino acid content and digestibility, represents an important basis to optimize skeletal muscle anabolism in response to meal in elderly subjects. Fast soluble proteins, i.e., whey proteins within a mixed meal, was able to induce a higher whole-body and postprandial protein retention than slow proteins, i.e., caseins, in older subjects [24,40]. At the muscle level, when casein, casein hydrolysate, or whey were ingested, a large increase of muscle protein synthesis was obtained with whey protein although protein intake was the same [24]. Interestingly, the peak leucine concentration in this study was strongly related to the stimulation of muscle protein synthesis, emphasizing that plasma AA availability for EAA and leucine is crucial. In a recent study, we have reported that fast soluble milk proteins consumed during 10 days improved postprandial muscle protein synthesis in healthy elderly [23]. The greater anabolic properties of whey protein than of casein is explained by both a rapid digestion and absorption rates and by the higher essential amino acids composition. After ingestion of 15 g of whey protein, muscle protein accretion was even greater than after ingestion of its constitutive EAA (B7 g), suggesting that the beneficial effect of whey on skeletal muscle protein might be attributed to other properties beyond its constitutive essential amino acids [41]. Indeed, whey protein properties are numerous, and its biological and physical qualities have been reviewed in several articles targeting numerous health goals [42,43]. Similarly, in healthy elderly men, a greater stimulation of myofibrillar protein synthesis rates after whey protein ingestion than after the equivalent protein ingested as micellar casein [44] was shown like other reports [24]. The impact of graded amount of whey protein on protein digestion and absorption kinetics and on postprandial muscle protein accretion indicated that the ingestion of 35 g whey protein resulted in a greater stimulation of skeletal muscle protein synthesis compared with the ingestion of 10 or 20 g whey protein in older men [45]. Therefore, it is speculated that the higher and acute but transient rise in essential amino acids concentrations after whey ingestion is the key anabolic factor to trigger muscle protein synthesis, as leucine coingestion with a bolus of pure dietary protein further stimulates postprandial muscle protein synthesis rates in elderly men [46]. A lot of reviews dedicated to the muscle anabolic action of whey protein in different pathophysiological conditions have been consequently published [47 49]. Of note, the stimulatory effect of whey protein on myofibrillar protein synthesis is potentiated by exercise

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in young resistance-training individuals [50] or in older adults [44,51,52], even if this observation is not shared by other authors [53 55], mainly to due methodological discrepancies (different types of proteins, timing of protein synthesis rates measurements, type of tracer infused) [44]. So, consuming whey protein after exercise might be a good strategy to optimize the anabolic effect of exercise on muscle in elderly subjects, since muscle protein synthesis response to exercise is attenuated in this population [56]. Practically, it seems that a 20-g dose of whey protein would be sufficient to reach a maximal stimulation of myofibrillar protein synthetic rate at rest, but it may still be activated up to 40 g in exercised muscle [52,57]. The recent idea is that fast digestive proteins are efficient not only on muscle mass but also on muscle function, notably after physical exercise. Aged rats supplemented with rapidly milk proteins during 2 months in association with moderate physical activity have an increase in spontaneous locomotor activity and improved dynamic and static gait parameters without increase in muscle mass [58]. In 60-year-old participants, significant increases in muscle mass and strength and a reduction in muscle fatigue has been observed following 4-month training coupled with the daily consumption of 10 g of soluble milk proteins [59]. In another study, whey protein concentrated supplementation during 6 months did not potentiate the beneficial effect of resistance training on muscle mass strength and power, and physical function in elderly subjects [53]. This observation might be not surprising since the elderly subjects included in this study presented limited mobility, and it is known that if physical exercise can improve muscle strength in frail old people, nutritional supplements may not have any additional effects [60]. In this context, fast digestive proteins might be of interest for elderly, mainly during the recovery period following diseases. Interestingly, fast-digestive proteins administrated as a bolus could limit muscle protein loss and speed up muscle recovery after a disease-related immobilization during aging [61,62]. Considering the complexity and multifactorial aspects of sarcopenia, several authors have investigated the combined effect of fast digestive protein such as whey protein with other nutrients able to directly or indirectly regulate the processes of muscle protein synthesis in order to limit sarcopenia. In sarcopenic older adults, 3-month intervention of a vitamin D and leucine-enriched whey protein oral nutritional supplement resulted in improvements in muscle mass and lower extremity function [63]. A recent randomized, double-blind, placebo-controlled supplementation trial [64] reported that after 12 weeks intervention, combining physical activity 5 times per week with 32 g of a supplement containing 22 g of whey protein (B4 g of leucine) and 2.5 g of vitamin D one time per day, 68% of the sarcopenic elderly became nonsarcopenic with a gain of B1.7 kg in fat-free mass. These results were concomitant with others improvements in health parameters, such as reduced inflammatory state, increased performance on activities of daily living, and enhanced insulin-like growth factor-1 (IGF-1) concentrations. So adding synergistically acting nutrients like vitamin D, which stimulate in vitro and in vivo muscle protein synthesis [65 68], or polyunsaturated fatty acids like omega-3, which improved insulin sensitivity of muscle protein synthesis to insulin stimulatory effect [69], to whey seems to be a winning strategy. More recently, we tested the hypothesis that all these effects could also be amplified by the timing of meal protein ingestion since we have demonstrated for many years that feeding pattern should be considered [70] but also that we would respect

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the meal protein content of the breakfast by focusing on protein intake during the first part of the day [71]. In a RCT involving older healthy subjects, supplementing breakfast with a vitamin D and leucine-enriched whey protein nutrition drink was able to stimulate short-term postprandial muscle protein synthesis and to increase muscle mass after 6 weeks of intervention in healthy older adults [72]. Altogether, these new nutritional strategies combining different but synergistic nutrients compounds acting on muscle protein synthesis may help to maintain the stimulating effect of fast digestive protein on muscle protein synthesis over a prolonged period by favoring long-term anabolic environment. Muscle protection may also be required in voluntary weight loss situations such as obesity in young or older adults [73]. The prevalence of obesity among older adults is rapidly increasing [74] raising new medical questions about the therapeutic approach where weight loss is sought to get metabolic and functional benefits but where anabolic action of meal is still expected [75,76]. A potential drawback of weight loss in older adults is the accompanying loss of lean body mass [77], which might potentially accelerate the development of sarcopenia [78,79]. Therefore, the nutritional management of sarcopenic obesity may benefit from the knowledge in the geriatric field described above, particularly using whey protein [80]. Recent data have shown that the combination of diet and physical exercise especially resistance plus endurance exercise was the most efficient approach [81,82]. However, the quality of dietary protein should also be tested in the near future to see if this action might be amplified in this population [83,84]. In this way, a high whey protein, leucine-, and vitamin D-enriched supplement compared with isocaloric control has been tested to preserve appendicular muscle mass in obese older adults under a hypocaloric diet and a resistance exercise program. This RCT showed that this synergistic approach may reduce the risk of muscle loss in sarcopenic obese elderly patients [85].

CONCLUSION Dietary protein quality is not only a function of amino acid score and protein digestibility but should also consider its impact on plasma amino acid availability as it is a key stimulatory factor triggering muscle protein synthesis. In line with this concept, a “fast” dietary protein such as whey protein, rich in high essential, branched-chain, and leucine amino acid content and rapidly digestible, has the ability to stimulate muscle protein synthesis in situations where a rapid muscle mass and function recovery is needed such as after critical situations or chronic disease promoting sarcopenia. This concept of “slow” and “fast” protein concept has led to numerous studies involved in optimizing postprandial muscle protein synthesis and muscle preservation in many physiological and clinical conditions. Interestingly, combining “fast” proteins like whey with synergistically metabolic activators of protein synthesis together with physical exercise in the best timing represents innovative multimodal strategies to counteract sarcopenia for the future.

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[23] Walrand S, Gryson C, Salles J, Giraudet C, Migne C, Bonhomme C, et al. Fast-digestive protein supplement for ten days overcomes muscle anabolic resistance in healthy elderly men. Clin Nutr. 2016;35(3):660 8 Epub 2015/05/27. [24] Pennings B, Boirie Y, Senden JM, Gijsen AP, Kuipers H, van Loon LJ. Whey protein stimulates postprandial muscle protein accretion more effectively than do casein and casein hydrolysate in older men. Am J Clin Nutr. 2011;93(5):997 1005 Epub 2011/03/04. [25] Barbe F, Menard O, Le Gouar Y, Buffiere C, Famelart MH, Laroche B, et al. Acid and rennet gels exhibit strong differences in the kinetics of milk protein digestion and amino acid bioavailability. Food Chem. 2014;143:1 8 Epub 2013/09/24. [26] Gorissen SH, Burd NA, Hamer HM, Gijsen AP, Groen BB, van Loon LJ. Carbohydrate coingestion delays dietary protein digestion and absorption but does not modulate postprandial muscle protein accretion. J Clin Endocrinol Metab. 2014;99(6):2250 8 Epub 2014/03/19. [27] Luiking YC, Abrahamse E, Ludwig T, Boirie Y, Verlaan S. Protein type and caloric density of protein supplements modulate postprandial amino acid profile through changes in gastrointestinal behaviour: a randomized trial. Clin Nutr. 2016;35(1):48 58 Epub 2015/03/21. [28] van den Braak CC, Klebach M, Abrahamse E, Minor M, Hofman Z, Knol J, et al. A novel protein mixture containing vegetable proteins renders enteral nutrition products non-coagulating after in vitro gastric digestion. Clin Nutr. 2013;32(5):765 71 Epub 2013/01/01. [29] Remond D, Machebeuf M, Yven C, Buffiere C, Mioche L, Mosoni L, et al. Postprandial whole-body protein metabolism after a meat meal is influenced by chewing efficiency in elderly subjects. Am J Clin Nutr. 2007;85 (5):1286 92 Epub 2007/05/11. [30] Bax ML, Buffiere C, Hafnaoui N, Gaudichon C, Savary-Auzeloux I, Dardevet D, et al. Effects of meat cooking, and of ingested amount, on protein digestion speed and entry of residual proteins into the colon: a study in minipigs. PLoS ONE. 2013;8(4):e61252 Epub2013/04/18. [31] Pennings B, Groen BB, van Dijk JW, de Lange A, Kiskini A, Kuklinski M, et al. Minced beef is more rapidly digested and absorbed than beef steak, resulting in greater postprandial protein retention in older men. Am J Clin Nutr. 2013;98(1):121 8 Epub 2013/05/03. [32] Rasmussen BB, Wolfe RR, Volpi E. Oral and intravenously administered amino acids produce similar effects on muscle protein synthesis in the elderly. J Nutr Health Aging 2002;6(6):358 62 Epub 2002/12/03. [33] Dardevet D, Remond D, Peyron MA, Papet I, Savary-Auzeloux I, Mosoni L. Muscle wasting and resistance of muscle anabolism: the “anabolic threshold concept” for adapted nutritional strategies during sarcopenia. The Scientific World Journal. 2012;2012 269531. Epub 2013/01/18. [34] Walrand S, Boirie Y. Optimizing protein intake in aging. Curr Opin Clin Nutr Metab Care. 2005;8(1):89 94 Epub 2004/12/09. [35] Volpi E, Kobayashi H, Sheffield-Moore M, Mittendorfer B, Wolfe RR. Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults. Am J Clin Nutr. 2003;78(2):250 8 Epub 2003/07/30. [36] Casperson SL, Sheffield-Moore M, Hewlings SJ, Paddon-Jones D. Leucine supplementation chronically improves muscle protein synthesis in older adults consuming the RDA for protein. Clin Nutr. 2012;31 (4):512 19 Epub 2012/02/24. [37] van Loon LJ. Leucine as a pharmaconutrient in health and disease. Curr Opin Clin Nutr Metab Care. 2012;15 (1):71 7 Epub 2011/11/01. [38] Lancha Jr AH, Zanella Jr R, Tanabe SG, Andriamihaja M, Blachier F. Dietary protein supplementation in the elderly for limiting muscle mass loss. Amino Acids. 2017;49(1):33 47 Epub 2016/11/04. [39] Cermak NM, Res PT, de Groot LC, Saris WH, van Loon LJ. Protein supplementation augments the adaptive response of skeletal muscle to resistance-type exercise training: a meta-analysis. Am J Clin Nutr 2012;96 (6):1454 64 Epub 2012/11/09. [40] Dangin M, Guillet C, Garcia-Rodenas C, Gachon P, Bouteloup-Demange C, Reiffers-Magnani K, et al. The rate of protein digestion affects protein gain differently during aging in humans. J Physiol. 2003;549(Pt 2):635 44 Epub 2003/04/01. [41] Katsanos CS, Chinkes DL, Paddon-Jones D, Zhang XJ, Aarsland A, Wolfe RR. Whey protein ingestion in elderly persons results in greater muscle protein accrual than ingestion of its constituent essential amino acid content. Nutr Res. 2008;28(10):651 8 Epub 2008/12/17.

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[61] Magne H, Savary-Auzeloux I, Migne C, Peyron MA, Combaret L, Remond D, et al. Contrarily to whey and high protein diets, dietary free leucine supplementation cannot reverse the lack of recovery of muscle mass after prolonged immobilization during ageing. J Physiol. 2012;590(8):2035 49 Epub 2012/02/22. [62] Martin V, Ratel S, Siracusa J, Le Ruyet P, Savary-Auzeloux I, Combaret L, et al. Whey proteins are more efficient than casein in the recovery of muscle functional properties following a casting induced muscle atrophy. PLoS ONE. 2013;8(9):e75408 Epub2013/09/27. [63] Bauer JM, Verlaan S, Bautmans I, Brandt K, Donini LM, Maggio M, et al. Effects of a vitamin D and leucineenriched whey protein nutritional supplement on measures of sarcopenia in older adults, the PROVIDE study: a randomized, double-blind, placebo-controlled trial. J Am Med Dir Assoc. 2015;16(9):740 7 Epub 2015/07/15. [64] Rondanelli M, Klersy C, Terracol G, Talluri J, Maugeri R, Guido D, et al. Whey protein, amino acids, and vitamin D supplementation with physical activity increases fat-free mass and strength, functionality, and quality of life and decreases inflammation in sarcopenic elderly. Am J Clin Nutr. 2016;103(3):830 40 Epub 2016/02/13. [65] Salles J, Chanet A, Giraudet C, Patrac V, Pierre P, Jourdan M, et al. 1,25(OH)2-vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes. Mol Nutr Food Res. 2013;57(12):2137 46 Epub 2013/08/10. [66] Chanet A, Salles J, Guillet C, Giraudet C, Berry A, Patrac V, et al. Vitamin D supplementation restores the blunted muscle protein synthesis response in deficient old rats through an impact on ectopic fat deposition. J Nutral biochemistry. 2017;46:30 8 Epub 2017/04/27. [67] Domingues-Faria C, Boirie Y, Walrand S. Vitamin D and muscle trophicity. Curr Opin Clin Nutr Metab Care. 2017;20(3):169 74 Epub 2017/03/04. [68] Verlaan S, Maier AB, Bauer JM, Bautmans I, Brandt K, Donini LM, et al. Sufficient levels of 25hydroxyvitamin D and protein intake required to increase muscle mass in sarcopenic older adults—the PROVIDE study. Clin Nutr. 2018;37(2):551 7 Epub 2017/01/31. [69] Smith GI, Julliand S, Reeds DN, Sinacore DR, Klein S, Mittendorfer B. Fish oil-derived n-3 PUFA therapy increases muscle mass and function in healthy older adults. Am J Clin Nutr. 2015;102(1):115 22 Epub 2015/ 05/23. [70] Arnal MA, Mosoni L, Boirie Y, Houlier ML, Morin L, Verdier E, et al. Protein pulse feeding improves protein retention in elderly women. Am J Clin Nutr. 1999;69(6):1202 8 Epub 1999/06/05. [71] Paddon-Jones D, Rasmussen BB. Dietary protein recommendations and the prevention of sarcopenia. Curr Opin Clin Nutr Metab Care. 2009;12(1):86 90 Epub 2008/12/06. [72] Chanet A, Verlann S, Salles J, Giraudet C, Patrac V, Pidou V, et al. Supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink enhances postprandial muscle protein synthesis and muscle mass in healthy older men. J Nutr. 2017;147(12):2262 71. [73] Guillet C, Masgrau A, Walrand S, Boirie Y. Impaired protein metabolism: interlinks between obesity, insulin resistance and inflammation. Obes Rev. 2012;13(Suppl 2):51 7 Epub 2012/11/01. [74] Mathus-Vliegen EM. Prevalence, pathophysiology, health consequences and treatment options of obesity in the elderly: a guideline. Obes Facts. 2012;5(3):460 83 Epub 2012/07/17. [75] Bales CW, Buhr G. Is obesity bad for older persons? A systematic review of the pros and cons of weight reduction in later life. J Am Med Dir Assoc. 2008;9(5):302 12 Epub 2008/06/04. [76] Bischoff SC, Boirie Y, Cederholm T, Chourdakis M, Cuerda C, Delzenne NM, et al. Towards a multidisciplinary approach to understand and manage obesity and related diseases. Clin Nutr 2017;36(4):917 38 Epub 2016/11/29. [77] Houston DK, Nicklas BJ, Zizza CA. Weighty concerns: the growing prevalence of obesity among older adults. J Am Diet Assoc 2009;109(11):1886 95 Epub 2009/10/28. [78] Villareal DT, Apovian CM, Kushner RF, Klein S. Obesity in older adults: technical review and position statement of the American Society for Nutrition and NAASO, The Obesity Society. Obes Res 2005;13(11):1849 63 Epub 2005/12/13. [79] Wolfe RR. The underappreciated role of muscle in health and disease. Am J Clin Nutr. 2006;84(3):475 82 Epub 2006/09/09. [80] Jakubowicz D, Froy O. Biochemical and metabolic mechanisms by which dietary whey protein may combat obesity and Type 2 diabetes. J Nutr Biochem. 2013;24(1):1 5 Epub 2012/09/22.

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[81] Villareal DT, Aguirre L, Gurney AB, Waters DL, Sinacore DR, Colombo E, et al. Aerobic or resistance exercise, or both, in dieting obese older adults. N Engl J Med 2017;376(20):1943 55 Epub 2017/05/18. [82] Villareal DT, Chode S, Parimi N, Sinacore DR, Hilton T, Armamento-Villareal R, et al. Weight loss, exercise, or both and physical function in obese older adults. N Engl J Med. 2011;364(13):1218 29 Epub 2011/04/01. [83] Walrand S, Zangarelli A, Guillet C, Salles J, Soulier K, Giraudet C, et al. Effect of fast dietary proteins on muscle protein synthesis rate and muscle strength in ad libitum-fed and energy-restricted old rats. Br J Nutr 2011;106(11):1683 90 Epub 2011/07/09. [84] Hector AJ, Marcotte GR, Churchward-Venne TA, Murphy CH, Breen L, von Allmen M, et al. Whey protein supplementation preserves postprandial myofibrillar protein synthesis during short-term energy restriction in overweight and obese adults. J Nutr. 2015;145(2):246 52 Epub 2015/02/04. [85] Verreijen AM, Verlaan S, Engberink MF, Swinkels S, de Vogel-van den Bosch J, Weijs PJ. A high whey protein-, leucine-, and vitamin D-enriched supplement preserves muscle mass during intentional weight loss in obese older adults: a double-blind randomized controlled trial. Am J Clin Nutr. 2015;101(2):279 86 Epub 2015/02/04.

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Branched-Chain Amino Acids (Leucine, Isoleucine, and Valine) and Skeletal Muscle Stefan H.M. Gorissen and Stuart M. Phillips Department of Kinesiology, McMaster University, Hamilton, ON, Canada

LIST OF ABBREVIATIONS 4E-BP1 BCAA CoA DEPTOR EAA eIF4E KIC KIV KMV mLST8 mTOR mTORC1 NEAA PRAS40 Raptor RDA rpS6 S6K1

eIF4E-binding protein 1 branched-chain amino acid Coenzyme A DEP domain containing mTOR-interacting protein essential amino acid eukaryotic translation initiation factor 4E α-keto acids α-ketoisocaproic acid α-ketoisovaleric acid α-keto-β-methylvaleric acid mammalian lethal with sec-13 protein 8 mechanistic target of rapamycin mTOR complex 1 nonessential amino acid proline-rich Akt substrate 40 kDa regulatory-associated protein of mechanistic target of rapamycin recommended dietary allowance ribosomal protein S6 S6 kinase 1

INTRODUCTION The branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, comprise B17% of total amino acids present in human skeletal muscle [1,2]. Besides containing an amino ( NH2) and carboxyl ( COOH) group, which are common to all amino acids,

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BCAAs contain an aliphatic side-chain that is nonlinear [3] (Fig. 16.1). BCAAs are essential amino acids (EAAs) that cannot be synthesized by the human body and thus must be provided by the diet [4]. Dietary protein provides amino acids that can be used for (whole body) protein synthesis. Following ingestion, dietary protein is digested and absorbed before dietary protein-derived amino acids become available in circulation [5]. Amino acids are subsequently available to peripheral tissues including skeletal muscle. Though multiple food sources contain protein, the amino acid composition (and BCAA content) can vary substantially between dietary protein sources. Food sources that provide a substantial amount of our daily protein intake include milk, eggs, corn, soy, fish, rice, beef, beans, wheat, and potatoes [6]. Table 16.1 provides an overview of these food sources and their respective leucine, isoleucine, valine, and total BCAA content [7]. The BCAA compoFIGURE 16.1 Molecular structure of the branched-chain amino acids. Besides containing an amino ( NH2) and carboxyl ( COOH) group, which is common to all amino acids, branched-chain amino acids contain an aliphatic sidechain that is nonlinear.

TABLE 16.1 Branched-Chain Amino Acid Composition of Various Food Sources and Skeletal Muscle Leucine, % of total protein

Isoleucine, % of total protein

Valine, % of total protein

Total BCAAs, % of total protein

Milk

12.3

6.3

7.3

26

Eggs

8.8

6.3

6.8

22

Maize

12.5

3.7

4.8

21

Soy beverage

8.7

5.3

5.1

19

Soybeans

8.5

5.0

5.3

19

Fish

7.7

4.8

6.1

19

Rice

8.6

4.0

5.8

18

Beef

8.1

4.8

5.0

18

Beans

7.6

4.2

4.6

16

Wheat

7.2

3.5

4.7

15

Potatoes

6.0

3.8

4.7

14

Skeletal muscle

7.5

4.0

5.0

17

Food source

Human skeletal muscle is shown as a reference value. Data are derived from FAO Nutritional Studies [7]. BCAAs, branchedchain amino acids.

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sition of human skeletal muscle is provided as a reference point. The BCAA content of these food sources ranges between 14% and 26% of total protein and is relatively low in potatoes, wheat, and beans (plant-based), whereas milk and eggs (animal-derived) have a high BCAA content. A high BCAA and/or leucine content is generally desirable for stimulating protein synthesis in skeletal muscle tissue. The main aim of this chapter is to provide an overview of the current understanding of the role of BCAAs in stimulating muscle protein synthesis and promoting muscle growth. Besides their role in anabolism, we will briefly discuss BCAA catabolism and the hypothesis that BCAAs may be involved in the etiology of insulin resistance.

BCAA REQUIREMENT AND TOLERABLE UPPER INTAKE LEVEL An important question is what quantity of BCAAs should be consumed to maintain muscle mass or, more importantly, body function? A method for determining amino acid requirements is the indicator amino acid oxidation method [8]. This technique is based on the concept that amino acids cannot be stored and therefore must either be incorporated into protein or deaminated and oxidized. When the amino acid of interest is deficient for protein synthesis, then all other amino acids (including the labeled indicator amino acid, often L-[1-13C]-phenylalanine) will be oxidized. Oxidation of the indicator amino acid can be determined by collecting breath samples and measuring 13CO2 in exhaled breath. With increasing intakes of the amino acid of interest, oxidation of the indicator amino acid will decrease, indicating increased incorporation of the amino acid of interest into protein. Once the requirement of the amino acid of interest is met, there will be no further change in oxidation of the indicator amino acid. The requirement for BCAAs has been determined by providing a mix of all three BCAAs with a composition as found in egg protein at doses ranging from 26 to 180 mg/kg/day [2]. The rate of 13CO2 release did not further decline after a BCAA intake of 144 mg/kg/day, indicating that the total BCAA requirement is 144 mg/kg/day [2]. Based on the BCAA requirement and the BCAA content of various food sources, we calculated as an illustrative example the amount of protein as well as the amount of food product that should be consumed per day to meet the requirements for BCAA intake (Table 16.2). For the selected food sources, between 42 and 75 g protein needs to be consumed per day, which translates to e.g., as little as B150 g soybeans to as much as 3.7 kg potatoes. Certainly, a normal diet is composed of a combination of various food products that provide BCAAs. Besides consuming food products that provide BCAAs, the use of BCAA supplements is becoming increasingly popular [9]. Particularly, supplements containing free leucine are often used by athletes to improve performance and by older or clinical populations to support muscle mass maintenance. It has been questioned whether excessive leucine intake may have adverse effects [9]. In an effort to determine the tolerable upper intake level of leucine, Elango et al. [10] provided adult men with graded doses of L-[1-13C]-leucine ranging from 50 to 1250 mg/kg/day. With increasing leucine intakes, the oxidation of leucine (determined by measuring 13CO2 in exhaled breath) increased until a plateau was reached. The plateau was reached at 550 mg/kg/day, which indicates the dose at which the oxidative process is unable to keep up with the increasing leucine provision. As a result,

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TABLE 16.2 Amount of Protein and Amount of Food Product that Provides the Daily Branched-Chain Amino Acid Requirement Food source

Amount of protein, g/day

Amount of food product, g/day

Milk

42

1194

Eggs

49

398 (B7 eggs)

Corn

51

540

Soy beverage

56

1760

Soybeans

58

152

Fish

58

309

Rice

59

782

Beef

60

340

Beans

66

298

Wheat

70

576

Potatoes

75

3729

Based on 75 kg body mass. Total branched-chain amino acid requirement is 144 mg/kg/day.

accretion of free leucine in body tissue pools will occur, which could potentially be harmful. Therefore, it has been suggested that intakes of leucine below 550 mg/kg/day are safe for human health.

CATABOLISM OF BCAAs Catabolism of amino acids involves the removal of the amino group, followed by the breakdown of the resulting carbon skeleton. In contrast to other amino acids, BCAAs are metabolized primarily by the peripheral tissues (particularly muscle), rather than by the liver [11]. The first step in the catabolism of the BCAAs is transamination to remove the amino group, which is catalyzed by BCAA aminotransferase. Transamination of leucine, isoleucine, and valine results in the production of the α-keto acids α-ketoisocaproic acid (KIC), α-keto-β-methylvaleric acid (KMV), and α-ketoisovaleric acid (KIV), respectively. The second step in BCAA catabolism is oxidative decarboxylation that results in the removal of the carboxyl group of the α-keto acids, which is catalyzed by the branchedchain α-keto acid dehydrogenase complex. Oxidative decarboxylation of KIC, KMV, and KIV results in the production of isovaleryl coenzyme A (CoA), α-methylbutyryl CoA, and isobutyryl CoA, respectively. Catabolism of leucine ultimately yields acetoacetate and acetyl CoA, isoleucine is metabolized to succinyl CoA and acetyl CoA, and valine yields succinyl CoA [11] (Fig. 16.2, [12]). The three intermediate products derived from BCAA catabolism (i.e., succinyl CoA, acetyl CoA, and acetoacetate) directly enter the pathways of intermediary metabolism,

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FIGURE 16.2 Branched-chain amino acid catabolism. Step 1, transamination (removal of the amino group): branched-chain amino acid aminotransferase catalyzes the conversion of leucine, isoleucine, and valine into α-ketoisocaproic acid, α-keto-β-methylvaleric acid, and α-ketoisovaleric acid, respectively. Step 2, oxidative decarboxylation (removal of the carboxyl group): the α-keto acids are further metabolized by the branched-chain α-keto acid dehydrogenase complex to form isovaleryl CoA, α-methylbutyryl CoA, and isobutyryl CoA, respectively. Catabolism of leucine ultimately yields acetoacetate and acetyl CoA, isoleucine is metabolized to succinyl CoA and acetyl CoA, and valine yields succinyl CoA. CoA, coenzyme A. Adapted from Block KP. Interactions among leucine, isoleucine, and valine with special reference to the branched chain amino acid antagonism. Volume 1 ed. Friedman M, editor. Boca Raton, FL: CRC Press; 1989.

resulting either in the synthesis of glucose (gluconeogenesis) or in the production of adenosine triphosphate (ATP) through their oxidation by the citric acid cycle [11]. Amino acids can be classified as glucogenic, ketogenic, or both based on which of the three intermediates are produced during their catabolism [11]. Catabolism of valine yields succinyl CoA, which is one of the intermediates of the citric acid cycle and a substrate for gluconeogenesis (can give rise to the formation of glucose in the liver and kidney). As such, valine is a glucogenic amino acid. Leucine catabolism yields acetyl CoA and acetoacetate, which feed into the citric acid cycle, but their carbon skeletons cannot be used for gluconeogenesis. Therefore, leucine is considered a ketogenic amino acid. Isoleucine catabolism yields succinyl CoA as well as acetyl CoA, rendering it both glucogenic and ketogenic [11] (Fig. 16.3).

mTOR SIGNALING AND MUSCLE PROTEIN SYNTHESIS Maintenance of skeletal muscle mass is of major importance to maintain functional capacity and metabolic health. Muscle mass maintenance is regulated by fluctuations in muscle protein synthesis and breakdown rates that are most often balanced such that net

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FIGURE 16.3 Metabolism of the intermediate products derived from branched-chain amino acid catabolism. Valine catabolism yields succinyl CoA, which is one of the intermediates of the citric acid cycle and a substrate for gluconeogenesis (can give rise to the formation of glucose). Leucine catabolism yields acetyl CoA and acetoacetate, which is used for the production of energy by the citric acid cycle. Isoleucine catabolism yields succinyl CoA as well as acetyl CoA. CoA, coenzyme A. Adapted from Harvey RA, Ferrier DR. Amino acid degradation and synthesis. 5th ed. Harvey RA, editor. Baltimore, MD: Wolters Kluwer|Lippincott Williams & Wilkins; 2011.

muscle protein balance remains zero by the end of each day [13]. In the postabsorptive or fasting state muscle protein breakdown is equal to or exceeds muscle protein synthesis, resulting in a zero or negative net protein balance. Food intake, and protein ingestion in particular, stimulates muscle protein synthesis by providing substrate (i.e., amino acids) for the production of new muscle proteins. Food intake also reduces muscle protein breakdown via the inhibitory effect of the postprandial rise in circulating insulin. The postprandial increase in muscle protein synthesis and decrease in muscle protein breakdown results in a positive net protein balance. Physical activity or exercise increases both muscle protein synthesis and breakdown rates, but synthetic rates are stimulated to a greater extent [14]. Moreover, physical activity or exercise further enhances the muscle protein synthetic response to food intake, thereby further improving net protein balance. A positive net protein balance over a prolonged period of time may result in gains in skeletal muscle mass. Recent research has aimed at identifying dietary factors responsible for stimulating muscle protein synthesis and thereby promoting gains in skeletal muscle mass in both resting and exercise conditions. Upon digestion and absorption, dietary protein provides amino acids for synthesis of new muscle proteins. Besides serving as substrate, certain amino acids can directly activate the muscle protein synthetic machinery by activating mechanistic target of rapamycin (mTOR) signaling. Activation of mTOR signaling regulates various cellular processes

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including protein synthesis, lipid synthesis, autophagy, lysosome biogenesis, and energy metabolism [15]. A central protein complex within this signaling pathway is the mTOR complex 1 (mTORC1), which is composed of the catalytic mTOR subunit, mammalian lethal with sec-13 protein 8 (mLST8), DEP domain containing mTOR-interacting protein (DEPTOR), the Tti1/Tel2 complex, regulatory-associated protein of mTOR (Raptor), and proline-rich Akt substrate 40 kDa (PRAS40) [15]. mTORC1 can sense a variety of signals including growth factors, stress, energy status, oxygen, and amino acids [15]. The exact mechanism by which mTORC1 can sense amino acids remains unknown, but it involves mitogen-activated protein kinase kinase kinase kinase 3, mammalian vacuolar protein sorting 34 homolog, inositol polyphosphate monokinase, the Rag Ragulator system, and the recently discovered Sestrin2 [15 18]. Sestrin2 contains a leucine-binding pocket with an additional lid-latch mechanism that helps lock leucine in place and is required for binding [17]. Leucine binding causes dissociation of Sestrin2 from GATOR2 [18]. The separate GATOR2 complex negatively regulates GATOR1, which allows the Rag Ragulator system to bring mTORC1 onto the lysosomal surface where mTORC1 can bind to and becomes activated [16] (Fig. 16.4). Upon activation, mTORC1 directly phosphorylates the eukaryotic

FIGURE 16.4 Leucine-induced activation of mTORC1. Sestrin2 contains a leucine-binding pocket with an additional lid-latch mechanism that helps lock leucine in place and is required for binding. Leucine binding causes dissociation of Sestrin2 from GATOR2. The separate GATOR2 complex negatively regulates GATOR1, which allows the Rag Ragulator system to bring mTORC1 onto the lysosomal surface where mTORC1 can bind to and becomes activated. mTORC1, mechanistic target of rapamycin complex 1. Adapted from Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012;149(2):274 93; Chantranupong L, Wolfson RL, Orozco JM, Saxton RA, Scaria SM, Bar-Peled L, et al. The Sestrins interact with GATOR2 to negatively regulate the amino-acidsensing pathway upstream of mTORC1. Cell Rep 2014;9(1):1 8; Saxton RA, Knockenhauer KE, Wolfson RL, Chantranupong L, Pacold ME, Wang T, et al. Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway. Science 2016;351(6268):53 8; Wolfson RL, Chantranupong L, Saxton RA, Shen K, Scaria SM, Cantor JR, et al. Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 2016;351(6268):43 8.

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translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1). Phosphorylation of 4E-BP1 prevents its binding to eIF4E, enabling eIF4E to participate in the formation of the eIF4F complex that is required for the translation initiation and phosphorylation of S6K1 leads, through a variety of effectors, to an increase in RNA biogenesis as well as translational initiation and elongation, both promoting protein synthesis [15]. Using murine C2C12 myocytes, Atherton et al. [3] observed that non-EAAs (NEAAs) had no effect in stimulating mTOR signaling. Of the EAAs, leucine induced a twofold increase in mTOR and 4E-BP1 phosphorylation and a fivefold increase in S6K1 phosphorylation [3]. Interestingly, other EAAs including lysine, methionine, phenylalanine, tryptophan, and threonine induced B2-fold increases in the phosphorylation of S6K1 and its downstream signaling target ribosomal protein S6, while isoleucine and valine did not have such effect [3]. This is in line with much earlier work by Buse and Reid [19] who assessed the incorporation of 14C-lysine into muscle protein (indicative of muscle protein synthesis) in rat diaphragm muscle following incubation with BCAAs. They observed that a mixture of all three BCAAs increased ex vivo muscle protein synthesis rates. In a separate experiment, they showed that leucine alone stimulated muscle protein synthesis as effectively, indicating that leucine, but not isoleucine and valine, stimulates muscle protein synthesis [19]. Recent work in humans confirms these findings and shows that the ingestion of leucine further increases the phosphorylation of mTOR and S6K1 and tends to stimulate muscle protein synthesis to a greater extent when compared with the ingestion of an EAA mixture devoid of leucine after resistance exercise [20]. Together, these data provide evidence that leucine is sensed by Sestrin2, activating mTORC1, and leading to activation of downstream signaling and subsequent stimulation of muscle protein synthesis.

ACUTE EFFECTS OF BCAA INTAKE ON MUSCLE PROTEIN SYNTHESIS Leucine has been shown to be one of the key regulators of mTOR signaling and subsequent translation initiation. To our knowledge, no studies have investigated the individual effects of isoleucine or valine or the effect of ingesting all three BCAAs on postprandial muscle protein synthesis rates, whereas leucine has been studied extensively. Katsanos et al. [21] assessed basal and postprandial muscle protein synthesis rates in both young and older individuals and showed that a mixture of all EAAs stimulated muscle protein synthesis in young individuals, whereas postprandial muscle protein synthesis rates were not elevated from basal values in the older individuals. The lower postprandial muscle protein synthetic response in older individuals has been referred to as anabolic resistance [22] and has been suggested to be one of the factors responsible for the age-related loss of skeletal muscle mass (i.e., sarcopenia) [23]. In an effort to enhance the postprandial muscle protein synthetic response, Katsanos et al. [21] increased the leucine content of an EAA mixture from 1.7 to 2.8 g. The ingestion of this dose of leucine-enriched EAAs resulted in a stimulation of muscle protein synthesis in older individuals [21]. Also, when lower quantities of dietary protein are enriched with free leucine, postprandial muscle protein synthesis rates [24] as well as the incorporation of dietary protein derived amino acids into skeletal muscle protein [25] are higher when compared with the ingestion of protein only. Based on these data and the

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dose of leucine provided in these studies, it is generally recommended for older individuals that at least 2.5 g leucine should be ingested per meal to ensure a significant stimulation of muscle protein synthesis per meal as a strategy to attenuate the loss of skeletal muscle mass with aging and delay the onset of sarcopenia [26].

THE LEUCINE CONTENT OF DIETARY PROTEIN: A DETERMINANT OF THE ANABOLIC POTENTIAL? The leucine content of a meal can depend on the source of protein consumed since the leucine content of dietary protein can vary substantially between various protein sources [27 30] (Table 16.1). Multiple studies have compared various dietary protein sources and observed that the postprandial muscle protein synthetic response can vary substantially between various protein sources [27 35]. The question arises whether these differences in the postprandial muscle protein synthetic response are caused by differences in the leucine content of the ingested protein source. A number of studies in which free leucine was added to the ingested protein source have shown an improved muscle protein synthetic response [21,24,25,28]. Another strategy to increase the leucine content of the meal is to increase the amount of protein consumed. Norton et al. [32] assessed muscle protein synthesis rates in rats fed a diet composed of wheat protein compared to whey protein (providing 6.8% compared to 10.9% leucine, respectively) and observed that muscle protein synthesis rates are lower on a wheat protein compared to whey protein diet. By increasing the amount of wheat protein in the diet, muscle protein synthesis rates increased and reached similar values as compared to whey protein [32]. We recently confirmed that the postprandial muscle protein synthetic response to wheat protein is lower when compared with dairy protein in humans [27]. Interestingly, increasing the amount of wheat protein consumed to match for the leucine content of whey protein resulted in an even greater postprandial stimulation of muscle protein synthesis. These data [27] suggest that the leucine content of the protein source may not be the only amino acid responsible for determining the postprandial increase in muscle protein synthesis rates. Nevertheless, the amount of protein consumed may be modified to match the anabolic properties of a certain protein source. It is not surprising that the leucine content of the dietary protein source is not the only factor determining the postprandial muscle protein synthetic response. Leucine is mainly responsible for translation initiation. When ample leucine is consumed (B2.5 g leucine) and the muscle protein synthetic machinery is activated, other amino acids would likely start to become important as they are utilized as substrates to support elongation of the polypeptide chain. The postprandial availability of amino acids for protein synthesis can be influenced by a number of processes. First, the rate of protein digestion and amino acid absorption may modulate postprandial amino acid availability. For example, a rapidly digestible protein such as whey protein may result in a greater hyperaminoacidemia for a shorter period, while a slowly digestible protein such as casein results in a more moderate release of amino acids over a more prolonged time period [29,36]. Second, utilization of dietary protein-derived amino acids by splanchnic tissues (which includes the gut and liver) modulates the postprandial availability of dietary protein-derived amino acids for

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muscle protein synthesis [37,38]. In addition, the postprandial rise in circulating insulin may play an important role by enhancing skeletal muscle perfusion and delivery of amino acids to the muscle [39 41]. Finally, uptake of amino acids via amino acid transporters located on the cell membrane of the muscle cell may regulate the intracellular availability of dietary protein-derived amino acids [42]. The type, source, and amount of protein consumed, other components of the meal such as carbohydrates or fat [43,44], as well as the level of physical activity may affect one or more of the aforementioned processes. As such, it is important to assess long-term effects of leucine supplementation on skeletal muscle mass and function.

CHRONIC EFFECTS OF BCAA SUPPLEMENTATION AND MUSCLE GROWTH Muscle protein synthesis rates fluctuate throughout the day, with the lower rates being observed on waking or between meals and the higher rates being observed after meal intake. The upper and lower level of muscle protein synthesis rates are generally measured in a laboratory setting where subjects arrive in the laboratory in the overnight fasting state and lie in a supine position to measure basal muscle protein synthesis rates. Next, amino acids or protein are consumed to measure postprandial muscle protein synthesis rates over a period of several hours. This method has been used extensively by multiple laboratories to compare interventions or populations on their capacity to stimulate muscle protein synthesis from basal rates. However, the values observed in a laboratory setting do not necessarily reflect muscle protein synthesis rates observed during freeliving conditions where people consume multiple meals or snacks per day and perform activities of daily living. Recent advances in the field of tracer methodology have introduced the use of deuterium-labeled water for measuring daily muscle protein synthesis rates in free-living conditions over a period of days or weeks. The integrated protein synthetic response incorporates all acute fluctuations in muscle protein synthesis rates throughout the day. In addition, the acute muscle protein synthetic response as measured in a laboratory setting may change over time following habituation to the intervention [45]. As such, longer term measurements of muscle protein synthesis rates under freeliving conditions are warranted. We have recently conducted an experiment to investigate the effects of leucine supplementation on integrated rates of muscle protein synthesis over a period of 3 days. We observed that coingestion of 5 g leucine with each of the three main meals enhances integrated muscle protein synthesis rates in free-living older men [46]. The observed leucine-induced increase in muscle protein synthesis was independent of the amount of protein being consumed (0.8 or 1.2 g protein per kg body mass per day). Thus, in addition to the acute stimulatory effect of leucine coingestion with a single meal, leucine supplementation appears to be an effective strategy to enhance daily muscle protein synthesis rates. All the evidence provided so far strongly suggests that leucine could be used as a nutraceutical to effectively increase skeletal muscle mass. It could be hypothesized that coingesting 2.5 g leucine with each meal maximizes the muscle protein synthetic response to each meal and, as such, results in gains in skeletal muscle mass over a

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prolonged period of time. Verhoeven et al. [47] tested this hypothesis and assessed muscle mass and strength in healthy older men before and after 3 months of supplementing 2.5 g leucine with each of the main meals. In contrast to the hypothesis, leucine supplementation did not increase skeletal muscle mass [47]. The authors speculated that the study population may have been too healthy and/or the intervention period too short to detect significant changes. Leenders et al. [48] performed a follow-up study applying the same intervention over a 6-month period in type 2 diabetes mellitus patients, who generally show an accelerated loss of skeletal muscle mass. Despite the more compromised study population and the more prolonged intervention period, no gains in skeletal muscle mass or strength were observed [48]. Participants in both studies consumed B1.0 g protein per kg body mass per day, which is above the recommended dietary allowance (RDA) for protein intake of 0.8 g/kg/day. This level of protein intake has been suggested to be sufficient for maintaining skeletal muscle mass, and leucine supplementation might not have been able to further enhance skeletal muscle mass. On the other hand, the acute muscle protein synthetic response to leucine coingestion with a meal may have declined over time following a prolonged period of habituation and/or the supplementation period was simply too short to detect a change in skeletal muscle. A higher or an increasing dose of leucine might have been required to promote gains in skeletal muscle mass. Certainly, leucine dose-response studies are warranted to determine the optimal dose of leucine. Protein supplementation has been shown to augment resistance training induced gains in skeletal muscle mass and strength [49]. However, protein supplementation in the absence of resistance exercise fails to enhance skeletal muscle mass [50]. Even though resistance exercise provides a strong stimulus for muscle protein synthesis and sensitizes skeletal muscle tissue to the anabolic properties of dietary protein [51], performing resistance exercise is not always desirable or feasible. There is an intense search for alternative strategies to enhance the anabolic properties of dietary protein in the absence of exercise. Based on numerous studies indicating leucine as a strong anabolic signal, nutrition companies have developed nutritional supplements combining protein and leucine. In theory, the free leucine in this supplement is rapidly absorbed by the gut, transported to, and taken up by the muscle where it reaches the leucine threshold for maximal activation of translation initiation. Subsequently, the protein is digested and absorbed, and the resultant aminoacidemia would provide a complete spectrum of amino acids for maintenance of elevated muscle protein synthesis rates. Bauer et al. [52] conducted a study to assess whether a leucine-enriched protein supplement had the capacity to promote gains in skeletal muscle mass and/or strength. Supplements were provided to frail older individuals twice daily before breakfast and lunch, as these meals generally contains a lower amount of protein [53]. After 13 weeks of supplementing leucine enriched protein, both muscle mass as well as chair-stand time (functional outcome measure) were significantly improved versus a group receiving placebo [52]. It should be noted that this nutritional supplement contained a variety of components including vitamin D. As such, this study does not allow a conclusion regarding the individual effects of the different dietary components. Nonetheless, this leucine-enriched protein supplement was shown to be effective in improving muscle mass and may be used as a nutritional strategy to combat muscle loss in health and disease. For example, this leucine-enriched protein supplement has

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successfully been used to preserve muscle mass during weight loss in obese older adults [54], while supplementing protein only is not able to preserve skeletal muscle mass during energy restriction [55]. It remains to be investigated whether this supplement could be applied in other settings such as immobilization or bed rest or in patient populations who suffer from accelerated muscle loss.

BCAAs AND INSULIN RESISTANCE Several decades ago, Felig et al. [56] were the first to discover that fasting concentrations of circulating BCAAs are higher in obese when compared with healthy individuals, and that BCAA concentrations directly correlated with insulin concentrations. Recently, Wang et al. [57] performed metabolite profiling in a longitudinal cohort of .2400 normoglycemic individuals, of which B200 developed diabetes within the 12-year follow-up period. Metabolite profiling on blood samples obtained at baseline revealed that concentrations of BCAAs were correlated with development of diabetes [57]. Based on these and many other studies [56 60], it has been speculated that BCAAs may be playing a role in the pathogenesis of type 2 diabetes. Such a thesis raises several questions. First, why do levels of BCAAs rise in obese insulin-resistant individuals? Plasma BCAA concentrations would be a function of the rate of appearance of BCAAs and the rate of disappearance from the circulation. Increased BCAA levels must be attributed to an imbalance between their rates of appearance and disappearance. Thus, an important question is whether there is an increased rate of appearance, reduced disappearance, or both? It has been suggested that the rise in circulating BCAAs is driven by a decline in BCAA catabolism in adipose tissue, possibly accompanied by contributions from the diet (increased intake of protein and fat), genetic differences in BCAA or protein turnover, and/or de novo synthesis of BCAAs by the gut microbiome [59]. Accumulation of BCAAs in the circulation increases flux of these amino acids into skeletal muscle and liver and through the catabolic pathway, which is supported by the finding that not only BCAAs but also intermediates of BCAA catabolism (including C3 and C5 acylcarnitines) are elevated in obese persons when compared with lean individuals and are associated with insulin resistance [60]. It has been speculated that the enhanced flux of BCAAs through catabolic pathways and the generation of catabolic intermediates in skeletal muscle and liver may play a role in the etiology of insulin resistance [59]. It should be noted, though, that this mechanism is only a working model and that a cause effect relationship between BCAAs and insulin resistance is far from proven. In fact, dietary BCAAs may have multiple positive effects on parameters associated with obesity and insulin resistance [58]. For example, BCAAs can act as satiety signals and may reduce food intake. Moreover, BCAAs can serve as insulin secretagogues, which can improve glycemic control [61] and inhibit protein breakdown. Inhibition of protein breakdown in combination with the leucine-induced activation of mTORC1 and subsequent stimulation of protein synthesis may result in a sustained mass of energyconsuming tissues (e.g., skeletal muscle). In addition, while dietary BCAAs may cause an increase in plasma concentrations of all BCAAs, supplementation of leucine alone causes an increase in plasma leucine concentrations but a relative decline in plasma concentrations of the other BCAAs. As such, supplementation of leucine alone as opposed to all

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three BCAAs might be preferred to exert the positive effects on parameters associated with obesity and diabetes while preventing an increase in total BCAAs.

KEY FACTS • In contrast to other amino acids, BCAAs are metabolized primarily by skeletal muscle, rather than by the liver. • Of the BCAAs, leucine has the highest anabolic potential. • In a Sestrin2-dependent manner, leucine is sensed by mTORC1 resulting in activation of downstream signaling leading to translation initiation. • Upon leucine-induced activation of translation initiation, a complete spectrum of amino acids is required to support muscle protein synthesis. • Plasma BCAA concentrations may serve as a marker for (the development of) insulin resistance, but a cause effect relationship between BCAAs and insulin resistance in humans is far from proven.

SUMMARY POINTS Maintenance of skeletal muscle mass is of major importance to maintain functional capacity and metabolic health. Muscle mass is for a large part regulated by the postprandial muscle protein synthetic response to the ingestion of protein. Dietary protein provides a complete spectrum of amino acids including the BCAAs leucine, isoleucine, and valine that are used as substrates for muscle protein synthesis. The content and composition of BCAAs in the diet can vary substantially between dietary protein sources. A high BCAA content is generally desirable for stimulating muscle protein synthesis. In particular, leucine has been shown to activate the muscle protein synthetic machinery through Sestrin2-dependent activation of mTORC1 leading to translation initiation. Indeed, leucine-enriched protein supplementation has been shown to promote muscle growth in frail elderly. In contrast, it has been observed that plasma BCAA concentrations are elevated in insulin-resistant individuals and that circulating BCAA levels may predict development of insulin resistance. This has led to the thesis that BCAAs may be involved in the etiology of insulin resistance. However, a causal relationship between BCAAs and insulin resistance has not been proven and, in fact, dietary BCAAs may have positive effects on food intake, glycemic control, and energy expenditure. This chapter provides evidence that leucine supplementation, up to a total leucine intake of 40 g/day, is safe and effective in supporting muscle mass maintenance.

Acknowledgments We would like to thank Dr. Chris McGlory for providing insightful debate during the preparation of this manuscript. S.M.P. is supported by the Canada Research Chairs program and grants from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Canadian Diabetes Association, and the Canadian Foundation for Innovation.

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[46] Murphy CH, Saddler NI, Devries MC, McGlory C, Baker SK, Phillips SM. Leucine supplementation enhances integrative myofibrillar protein synthesis in free-living older men consuming lower- and higher-protein diets: a parallel-group crossover study. Am J Clin Nutr. 2016 Dec;104(6):1594 606. [47] Verhoeven S, Vanschoonbeek K, Verdijk LB, Koopman R, Wodzig WK, Dendale P, et al. Long-term leucine supplementation does not increase muscle mass or strength in healthy elderly men. Am J Clin Nutr. 2009;89 (5):1468 75. [48] Leenders M, Verdijk LB, van der Hoeven L, van Kranenburg J, Hartgens F, Wodzig WK, et al. Prolonged leucine supplementation does not augment muscle mass or affect glycemic control in elderly type 2 diabetic men. J Nutr. 2011;141(6):1070 6. [49] Cermak NM, Res PT, de Groot LC, Saris WH, van Loon LJ. Protein supplementation augments the adaptive response of skeletal muscle to resistance-type exercise training: a meta-analysis. Am J Clin Nutr 2012;96 (6):1454 64. [50] Tieland M, van de Rest O, Dirks ML, van der Zwaluw N, Mensink M, van Loon LJ, et al. Protein supplementation improves physical performance in frail elderly people: a randomized, double-blind, placebo-controlled trial. J Am Med Dir Assoc. 2012;13(8):720 6. [51] Burd NA, West DW, Moore DR, Atherton PJ, Staples AW, Prior T, et al. Enhanced amino acid sensitivity of myofibrillar protein synthesis persists for up to 24 h after resistance exercise in young men. J Nutr. 2011;141 (4):568 73. [52] Bauer JM, Verlaan S, Bautmans I, Brandt K, Donini LM, Maggio M, et al. Effects of a vitamin D and leucineenriched whey protein nutritional supplement on measures of sarcopenia in older adults, the PROVIDE study: a randomized, double-blind, placebo-controlled trial. J Am Med Dir Assoc. 2015;16(9):740 7. [53] Tieland M, Borgonjen-Van den Berg KJ, van Loon LJ, de Groot LC. Dietary protein intake in communitydwelling, frail, and institutionalized elderly people: scope for improvement. Eur J Nutr. 2012;51(2):173 9. [54] Verreijen AM, Verlaan S, Engberink MF, Swinkels S, de Vogel-van den Bosch J, Weijs PJ. A high whey protein-, leucine-, and vitamin D-enriched supplement preserves muscle mass during intentional weight loss in obese older adults: a double-blind randomized controlled trial. Am J Clin Nutr. 2015;101(2):279 86. [55] Backx EM, Tieland M, Borgonjen-van den Berg KJ, Claessen PR, van Loon LJ, de Groot LC. Protein intake and lean body mass preservation during energy intake restriction in overweight older adults. Int J Obes. 2016;40(2):299 304. [56] Felig P, Marliss E, Cahill Jr. GF. Plasma amino acid levels and insulin secretion in obesity. N Engl J Med. 1969;281(15):811 16. [57] Wang TJ, Larson MG, Vasan RS, Cheng S, Rhee EP, McCabe E, et al. Metabolite profiles and the risk of developing diabetes. Nat Med. 2011;17(4):448 53. [58] Lynch CJ, Adams SH. Branched-chain amino acids in metabolic signalling and insulin resistance. Nat Rev Endocrinol. 2014;10(12):723 36. [59] Newgard CB. Interplay between lipids and branched-chain amino acids in development of insulin resistance. Cell Metab. 2012;15(5):606 14. [60] Newgard CB, An J, Bain JR, Muehlbauer MJ, Stevens RD, Lien LF, et al. A branched-chain amino acidrelated metabolic signature that differentiates obese and lean humans and contributes to insulin resistance. Cell Metab. 2009;9(4):311 26. [61] Leenders M, van Loon LJ. Leucine as a pharmaconutrient to prevent and treat sarcopenia and type 2 diabetes. Nutrition reviews. 2011;69(11):675 89.

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Glutamine and Skeletal Muscle Vinicius F. Cruzat School of Pharmacy and Biomedical Sciences, Curtin University, Perth, Western Australia, Australia; Faculty of Health, Torrens University, Melbourne, Victoria, Australia

AN INTRODUCTION TO GLUTAMINE METABOLIC BIOCHEMISTRY Amino acids are the building blocks of proteins in our cells and tissues and after water are the second most abundant compound in mammals. Amino acids impact on innumerable metabolic pathways, such as protein synthesis and the production of hormones, antioxidants, and low-molecular weight substances including nitric oxide (NO), polyamines, and small peptides. Among the 20 amino acids that appear in the genetic code, the 5-carbon amino acid glutamine (C5H10N2O3) is considered the most versatile [1]. In both health and disease situations, glutamine is of fundamental importance to the intermediary metabolism, the interorgan nitrogen exchange and pH homeostasis, and can be used as a substrate for nucleotide synthesis (purines, pyrimidines, and amino sugar), antioxidants, and chaperone protein modulation [2,3]. Some cell types, such as immune cells, can use glutamine as an oxidizable fuel at high rates similar to or greater than glucose (e.g., lymphocytes and macrophages) [4 6], and possibly for this reason glutamine is the most widely recognized immunonutrient. In a health situation, glutamine is nutritionally classified as a nonessential amino acid, which means that the organism can synthesize and release glutamine into the blood in sufficient quantities from endogenous nitrogen compounds (e.g., de novo glutamine synthesis, peptides degradation, amino acids transamination) or dietary sources. However, in many catabolic situations, including sepsis [7,8], infection [9], surgery [10], trauma [11], and exhaustive physical exercise [12,13], the cellular demand for glutamine exceeds its synthesis and supply, and glutamine becomes an essential nutrient. As a result, glutamine is now commonly described as a conditionally essential amino acid [14]. All body tissues are important for glutamine metabolism and homeostasis; however, the liver and skeletal muscles play a key role in the maintenance of glutamine availability. The liver exhibits the highest capacity for glutamine production, and the skeletal muscles are the main tissue of glutamine synthesis, storage, and release [15]. This is due to the fact

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00017-8

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that skeletal muscles represent an average of 40% of the total body mass and demonstrate capability to metabolize several glutamine precursors, including the branched-chain amino acids (BCAA), leucine, isoleucine, and valine. Although glutamine was first described in beet juice by Hlaziwetz and Habermann in 1873, its synthesis and metabolic role in mammals was elucidated due to the combined efforts of Schulze and Bosshard in 1883 and Damodaran in 1932 [16]. The number of studies describing the importance of glutamine metabolism for the whole body only increased after the papers produced by Sir Hans Adolf Krebs in 1935 (Nobel prize in Physiology or Medicine in 1953). Hans Krebs (1900 1981), widely known for his discovery of the tricarboxylic acid (TCA) cycle (also known as Krebs cycle), also described for the first time the key aspects of glutamine synthesis and degradation, as well as the energy-requiring process involved [17]. In addition, a major contribution to our current understanding of glutamine metabolic pathways and exogenous supply came from Eric Newsholme’s laboratory. Eric Arthur Newsholme (1935 2011) created seminal concepts in metabolic regulation and intertissue metabolic flux [14] that were experimentally confirmed by his own research group and subsequently by many other laboratories worldwide [8,11,18]. For example, he speculated that at maximal rates of glutamine utilization by the kidney, all of the glutamine in the blood would be consumed in less than 30 minutes if no glutamine was released from the skeletal muscle [14]. This would have a major impact on the whole body, especially for immune cell function and survival. Subsequently, researchers hypothesized the possible benefits of glutamine supplementation in catabolic situations, which is still an area that deserves some consideration. In the present chapter, the underlying mechanisms of glutamine metabolic biochemistry in skeletal muscle and importance for the whole body are discussed, as well as the increased interest in the effects of glutamine supplementation.

GLUTAMINE METABOLIC BIOCHEMISTRY IN SKELETAL MUSCLES The endogenous production of glutamine is between 40 and 80 g/24, as estimated by isotopic and pharmacokinetic techniques [19,20]. As a result, healthy individuals weighing about 70 kg show approximately 70 80 g of glutamine distributed in the whole body [15]. Plasma glutamine concentration is B500 to 800 μM, which represents about 20% of the total free amino acids pool in the blood. This concentration is 10 100-fold in excess of any other amino acid, and for this reason, glutamine is considered the body’s most abundant amino acid. In skeletal muscles, glutamine concentration is even higher, representing about 40% 60% of the total amino acid pool (e.g., Human Vastus Lateralis— 12.8 18.2 μmol/g fresh tissue) [21]. Glutamine is also a proteinogenic amino acid, i.e., amino acids that are incorporated biosynthetically into proteins and accounts for 5% 6% of bound amino acids [18]. Glutamine is an L-α-amino acid biochemically classified as a polar amino acid, i.e., the carboxyl group carries a negative charge, whereas the amino group is protonated [22]. The side-chain amide group is easily removed by various enzymatic reactions or even by spontaneous hydrolysis in water at room temperature [23]. Interestingly, when long stretches of glutamine residues are synthesized (also called polyglutamines) by the expansion of the

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trinucleotide repeat cytosine adenine guanine (which encodes the amino acid glutamine in the translation process), the glutamine hydrogen bonding tendencies contribute to polyglutamines aggregation inside the cell and cause many toxic effects. Mutant polyglutamines protein aggregation may lead to neurodegenerative disorders like Huntington’s disease and several spinocerebellar ataxias [24]. Glutamine availability in blood and tissues is mainly regulated through the activity of two enzymes; phosphate-dependent glutaminase (GLS, EC 3.5.1.2) and glutamine synthetase (GS, EC 6.3.1.2, Fig. 17.1). GLS is a mitochondrial enzyme responsible for glutamine hydrolysis to glutamate (GLU), releasing ammonia (NH3) products [17,25]. GLU is a compound that carries a negative charge and cannot freely cross the plasma membrane. In this way, GLU is converted to intermediary substrates of the TCA cycle (e.g., 2-oxoglutarate, succinate, fumarate, malate, and oxaloacetate to phosphoenolpyruvate, or even malate to pyruvate directly) to produce several other compounds, such as nucleotides, lipids, and amino acids. This process is also known as glutamine-dependent anaplerosis and is critically important for predominantly glutamine consuming tissues, such as the cells of the immune system, kidneys (key role in acid base balance), and the gut [26]. Conversely, GS activity is characteristic of ammonia-producing tissues, such as the skeletal muscle and liver, and promotes glutamine synthesis via an endergonic reaction of ΔG , 0, using Adenosine triphosphate (ATP) and GLU synthesized from alpha-ketoglutarate and NH3. Although GLS and GS can be found in many organs and tissues, its predominant activity depends of many factors, including the metabolic requirement, fed state, and tissue specificity [15,27]. For example, in response to catabolic stimulus, such as starvation, exhaustive exercise, infection, and uncontrolled diabetes, a number of organs and tissues

FIGURE 17.1 Biochemistry of glutamine synthesis and hydrolysis. Although many enzymes are involved in glutamine metabolism, glutamine synthesis and hydrolysis is mainly coordinated by GS and GLS, respectively. GS catalyzes glutamine biosynthesis from glutamate and ammonia (NH3) using ATP, whereas GLS is responsible for glutamine hydrolysis to glutamate and NH4. Both enzymes appear to be expressed in most cells and tissues; however, their predominant activity will determine if the tissue is more likely to produce or consume glutamine. GS, glutamine synthetase; GLS, glutaminase; NH4, ammonium ion.

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FIGURE 17.2 The importance of muscle glutamine stores in health and catabolic situations. Through the intertissue metabolic flux, skeletal muscles constantly release GLN into the blood and feed many other cells and tissues, such as the immune system, liver, and the gut. In health, the gut is the principal organ of glutamine uptake, and muscle and liver are the main organs of glutamine synthesis and release. Conversely, during catabolic situations, the liver and immune system cells become major sites of glutamine consumption. In this situation, the reduced glutamine uptake by the gut can facilitate bacterial translocation into the blood and systemic inflammation. It is important to stress that because the skeletal muscle serve as the major glutamine exporter during catabolism, plasma glutamine might not be sufficiently depleted in overtrained athletes or individuals involved in exhaustive exercise situations. GLN, glutamine.

(e.g., cells of the immune system, liver, and kidneys) show an increase in GLS activity, as well as demand for plasma glutamine [26]. This is due to the action of catabolic hormones/catecholamine (e.g., adrenocorticotropic hormone, adrenaline, and cortisol) and/or cytokines tumor necrosis factor (e.g., TNF-α, IL-6, IL-1β) [28,29]. Simultaneously, GS activity, and mRNA increase in skeletal muscles, which helps to prevent the muscle glutamine pool from being completely depleted in a short period of time [26]. In addition, skeletal muscle cells are also able to maximize their BCAA utilization to produce and supply glutamine to the whole body. Through the action of BCAA aminotransferase, BCAA can be transaminated and release α-ketoglutarate and GLU. GLU can donate its amino group to pyruvate, generating alanine or incorporate free NH3 for de novo synthesis of glutamine. This metabolic pathway significantly contributes to glutamine metabolism in the whole body [25,30]. BCAA can also be deaminated in the skeletal muscle, feeding a critical portion of the TCA cycle (i.e., acetyl-CoA 1 oxaloacetate - citrate - aconitate - isocitrate - oxoglutarate), which is responsible for providing additional carbon atoms for glutamine synthesis [14]. Importantly, the ratio of glutamine synthesis in the skeletal muscle is approximately 50 mmol/hours, higher than any other amino acid [23]. Hence, in catabolic situations, all glutamine-dependent tissues rely on the ability of the skeletal muscles to supply sufficient quantities of glutamine through the blood [25] (Fig. 17.2). However, this

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process is accompanied by an intense muscle proteolysis. In nonsurviving septic patients, for example, the amount of free glutamine in skeletal muscle can be reduced by 90%, which correlates with the patient outcome [11,18,31]. Glutamine released from the skeletal muscle can be utilized by cells of the immune system in addition to glucose. Both glucose and glutamine metabolism may lead to an increase in pyruvate production, and its conversion into CO2, which is associated with stimulation of pyruvate carboxylase activity rather than pyruvate dehydrogenase. The provision of glutamine also promotes Nicotinamide adenine dinucleotide phosphate (NADPH) synthesis, which participates in the intracellular substrate energy production. This pathway occurs via NADP1-dependent malic enzyme action, which catalyzes the conversion of malate (derived from GLU via formation of 2-oxoglutarate, succinate, and fumarate) to pyruvate. NADPH is required for biosynthetic reactions such as fatty acid synthesis and for the production of free radicals such as superoxide anion (O22) or nitric oxide (NO). O22 production occurs through the NADPH oxidase (NOX) system, and NO is produced by inducible NO synthase. Moreover, NADPH is also required for glutathione (GSH) synthesis via GSH reductase activity [32], which is discussed later in this chapter. In addition, glutamine generates GLU and subsequently may promote carbamoylphosphate synthesis by mitochondrial carbamoyl phosphate synthetase I (CPS I) or CPS II in the cytosol. Carbamoyl phosphate may combine with ornithine in the urea cycle to produce citrulline and is subsequently converted to argininosuccinate and then arginine (ARG). ARG is subsequently cleaved by arginase to produce urea and ornithine [33,34]. The utilization of the amide nitrogen atom of glutamine, catalyzed by the glutamine amidotransferases, leads to the synthesis of such important biological compounds as the pyridine nucleotide coenzymes, purines, pyrimidines, glucosamine-6-phosphate, and asparagine. This is a remarkable example of intertissue metabolic flux between the skeletal muscle’s GLU glutamine synthesis and the glutamine GLU consumers (e.g., immune cells, kidneys, liver, gut) during catabolic situations. On the other hand, hormones like insulin and insulin growth factors stimulate glutamine transport to the intracellular medium [35]. However, in many tissues which include the skeletal muscle, the transmembrane gradient for glutamine is high and its diffusion is restricted. The intracellular concentration of glutamine ranges from 2 to 20 mM, while the extracellular concentration ranges is between 0.2 and 0.8 mM. Hence, glutamine transport into the cells is dependent on active transporters (e.g., coupling with Na1 or H1 dependent channel) using ATP as an energy source. Many glutamine transporters have been studied and described in the literature, and their classification is clustered in “systems.” Most transporters share specificity for other neutral or cationic amino acids, and while Na1-dependent cotransporters efficiently accumulate glutamine, antiporters regulate the glutamine pool, as well as other amino acids, like GLU[36]. In the skeletal muscle, the most wellknown glutamine transporters are systems L (e.g., LAT2), N (e.g., SNAT3), and alanineserine-cysteine transporter 2 (ASC) (e.g., ASCT2), and all belong to the SLC gene family [37]. Importantly, the presence of transport systems and type is tissue specific, and many other systems with specificity for glutamine transport have been reported in the brain, gut (apical and basolateral membrane), placenta, liver, pancreas, lung, kidneys, and other tissues [38,39]. Interestingly, when glutamine is transported inside the cell, it promotes water absorption and potassium (K1) release, which increases the cell hydration state and

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influences its volume [40]. The volume of the cell is an important factor that stimulates skeletal muscle protein synthesis, and therefore it’s considered an anabolic signal or resistance to injury, even during illness or extreme exercise situations [40]. Further study is needed to identify the molecular mechanisms of glutamine transport and regulation.

GLUTAMINE NUTRITIONAL PROPERTIES IN SKELETAL MUSCLES Glutathione Antioxidant System Glutamine (via GLU), cysteine, and glycine are the precursor amino acids for the synthesis of GSH (γ-L-glutamyl-L-cysteinylglycine). GSH is the most important and concentrated (0.5 10 mmol/L) nonenzymatic antioxidant in the cells. Around 85% 90% of GSH is found in the cytosol, and the remainder is located in organelles (including mitochondria, nuclear matrix, and peroxisomes). GSH can directly react with reactive oxygen species (ROS), generating oxidized GSH (GSSG), and can act as an electron donor in peroxide reduction, catalyzed by glutathione peroxidase enzyme [11,12]. GSH self-oxidation can also be catalyzed by iron and copper ions, leading to the formation of intermediate radicals and the generation of glutathione disulfide (GSSG) and GSH, the superoxide radical (O2•2), hydroxyl radical (OH•), and hydrogen peroxide (H2O2). The redox state of the cell can be obtained from the ratio between the intracellular concentration of GSSG and GSH, which the ratio is [GSSG]/[GSH], resulting in a reduction of GSH and an increase in the amounts of GSSG [41] (Fig. 17.3). The redox state of the cells is consequently related to GSH concentrations, which are also influenced by the availability of amino acids. A higher glutamine/GLU ratio reinforces the substrates availability for GSH synthesis [42]. During high catabolism, there is a raise in the intracellular redox state, and all cell compartments are vulnerable to oxidative stress (characterized by increased levels of ROS and low removal by the antioxidant system) [10,42]. In this situation, catabolic hormones, such as glucagon, vasopressin, and catecholamines, induce the liver to export GSH (main organ for the de novo synthesis of GSH) to the plasma. In order to compensate the increased levels of ROS, i.e., oxidative stress, the skeletal muscle is one of the main tissues responsible for the increased capture of GSH from plasma. However, GSH intertissue metabolic flux is greatly dependent on glutamine availably, which is severely compromised during high catabolic situations/diseases. The skeletal muscle glutamine-GSH axis is critically important to attenuate oxidative stress and for caspase-independent signaling, proteolysis, inflammation, and mitochondrial-associated apoptotic events. It is important to note that nutritional alternatives to increase the GSH concentration through GSH supplements or supplements of GLU are not effective and can be toxic, sometimes accelerating the cell senescence process [43].

Heat Shock Response At the most basic level, mammalian cells developed a range of adaptations to survive and respond to different types of hostile situations such as heat shock (HS), toxins, exacerbated ROS production, and infection/inflammation by changing the expression of stress-related

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FIGURE 17.3 Glutamine: molecular mechanisms and metabolic biochemistry. Free GLN and ALA, or Ala-Gln supplementations can enter inside the cell through specific transporters (amino acid specific transporters, PepT1, respectively) and enhance the antioxidant status by increasing the synthesis of γ-L-glutamyl-Lcysteinylglycine (GSH). Made up of CYS residues, GLY and especially GLU, GSH is the most important and more concentrated nonenzymatic antioxidant in the cells. Glutamine is also important for the modulation of chaperone function mediated by HS protein 70 (HSP70). This mechanism occurs via cellular proteins and transcription factors O-glycosylated by the HBP, such as the nutrient sensor SIRT1. SIRT1 activate the main thermal shock eukaryotic factor (HSF1) and lead to the expression of HSP70. HSP70 is widely accepted as antiinflammatory protein by virtue of turning NF-κB/IkBα downstream pathway off, thus extinguishing the production of inflammatory mediators. In addition, HSP70 prevent protein misfolding during ER stress. On the other hand, glutamine hydrolysis to GLU leads to the production of ARG, and subsequently nitric oxide (NO) via NO synthase 2. In the mitochondrial metabolism, GLU from GLN can serve as a substrate for NADPH synthesis. NADPH is essential for the NOX system, and for the recycling of GSH disulfide (GSSG) to GSH via glutathione S-reductase. GLU can also be converted to intermediary substrates of the Tricarboxylic acid (TCA) cycle and produce several other substances, such as nucleotides, lipids, and amino acids. Other abbreviations: GS, glutamine synthetase; GLS, glutaminase; NH3, ammonia; GDH, glutamate dehydrogenase; GLN, glutamine; ALA, free alanine; Ala-Gln, L-alanyl-L-glutamine; PepT1, Peptide transporter 1; GSH, glutathione; CYS, cysteine; GLY, glycine; HSP, heat shock protein; HBP, hexosamine biosynthetic pathway; SIRT1, Sirtuin 1; HSF, heath shock factor; ER, endoplasmic reticulum; GLU, glutamate; ARG arginine; NOS2, NO synthase 2; NOX, NADPH oxidase; GSSG, GSH disulfide; GSR, glutathione S-reductase.

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genes, also known as HS genes. Once activated by transcriptional factors, the HS genes lead to the production of specific cytoprotective proteins, also known as HS proteins (HSPs). HSPs are a family of polypeptides clustered according to their molecular weight which have many intracellular functions, the most important being acting as a molecular chaperone [44]. Although several HSP families have been studied in the last couple of years (e.g., HSP10, HSP25, HSP27, HSP90), the most famous and well described in the literature is the HSP70 (i.e., HSP72 1 HSP73) family [44 46]. HSP70 acts as antiinflammatory protein by virtue of turning NF-κB off, and attenuating the production of inflammatory mediators [47]. Moreover, HSP70 modulate autophagy by regulating mTOR/Akt pathway and block signaling pathways associated with protein degradation [45]. Glutamine is a potent modulator of HSPs expression through the hexosamine biosynthetic pathway (HBP). HBP is a metabolic pathway that can be induced by glutamine: fructose-6-phosphate amidotransferase (the first and rate-limiting step of HBP), which leads to the production of uridine diphosphate (UDP)-N-acetylglucosamine and (UDP)-Nacetylgalactosamine (UDP-GlcNAc and UDP-GalNAc, respectively). Both UDP-GlcNAc and UDP-GalNAc may bind to serine or threonine residues in nuclear and cytoplasmic proteins in a process called O-linked-N-acetylglucosamine (O-GlcNAc) [48]. Once glutamine is metabolized, the amino acid leads to the O-GlcNAc of several transcriptional factors, such as Sp1, phosphorylation of eukaryotic initiation factor 2 (eIF2), and sirtuin-1 (SIRT1) [49]. Both Sp1 and eIF2 are key transcription factors for the induction of the main thermal shock eukaryotic factor, heath shock factor 1 (HSF-1). In addition, SIRT1 enhances HSF1 expression and prolongs its activation by binding to the promoters of HS genes, leading to HSPs expression [48,50] (Fig. 17.3). Interestingly, HSF1 is activated in response to nutrients, stress and/or inflammation [51], however, H2O2-induced oxidative stress disrupts SIRT1 to promote HSPs response [52].

Glutamine Intertissue Metabolic Flux, Immune System, and Inflammation In physiological situations and during catabolism, skeletal muscle cells constantly synthesize and release glutamine into the blood, which is utilized in cells of the immune system, such as neutrophils, macrophages, and lymphocytes. This is of fundamental importance for maintaining immune cells integrity and function in order to prevent and limit infections (immune activation), and also in the overall process of repair and recovery from injury (i.e., resolution of inflammation). Acting as antibacterial effector cells, neutrophils are part of the first line of defense against pathogens. The capacity to recognize and kill invaders largely depends on protein factors induced by inflammation, which includes the neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) enzyme activity, respectively [53]. Both NETs and MPO utilize ROS, such as O2•2 (from molecular oxygen) and H2O2 (via superoxide dismutase— Superoxide dismutase (SOD) activity) produced by NOX2 enzyme [54]. Although in immune cells glutamine is usually converted to GLU, aspartate (via TCA cycle activity)

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and lactate, neutrophils also utilized large amounts of glutamine to generate NADPH (for NOX2 activity and antimicrobial activity) via malate generation and action of malic enzyme. In fact, in cultured neutrophils, the maintenance of 2 mmol glutamine (twofold physiological levels) attenuated the immune suppression induced by catabolic hormones such as adrenaline, by restoring ROS production and the oxidative burst against pathogens [55]. In addition, it is already stablished that in neutrophils and other cell types (e.g., pancreatic beta-cells), glutamine can also regulate several components of the NOX complex, such as the expression of gp91phox, p22phox, and p47phox [56 58]. Like neutrophils, macrophages utilize large amounts of glutamine to maintain the antibacterial and antitumor activity. Macrophages are one of the main cells of the mononuclear phagocyte system and are found in the lymphoid tissue, peritoneal, pleural, and pericardial cavities. During the process of phagocytosis, macrophages utilize ARG to produce NO and citrulline via the NADPH-dependent inducible NOS enzyme, H2O2, and proinflammatory cytokines [54]. However, activated macrophages can secrete arginase and deplete local exogenous ARG concentration, because ARG is also an important fuel for pathogens. Hence, alternative ARG sources, such as glutamine is of fundamental importance for macrophage function. In addition, macrophages utilize glucose to generate ATP for cell maintenance and function, and glycerol 3-phosphate for lipid (triglyceride and phospholipid) synthesis through esterification pathways. However, only a small amount (10%) of glucose is oxidized through the TCA cycle [6]. Conversely, glutamine supply several TCA cycle substrates, which can be metabolized and contribute to ATP production, purines, and pyrimidines, and thus RNA synthesis, as well as cytokine production [59]. Interestingly, Eric (Eric Newsholme lab) and his son, Philip Newsholme, were pioneers to show glutamine enzymes distribution and metabolism in macrophages. For example, the production of TNF-α, IL-1β, IL-6, and IL-8, quantitatively the most important cytokines produced by LPS-stimulated macrophages, is completely dependent upon extracellular glutamine availability [6,60 62]. Up until the early 1980, it was believed that lymphocytes obtained most of their energy by metabolism and oxidation of glucose [63]. However, under stress/catabolism and inflammatory situations, activated lymphocytes (T and B) switch the metabolic requirements by increasing not only the rate of glycolysis but also glutamine consumption and utilization. This is associated with increased mitochondrial respiration to supply substrates for the biosynthesis of nucleotides, amino acids, proteins, and lipids, which are all important for intracellular pathways, enzyme activation/regulation, and cell proliferation [62,64]. In fact, the rate of lymphocyte proliferation is dependent on extracellular glutamine levels, which can be dramatically affected during illness [65]. Conversely, glutamine replacement induced an increase in lymphocyte proliferation (T and B) in septic animals [42]. Glutamine also impact in the expression of key lymphocyte cell surface markers such as CD25, CD45RO, CD71, and the production of many cytokines, including interferon-gamma (IFN-γ), TNF-α, and IL-6 [2,66 68]. In addition, glutamine availability is also important for lymphokine activated killer cells, affecting tumors arising from solid organs, cytokine synthesis and release, and antibody (B-lymphocytes) production [7].

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IMPORTANT CONSIDERATIONS OF GLUTAMINE SUPPLEMENTATION The main rationale for glutamine supplementation in catabolic situations is the lack of glutamine availability. Although a balanced diet with high quality and sufficient quantity of proteins provide all the required amino acids for physiological processes and health maintenance, this approach is not always feasible in many catabolic situations. For example, during major illness, sepsis, trauma, and postsurgery circumstances, patients have several nutritional limitations, including state of unconsciousness, gastrointestinal disturbances, and/or chew-related problems. In addition, the consumption of protein sources, such as dairy products, red meat, chicken, and fish is also a critical issue in overtrained athletes or individuals involved in exhaustive exercise situations. For these individuals, protein intake is hampered by anorexic behaviors induced by excessive exercise stress, and therefore the nutritional adequacy of the menu is not suitable. Conversely, there is growing evidence that some nonsynthetic supplements, including free glutamine or as a dipeptide, can assist optimal nutrition during the recovery period [1,69]. Glutamine supplementation is usually made by utilizing its free form, as an isolated amino acid, or bonded with another amino acid, also known as the dipeptide form, such as L-alanyl-L-glutamine (Ala-Gln), L-glycyl-L-glutamine (Gly-Gln), or L-arginyl-L-glutamine. In most cases, the choice of glutamine supplementation partially depends on the patient catabolic condition, and the route of administration. For instance, in patients receiving total parenteral nutrition (TPN), glutamine dipeptides are an advantageous treatment due to its stability during sterilization procedures and prolonged storage. Furthermore, dipeptides like Gly-Gln and Ala-Gln have a high range of solubility (154 g/L H2O at 20 , 568 g/L H2O at 20 , respectively), when compared to the free glutamine form (36 g/L H2O at 20 ). If glutamine dipeptides are not available, free glutamine can be offered to the patient by adding the crystalline amino acid powder to a commercially available amino acid solution. However, it should be noted that this solution requires daily preparations at controlled temperature (i.e., 14 C) and aseptic conditions followed by sterilization through membrane filtration. Furthermore, to reduce the risk of precipitation, the concentration of free glutamine solution should not exceed 1% 2%, which can be a severe burden for volume restricted patients [69]. Glutamine dipeptides supplementation is considered an important therapeutic strategy to reverse and stabilize the dramatic glutamine fall in intra- and/or extracellular glutamine pools and its consequences for the whole body. Following i.v. bolus injection or TPN containing glutamine dipeptides, Gly-Gln, or Ala-Gln can be quickly (2.4 and 8.6 minutes, respectively) metabolized and hydrolyzed by the liver. This rapid effect increases the availability of free amino acids into the blood and makes them available for other tissues, such as the skeletal muscles and the immune system (Fig. 17.3). Glutamine elimination kinetics is usually within 8 hours after its free or dipeptide i.v. infusion. Many clinical, experimental, and review studies [12,18,19] concluded that glutamine dipeptides given as i.v. bolus, or added to TPN formulas improve GSH antioxidant defense [11], and intracellular chaperone function mediated by HSP [47]. In addition, these dipeptides can favor

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cell growth and nitrogen retention [70], which is important to attenuate muscle proteolysis and atrophy during major catabolic situations [11,47,71,72]. The combination of these effects can also reduce the length of hospital stay and the risk of death [11,18]. However, it should be noted that i.v. or parenteral routes are always invasive and might increase the risk of infections per se. Hence, the decision for use i.v. or TPN should be based on several parameters, such as poor nutritional status, dramatic reduction of body weight and body mass index, low plasma albumin, and severe loss of nitrogen and tissue function. On the other hand, oral or enteral routes are always more physiological and represents a noninvasive way of glutamine supplementation. This is especially important for patients where i.v. or TPN solutions are not appropriate, such as individuals with regular enteral feeds at home or hospitals, and also for overtrained athletes and individuals involved in exhaustive exercise situations. It has been reported that oral glutamine supplementation (i.e., free and Ala-Gln) promotes an increase in the plasmatic glutamine concentration between 30 and 120 minutes after administration [73]. However, the peak concentration and the area under the curve of the dipeptide solution (i.e., Ala-Gln) tend to be higher when compared to free glutamine. This is due to the glycopeptide Peptide transporter 1 (PepT-1), which actively transport di- and tripeptides from the brush border membrane into the enterocytes. Subsequently, dipeptides can be hydrolyzed in free glutamine and/or glutamine precursors, such as alanine, citrulline, and ARG. In both oral/enteral or parenteral nutrition, the typical daily administration ranges from a fixed dose of 20 35 g/24 hours, or variable doses of ,1.0 (usually 0.2 0.3) g/kg of body weight. At these dosages, there are no contraindications to glutamine supplementation in any form. In addition, there are no scientific evidences that glutamine supplementation can suppress or inhibit the endogenous production and the glutamine de novo synthesis. However, the efficacy of oral free glutamine supplementation is frequently questioned due to the high glutamine consumption by the small intestine. Conversely, studies in animal models exposed to Lipopolysaccharides (LPS)-induced infection have shown that oral supplementation with Ala-Gln dipeptide or free glutamine plus free alanine can reestablish plasma and muscle glutamine levels and have antiinflammatory properties (e.g., attenuating TNF-α and IL-1β release, NF-κB downstream activation pathways). Furthermore, both types of supplementation restored liver and muscle (i.e., soleus and gastrocnemius) glutamine levels in animal models of endurance [74 77] and resistance exercise training [48,78]. The effects of oral Ala-Gln dipeptide or free glutamine plus free alanine are also associated with an increase in muscle antioxidant defense promoted by GSH, and chaperone function mediated by HSP70 [8,42]. As a whole, a considerable amount of scientific evidences suggest that oral glutamine supplementation is beneficial for the recovery of the immune system, skeletal muscle, liver, and other several tissues during catabolic circumstances. Nevertheless, the efficacy of this nutritional method is still debatable due to studies that did not report similar outcomes. Future studies in humans are required to determine key aspects of glutamine supplementation, such as frequency of the nutritional intervention, and optimal doses for specific catabolic/disease situation. Moreover, more research is needed to determine novel free and dipeptide forms of glutamine supplementation.

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CONCLUSION In health and disease situations, the conditional essential amino acid glutamine is of fundamental importance for all cells in the human body. Like glycemia, plasma glutamine and the intertissue metabolic flux must also be maintained at constant levels to ensure the functioning of vital systems. The skeletal muscle tissue is the major site of glutamine synthesis and release and plays a key role maintaining glutamine supply for all glutamine dependent tissues. However, during catabolic situations, glutamine stores and availability are severely compromised due to the increased glutamine requirements by cells of the immune system, liver, and kidneys. In order to reverse this scenario and restore glutamine levels, the exogenous supplementation is recommended. The effects of glutamine supplementation include improvements in the cellular redox state mediated by GSH, modulation of HSP expression, attenuation of the uncontrolled inflammatory response, and muscle proteolysis (Fig. 17.3). Most of these effects can be achieved in patients receiving i.v. or TPN containing Ala-Gln or Gly-Gln dipeptides, and free glutamine (prepared in appropriate conditions). In oral or enteral nutrition, similar benefits of glutamine administration can also be achieved with Ala-Gln, Gly-Gln, and free glutamine plus free alanine supplementation. Importantly, oral nutritional therapies are more physiological and have the potential to be applied in the field of clinical and sports nutrition. Future studies have to identify the frequency of the nutritional intervention and optimal doses for specific catabolic/disease situation.

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Arginine and Skeletal Muscle Jean-Pascal De Bandt EA4466 PRETRAM Centre de recherche pharmaceutique de Paris Faculte´ de Pharmacie de Paris, Universite´ Paris Descartes 4, avenue de l’observatoire 75270 Paris cedex 06, APHP, Paris, France

LIST OF ABBREVIATIONS AGAT Akt Arg ASL ASS CASTOR1 CAT Cit eIF 4EBP1 GAMT GAP GATOR GCN2 Gln GH GTP IED l-NAME mTOR mTORC1 NMMA NO NOS OAT OCT ODC Orn p70S6K1 PI3K PAT-1

arginine:glycine amidinotransferase protein kinase B/RAC-alpha serine/threonine-protein kinase arginine argininosuccinate lyase argininosuccinate synthase cellular arginine sensor for mTORC1 Cationic amino acid transporter citrulline eukaryotic translation initiation factor eIF4E-binding protein 1 guanidinoacetate methyltransferase GTPase-activating protein GAP activity toward rags general control nonderepressible 2 glutamine growth hormone guanosine trriphosphate immune-enhancing diet l-nitro-arginine methyl ester Leu leucine mammalian/mechanistic target of rapamycin mTOR complex 1 N-monomethyl-Arginine nitric oxide NO-synthases ornithine aminotransferase ornithine carbamyl transferase ornithine decarboxylase ornithine 70 kDa ribosomal protein S6 kinase 1 phosphatidylinositol 3-kinase proton-assisted amino acid transporter 1

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00018-X

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Rag Rheb TSC REDD1 SNAT2 vATPase

Ras-related GTPase Ras homolog enriched in brain tuberous sclerosis complex regulated in development and DNA damage responses 1 sodium-coupled neutral amino acid transporter 2 vacuolar H 1 -ATPase.

Arginine (Arg) is classically considered to be a conditionally essential amino acid as it is essential, for example, in newborns; however, various studies have shown that its availability may also be limited in several situations. Its role in nitrogen metabolism and ureagenesis, in cellular multiplication via aliphatic polyamines, and in energy metabolism as a precursor of creatine (Cr) has long been known. Interest in this amino acid increased in the late 1970s and early 1980s, on the one hand with different studies, including from Barbul’s team [1,2], showing the importance of Arg supplementation in injury situations to improve survival, healing, and growth and, on the other hand, with studies on atherosclerosis highlighting the role of Arg in endothelial function. Our knowledge has grown considerably thanks to the identification of nitric oxide ( NO) as the endothelium-derived relaxing factor and the demonstration of the precursor role of Arg for this  NO synthesis [3]. The importance of Arg in muscle function is underlined by its role both direct, on muscle protein synthesis, and indirect via increased availability of anabolic factors, and by its involvement in energy/Cr metabolism. However, its ability to promote muscle function is debated as most of its beneficial properties are demonstrated only in situations of altered availability (injury, insulin resistance).

ARGININE METABOLISM Before detailing the effects of Arg on muscle, it is important to recall some aspects of its metabolism (Fig. 18.1). FIGURE 18.1 Arginine metabolism. P5C, pyrroline-5-carboxylate.

Agmatine Argininosuccinate Glycine Guanidinoacetate

Arginine

°NO Citrulline

S-Adenosyl methionine S-Adenosyl homocysteine

Urea Creatine

Ornithine

Putrescine Spermidine Spermine

Carbamyl-P

Glutamate

P5C

Proline

α-Ketoglutarate CO2

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Blood Arg is determined by the equilibrium between its appearance (from the diet, from endogenous synthesis, and from protein degradation) and its utilization. The liver does not contribute significantly since, in the liver, Arg is tightly channeled in the urea cycle. Mean Arg intake is of the order of 4 5 g/day [4]. However, less than 50% of dietary Arg enters the systemic circulation due to its visceral metabolism [5]. Endogenous synthesis arises mainly from the recycling of citrulline (Cit) released by  NO synthases (NOS) and from ornithine (Orn) by the successive action of Orn carbamyl transferase (OCT), argininosuccinate synthase (ASS), and argininosuccinate lyase (ASL) [6]. Given the tissue distribution of these enzymes, an important component of Arg homeostasis is the interorgan cooperation involving the intestine and the kidney (Fig. 18.2). Indeed, the enterocytes utilize Orn, produced from dietary Arg by arginase or from glutamine via glutaminase and Orn aminotransferase (OAT), and release Cit as a result of OCT action since in adult the enterocyte does not express ASS and ASL. This Cit is mainly used by the kidney for Arg synthesis via ASS and ASL. In a normal situation, endogenous Arg synthesis seems to adequately cover needs [7]; however, in some clinical circumstances (trauma, burn, sepsis), its demand may be not fully met by de novo synthesis making that Arg is considered as a conditionally essential amino acid [6]. Besides its utilization for protein synthesis, quantitatively the main metabolic pathway of Arg, and for protein arginylation, the quantitatively main routes of Arg disappearance are its degradation: • By arginases into Orn and urea, Orn being possibly converted into glutamate and proline via OAT, and into polyamines via Orn decarboxylase. Arginases exist as two isoforms: cytosolic type I and mitochondrial type II; they can be expressed by a large number of cell types and their expression may be increased in several pathological situations such as hypertension, atherosclerosis, hyperuricemia, etc. [8]. OAT seems to be only poorly expressed in muscle cells even if, when corrected for muscle mass, it may represent a significant contribution to Arg disposal [9] • By NOS into  NO and Cit. Quantitatively, this pathway accounts for about 1% of plasma Arg flux in healthy humans [10]. NOS exist as three isoforms: type 1 or neuronal NOS (nNOS), type 2 or inducible NOS (iNOS), and type 3 or endothelial NOS (eNOS). Types 1 and 3 are constitutively expressed by a large number of cells and are calcium dependent. Type 2 expression is induced, for example, by inflammatory processes in not only immune cells and vascular endothelial cells but also other cell Enterocyte Arg

Arg

Arg

ARGase

Glu OAT GLNase

Gln

FIGURE 18.2 Interorgan cooperation for arginine homeostasis. ARGase, arginase; OAT, ornithine aminotransferase; OCT, ornithine carbamyl transferase.

Liver Urea Arg

Arg

Arg

Arg

Kidney Cit

Orn

Cit

OCT

+

Muscle Cit Gln

Endothelial function

Arg

+

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types such as the hepatocyte and may be also constitutively expressed in some [11]. All three isoforms are present in skeletal muscle with NOS 1 associated with muscle cell membrane and NOS 2 and 3 located in the cytosol [11]. NOS 1 seems to be the main isoform involved for muscle contraction [12] • By Arg:glycine amidinotransferase (AGAT) which transfers Arg guanidino group on glycine to form guanidinoacetate thereafter methylated into Cr by guanidinoacetate methyltransferase; Orn and Cr exert negative feedback on AGAT [13]. Note that a small amount of Arg may undergo decarboxylation into agmatine which displays neuroprotective effects. However, the reality of endogenous agmatine synthesis and its function in human are still unknown [14]. A last aspect of Arg metabolism must be mentioned: its methylation during posttranslational modification of proteins by methyl transferases. Mono or dimethylated Arg is released when these proteins are degraded and both asymmetric dimethylarginine (ADMA) and N-monomethylarginine (NMMA) are competitive inhibitors of NOS. High plasma ADMA or high Arg-to-ADMA ratio are associated with vascular dysfunction. NMMA and ADMA are degraded mainly by dimethylarginine dimethylaminohydrolase into Cit and mono or dimethylamine [8].

ARGININE AND MUSCLE CREATINE Cr is an important component of the coupling between energy metabolism and muscle function as an intracellular storage form, as phosphocreatine (PCr), of the energy of high energy phosphate bond of adenosine triphosphate (ATP), PCr/Cr interconversion being catalyzed by creatine kinase (CK). PCr/Cr interconversion serves as an energy shuttle between the mitochondria and the myofilaments: mitochondrial CK enables Cr phosphorylation from ATP, whereas cytosolic CK enables to generate ATP from CrP at the site of myofilament contraction [13]. While nearly 50% of body Cr is provided by the diet in omnivorous, the remainder comes from de novo synthesis from Arg. Owing to enzyme localization, guanidinoacetate is mainly synthesized in the kidney and its methylation occurs in the liver. Thereafter, Cr enters in various cells via a specific membrane transporter (SLC6A8) [13] (Fig. 18.3).

Kidney

Arg Gly

AGAT

Orn

Liver SAM

GA

GA

SAH

GAMT

Cr

Dietary Cr

FIGURE 18.3 Kidney-liver cooperation for endogenous creatine synthesis and creatine provision to the muscle. AGAT, arginine:glycine amidinotransferase; Cr, creatine; PCr, phosphocreatine; GA, guanidine acetate; GAMT, guanidinoacetate methyltransferase; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine.

Muscle Creatinine

PCr

Cr

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ARGININE AND ENDOTHELIAL FUNCTION As already mentioned Arg is a substrate of eNOS and thus plays an important role in endothelium-dependent vasodilation. In fact, eNOS is a highly regulated enzyme. In basal conditions, it is inactive due to its association with caveolin-1, an inhibitory protein within the caveolae at the plasma membrane. Stimulation by various agonists induces its release from the plasma membrane. eNOS activity is also regulated at the transcriptional level, for example, by insulin, and by posttranslational phosphorylation. Last, eNOS activity is closely regulated by the bioavailability of its unique substrate Arg and that of its cofactor, tetrabiopterin (BH4). When they are lacking, eNOS, a dimeric enzyme, functions in an uncoupled mode producing simultaneously  NO and the superoxide anion which combine into the very toxic peroxynitrite anion. Interestingly, eNOS activity varies according to the extracellular concentration of Arg, whereas the intracellular concentration of Arg exceeds its Km. This Arg paradox is explained by the compartmentalization of its metabolism, a competition between eNOS and arginase for its use, and the formation of ADMA, an endogenous competitive inhibitor of eNOS [15]. One of the very early manifestations of the metabolic syndrome is an alteration of endothelial function in particular as insulin resistance is associated with decreased  NO synthesis and eNOS activity [16]. Experimentally, in models of endothelial dysfunction, Arg supplementation induces an increase in the Arg/ADMA ratio, a reduction in reactive oxygen species production, an improvement in endothelial function, a reduction in the size of vascular lesions [17]. However, in humans, contrary to the animal models, the benefits of Arg on cardiovascular function are more debated as some studies failed to demonstrate any beneficial effect of Arg on endothelial function [16,18]. Taken as a whole, literature data indicate that Arg is effective mainly on previously deteriorated endothelial function and that one of the determinant of the response to Arg is elevated ADMA level. Note also that, while ineffective in healthy subjects, prolonged oral administration of Cit, the Arg precursor, may positively influence endothelial function in patients with cardiovascular risk factors and diseases. This is associated in hypertensive patients with lower blood pressure [19]. Alternatively, some studies have demonstrated beneficial effects of Arg supplementation on other aspects of the metabolic syndrome such as decreased fat mass in obese diabetic patients [20] and improves insulin sensitivity [21,22], which in the long term means an improvement in endothelial function.

ARGININE AND MUSCLE PROTEIN HOMEOSTASIS Increased Substrate Delivery Given the effects of Arg on endothelial function, the question arises of a possible increase in muscle anabolism via an Arg-dependent increase in peripheral blood flow. It has been suggested that Arg may promote muscle protein synthesis via a  NO-dependent improvement in muscle blood flow enabling enhanced delivery of amino acids to the muscle [7]. Indeed, the vasodilatory properties of insulin are the basis of the coupling between hemodynamic and metabolism. The anabolic effect of feeding on the skeletal muscle

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requires an increase in blood flow in order to supply anabolic factors and substrates to the muscle. In contrast, the use of eNOS inhibitors blocks insulin mediated capillary recruitment and results in a reduction in nutrient availability. As mentioned above, Arg may thus promote muscle anabolism via this mechanism, only in conditions of limited Arg availability for endothelial function.

Increased Secretion of Growth Factors Arg has an activating effect on the secretion of anabolic factor such as growth hormone (GH) and insulin. Concerning GH, this property has been used for a long time to test the pituitary axis [7]. However, while oral Arg induces a nearly doubling of basal plasma GH, it greatly reduces exercise-induced GH response. Moreover Arg supplementation does not appear to enhance exercise performance [23]. Arg is a potent inducer of insulin release through several mechanisms. Its electrogenic transport into the β-cell may lead to membrane depolarization and insulin secretion via a mechanism similar to that of glucose. In addition, Arg may also lead to the synthesis of glutamate and stimulus secretion coupling metabolites [24]. In addition, Arg-induced insulin secretion also involves  NO as shown by the inhibitory effect of l-nitro-arginine methyl ester (L-NAME) in vitro [25].

Arginine, mTOR, and Protein Synthesis Arg may directly act on muscle protein synthesis via mammalian/mechanistic target of rapamycin (mTOR) (Fig. 18.4). mTOR is a serine/threonine protein kinase present in the AA availability

GCN2

Cytosolic Arg Cytosolic Leu

Arg

Sestrin2

ATP

REDD1

TSC2 TSC1

ADP Ragulator

vATPase

Leu

GADD34

Gator2

Gator1

H+

Leu

P A T 1

S L C 3 8 A 9

Castor1

Raptor RagA--GTP mTOR RagC--GDP

Leu

Rheb-GTP

mLST8

VPS34

?

L A T 1

Leu

Late endosomes/ lysosomes

Arg

FIGURE 18.4 Arginine as a direct activator of mechanistic Target of rapamycine. CASTOR1, cellular arginine sensor for mTORC1; GADD34, growth arrest and DNA damage-inducible 34; GATOR, GAP activity toward rags; GCN2, general control nonderepressible 2; mTOR, mammalian/mechanistic target of rapamycin; PAT-1, proton-assisted amino acid transporter 1; Rag, Ras-related GTPase; Rheb, Ras homolog enriched in brain; TSC, tuberous sclerosis complex; REDD1, regulated in development and DNA damage responses 1; SNAT2, sodium-coupled neutral amino acid transporter 2; vATPase, vacuolar H 1 -ATPase.

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cell as two protein complexes, mTOR complex 1 (mTORC1) and 2, the former being involved in the response of protein synthesis to feeding and increased nutrient availability. mTORC1 promotes protein synthesis by acting on the initiation step of cap-dependent mRNA translation and more precisely the formation of the 48S preinitiation complex via the phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 and the 70 kDa ribosomal protein S6 kinase. While mTORC1 may be directly activated by protein kinase B/RAC-alpha serine/threonine-protein kinase (Akt), it is mainly regulated by its interaction with the guanosine trriphosphate (GTP)-bound form of Ras homolog enriched in brain (Rheb), a G protein at the lysosome membrane. Rheb is itself submitted to a negative regulation by the tuberous sclerosis complex (TSC) 1 and 2. Growth factors lead to TSC2 phosphorylation via phosphatidylinositol 3-kinase/Akt and Ras/mitogenactivated protein kinase pathways and TSC1 TSC2 complex inhibition thus enabling mTORC1/Rheb interaction. Adversely, TSC1 TSC2 complex is activated by hypoxia or glucose deficiency via the regulated in development and DNA damage responses 1 (REDD1) protein or by amino acid deficiency via general control nonderepressible 2 (GCN2) [26]. Arg may first act on muscle protein synthesis at the level of GCN2 control. On the other hand, mTORC1/Rheb interaction is controlled by the availability of specific amino acids, present at the luminal side of the lysosomes, via Ras-related GTPase (Rag), G proteins found at the membrane of lysosomes that enable mTORC1 interaction with Rheb. The underlying mechanism is complex as it involves vacuolar H 1 -ATPase (vATPase), which senses intralysosomal amino acid level, and functional lysosomal amino acid transporters enabling outward amino acid transport. While vATPase signals to Rag via the Ragulator protein complex, Leu and Arg transport outside the lysosome, via the proton-assisted amino acid transporter 1 (PAT1) and sodium-coupled neutral amino acid transporter 2 (SNAT2), lifts the inhibition of Rag via the Ragulator complex [26]. Last intracytoplasmic Leu and Arg lead to an inhibition of the GTPase-activating protein (GAP) activity toward rags (GATOR)1 GATOR2 complex via Sestrin 2 and the cellular Arg sensor for mTORC1 protein complex (CASTOR), respectively [26,27]. The question thus arises of the influence of variations in Arg availability on protein synthesis. Indeed, in nutrient and growth factor deprived C2C12 myotubes, Arg attenuates the deprivation-induced reduction in protein synthesis and myotube diameter via a mTOR-dependent and  NO-independent mechanism [28]. However, in normal conditions, endogenous Arg production is not rate limiting, for example, for the response of protein synthesis to a load of essential amino acids [7]. From the abovementioned studies, it appears that Arg availability is not a limiting factor for protein homeostasis in a healthy situation. This is illustrated, for example, by a recent study in healthy rodents where the animals received a 5% Arg-diet for 2 months. Compared with control-fed rats, Arg had no effect on muscle mass and protein content [29]. In the same way, in a model of intramuscular pegylated Arg deiminase injection in mice, it has been shown that plasma Arg depletion (,1 μmol/L) does not affect body weight, body composition, or protein synthesis and breakdown thanks to tissue conversion of Cit into Arg [30]. Adversely, in situations of insulin resistance, prolonged administration of Cit restores Arg availability and improves lean body mass [31,32]. A study in swine demonstrated that a 2-month Arg supplementation was associated with higher muscle gain and lower fat

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gain compared with an isonitrogenous diet [33]. In obese type 2 diabetes patients submitted to an exercise training program and a hypocaloric diet for 3 weeks, Arg supplementation led to a higher weight loss and fat mass loss than with the control diet and preserved lean body mass [20].

ARGININE AND MUSCLE FUNCTION Arginine in Exercise Arg may play a role in the adaptation to exercise through different mechanisms. nNOS seems to be the main NOS isoform involved for muscle contraction [12]. It is associated with syntrophin α1 and dystrophin complex at the sarcolemma, involved in cytoskeletal integrity and contraction transduction.  NO is suspected to modulate the effects of mechanotransduction on muscle adaptation to exercise [34]. However, results are contradictory [35]. On the other hand, some studies show a role of Arg in energy metabolism and mitochondrial function. In heart failure (HF), Williams et al. [36] demonstrated reduced expression of Arg transporter Cationic amino acid transporter 1 (CAT-1) and reduced Arg uptake. In cardiomyocytes, they showed that specific mitochondrial overexpression of CAT-1 significantly improved mitochondrial function and reduced hypoxia-reoxygenation associated oxidative stress.

Acute Arginine Supplementation and Exercise Intravenous Arg administration during exercise does not seem to influence heart rate, blood pressure, or blood flow but increases muscle glucose uptake. This effect is independent of insulin secretion. This is in keeping with the involvement of muscle NOS activity in glucose uptake during muscle contraction [12]. However, in a model of muscle contraction/exercise in healthy and type 2 diabetes Sprague-Dawley rats, Hong et al. [37] did not observe any contraction-induced increase in muscle NOS activity. In addition, they observed that local NOS inhibition had no effect on muscle contraction force, on contraction-induced increase in muscle capillary recruitment, and on glucose uptake. These authors explained this discrepancy not only from other data from the literature by strain and exercise related specificities but also by the fact that they measured glucose uptake during contraction at variance with several studies. Arg infusion has no effect on exercise performance in endurance trained volunteers, and in patients with chronic HF or hypercholesterolemia [12].

Chronic Arginine Supplementation and Exercise Some experimental studies show beneficial effect of prolonged Arg supplementation. For example, Valgas da Silva et al. [38] showed in rats that a 8-week Arg supplementation associated with physical training was effective in improving performance and markers of mitochondrial biogenesis.

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In contrast, a metaanalysis of studies in healthy active volunteers and in trained subjects did not find any benefit of Arg supplementation on exercise performance [39]. Prolonged oral Arg supplementation may increase maximal aerobic capacity in patients with cardiovascular diseases but not in healthy individuals [12]. In healthy subjects, acute Cit supplementation before high intensity exercise does not affect peripheral blood, glucose metabolism, or exercise performance. Adversely, prolonged Cit supplementation is associated with improved muscle oxygenation and may improve performance [19].

ARGININE AND TISSUE HEALING The role of Arg in tissue repair has long been known. It involves both  NO and aliphatic polyamines. First data came from studies of Barbul’s team on wound healing in a model of standardized skin wound showing impaired wound healing with an Arg-deficient diet and improved healing and collagen deposition with Arg supplementation [1]. Later on, it was proposed [40] that in the early phases of wound healing, macrophages and neutrophils produce various mediators including  NO, responsible for local hemodynamic changes and microbial killing. Secondarily, high  NO concentration leads to leukocyte death and arginase release enabling Orn accumulation from Arg and conversion of Orn into polyamines, required for fibroblast proliferation, and into proline, required for collagen synthesis (Fig. 18.5). Inflammatory signal Effects on blood flow and hemostasis

Polymorphonuclear Macrophages ↑iNOS Arginine

Arginine

NO°

Citrulline Polyamines

Ornithine

Polymorphonuclear Macrophages Cell apoptosis and arginase release Extracellular arginine

Ornithine Collagen

Urea

Proline Ornithine

Fibroblasts Cell proliferation and connective tissue synthesis

Polyamines

Lymphocytes Antiinflammatory response

FIGURE 18.5 Arginine in local inflammation and tissue healing.

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Data also support the role of Arg in intestinal mucosa reparation. It must be mentioned that mucosal healing proceeds via cell migration and proliferation. While polyamine synthesis is required for quiescent epithelial cells to resume cycling [41], it has been shown that  NO donors may partly reproduce the effect of Arg on cell migration via activation of focal adhesion kinase [42]. Only few data are available on the role of Arg in muscle repair and mainly focus of the role of  NO synthesis. For example, in a model of blunt trauma in rats, it has been shown that  NO synthesis was required in the initiation of the myogenic process enabling satellite cell activation, cell proliferation, and an increase satellite cell number [43]; adversely, LNAME treatment was associated with decreased cell proliferation and fibrosis [44]. One mechanism could be increased Arg utilization by infiltrating macrophages as shown by persistent muscle alterations in a model of muscle injury in iNOS knockout mice [45].

ARGININE AND MUSCLE PROTEIN HOMEOSTASIS IN ACUTE INJURY Another aspect of Arg properties relates with the metabolic response to injury and its ability to prevent muscle mass loss in hypercatabolic situations. Theoretically, Arg could reduce injury-induced hypercatabolism by several mechanisms. First, it could improve immune function and intestinal barrier function thus reducing infectious complications. Second, it could act on tissue healing as a collagen and polyamine precursor. Third, it could promote muscle protein anabolism either via an improvement in the secretion and action of growth factor or through a direct action on the muscle. Going back to the studies from Barbul’s team, it is worth to remember that, in their model of standardized wound, Arg supplementation was associated with improved wound healing but also improved immune response, survival, growth, and nitrogen homeostasis. It must be noted that in this relatively moderate catabolic model, part of the effects was pituitary-dependent [46]. On the other hand, as described above, Arg has been shown in vitro to attenuate deprivation-induced reduction in protein synthesis [28]. The ability of Arg to improve muscle protein turnover in catabolic conditions remains to be established. In a model of endotoxemia induced by a 24-hour continuous IV endotoxin perfusion in pigs, Arg was shown to increase protein turnover without net protein accretion [47]. Only few clinical data are available in terms of Arg effects on muscle protein metabolism especially since almost all the studies dealt with immune-enhancing diets (IED), i.e., enteral nutrition formulas enriched not only in Arg but also ω3 fatty acids and micronutrients. This makes it thus very difficult to evaluate the respective roles of the different pharmaconutrients. Moreover, clinical studies mostly focus on infectious complications and little on protein homeostasis. Only one study has evaluated the effects of Arg alone in gastrointestinal cancer surgery and showed only limited improvement in nitrogen balance [48]. In terms of immune response, metaanalysis repeatedly concluded on a positive effect of IED in gastrointestinal cancer surgery with a reduction of infectious complications and in hospital length of stay [49]. We will not enter the debate about the administration of IEDs

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to severely ill critical care patients [49]; however, available studies have not shown any anabolic/anticatabolic effects of IEDs in these clinical situations. Last, at variance with experimental studies, available evidence does not enable to recommend Arg administration in order to improve tissue healing. Globally, evidence of an improvement of muscle protein turnover with IED is lacking.

CONCLUSION Arg is an amino acid that contributes very significantly to muscle metabolism by direct effects on protein metabolism, by the synthesis of  NO, locally or at the vascular level, and by its role as a precursor of Cr. Moreover, this amino acid contributes greatly to the fight against infectious challenges. However, studies in healthy adult men show only limited effects of its supplementation, highlighting the body’s high ability to synthesize this amino acid or to recycle it from Cit. Conversely, various studies highlight the interest of Arg supplementation in situations of chronic metabolic disorders with a decrease in fat mass and a gain in lean mass, and an improvement in endothelial function and muscle function. However, some studies have moderated the enthusiasm, probably because Arg is an inducer of ureagenesis and oxidation of amino acids thus limiting its efficacy. A better knowledge of the anabolic effects of this amino acid could make it possible to precise its therapeutic indications. An alternative would also be the use of its precursor, Cit, as pointed out by various recent data [19,50]. Last, in acute situations, studies in surgical patients have focused on the effects on the infection, and we have very little data on a possible positive effect on the muscle.

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[37] Hong YH, Betik AC, Premilovac D, Dwyer RM, Keske MA, Rattigan S, et al. No effect of NOS inhibition on skeletal muscle glucose uptake during in situ hindlimb contraction in healthy and diabetic Sprague-Dawley rats. Am J Physiol Regul Integr Comp Physiol 2015;308:R862 71. [38] Valgas da Silva CP, Delbin MA, La Guardia PG, Moura CS, Davel APC, Priviero FB, et al. Improvement of the physical performance is associated with activation of NO/PGC-1α/mtTFA signaling pathway and increased protein expressions of electron transport chain in gastrocnemius muscle from rats supplemented with L-arginine. Life Sci 2015;125:63 70. [39] Brooks JR, Oketch-Rabah H, Low Dog T, Gorecki DKJ, Barrett ML, Cantilena L, et al. Safety and performance benefits of arginine supplements for military personnel: a systematic review. Nutr Rev 2016;74:708 21. [40] Shearer JD, Richards JR, Mills CD, Caldwell MD. Differential regulation of macrophage arginine metabolism: a proposed role in wound healing. Am J Physiol 1997;272:E181 90. [41] Desforges B, Curmi PA, Bounedjah O, Nakib S, Hamon L, De Bandt J-P, et al. An intercellular polyamine transfer via gap junctions regulates proliferation and response to stress in epithelial cells. Mol Biol Cell 2013;24:1529 43. [42] Marc Rhoads J, Wu G. Glutamine, arginine, and leucine signaling in the intestine. Amino Acids 2009;37:111 22. [43] Anderson JE. A role for nitric oxide in muscle repair: nitric oxide-mediated activation of muscle satellite cells. Mol Biol Cell 2000;11:1859 74. [44] Filippin LI, Cuevas MJ, Lima E, Marroni NP, Gonzalez-Gallego J, Xavier RM. Nitric oxide regulates the repair of injured skeletal muscle. Nitric Oxide 2011;24:43 9. [45] Rigamonti E, Touvier T, Clementi E, Manfredi AA, Brunelli S, Rovere-Querini P. Requirement of inducible nitric oxide synthase for skeletal muscle regeneration after acute damage. J Immunol 2013;190:1767 77. [46] Barbul A, Rettura G, Levenson SM, Seifter E. Wound healing and thymotropic effects of arginine: a pituitary mechanism of action. Am J Clin Nutr 1983;37:786 94. [47] Bruins MJ, Soeters PB, Lamers WH, Deutz NEP. L-Arginine supplementation in pigs decreases liver protein turnover and increases hindquarter protein turnover both during and after endotoxemia. Am J Clin Nutr 2002;75:1031 44. [48] Daly JM, Reynolds J, Thom A, Kinsley L, Dietrick-Gallagher M, Shou J, et al. Immune and metabolic effects of arginine in the surgical patient. Ann Surg 1988;208:512 23. [49] De Bandt J-P, Cynober L. Pharmaconutriments azote´s: glutamine, leucine, arginine, α-ce´toglutarate ˇ dornithine et citrulline. Traite´ de nutrition artificielle de l’adulte. SFNEP. Paris: Springer; 2016. p. 941 64. [50] Jegatheesan P, De Bandt J-P. Hepatic steatosis: a role for citrulline. Curr Opin Clin Nutr Metab Care 2016;19:360 5.

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Citrulline and Skeletal Muscle Charlotte Breuillard, Arthur Goron and Christophe Moinard Laboratoire de bioenerge´tique fondamentale et applique´e, Universite´ Grenoble Alpes, Grenoble, France

Citrulline is a nonessential and nonproteinogenic amino acid that takes its name from watermelon (Citrullus vulgaris). For decades, citrulline was considered an intermediate of the urea cycle and as just one among many other amino acids. However, it began to arouse interest when Windmueller and Spaeth [1] showed the significance of intestinally produced citrulline. This particular feature was first used for diagnostic purposes, as citrulline proved to be the best marker of intestinal function [2]. Metabolic properties of this amino acid were subsequently demonstrated: first at cardiovascular level [as a precursor of nitric oxide (NO); see [3,4] for reviews] and then as a modulator of nitrogen homeostasis (see [5] for a recent review) when Osowska et al. [6] showed in a short bowel syndrome model that citrulline supplementation improves nitrogen balance. Moving forward, it was proposed that citrulline would be a regulator of muscle protein synthesis: in 2006, the same authors demonstrated, for the first time, the stimulatory effect of citrulline on muscle protein synthesis [7] in aged undernourished rats, using a model of protein energy restriction. In this model, the rats fed only 50% of spontaneous food intake for 12 weeks are then refed for 1 week with a diet corresponding to 90% of their spontaneous food intake and enriched with citrulline or a mix of nonessential amino acids (in order to make the diets isonitrogenous). Rats receiving the citrulline-enriched diet showed 80% increased muscle protein synthesis and a net muscle protein gain of 20%, without affecting myofibrillar proteolysis (estimated by urinary excretion of 3-methylhistidine). This action of citrulline on muscle protein synthesis is not limited to the aged rat: it has been demonstrated in another model of protein energy deficiency, a short fasting model in adult rats (fasted for 18 hours). Fasting results in a decrease of muscle protein synthesis (240%) that is fully restored by an oral bolus of citrulline [8]. In the same way, Ventura et al. [9] showed in adult female rats supplemented with citrulline and moderately feed-restricted (60% of their spontaneous food intake) for 2 weeks that citrulline increases the synthesis of myofibrillar proteins, unlike in feed-restricted rats not given citrulline supplementation.

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Finally, in a model of intrauterine growth restriction induced by maternal food restriction in rats, Bourdon et al. [10] demonstrated that supplementation with citrulline stimulates fetal growth (associated to an increase of protein synthesis). Furthermore, it appears that the positive effect of citrulline on muscular gain is maintained over time, since citrulline supplementation for 3 months in “healthy” aged (20month-old) rats enables an average 25% muscle gain specifically related to protein accretion and to an increase in muscle fiber size [11]. Interestingly, the protein accretion associated with citrulline intake is accompanied by an improvement of muscular function, thus establishing a continuum between metabolic action and clinical repercussions. In the protein energy restriction model in aged rats, citrulline increases maximum strength as well as animal motricity [12]. Muscular strength is preserved when moderately restricted adult female rats are supplemented with citrulline, but not without citrulline supplementation [9]. Nevertheless, all these data on the effects of citrulline on muscle protein synthesis and muscular function were observed in protein energy deficiency or malnutrition models, so confirmation was needed in healthy animals. Goron et al. [13] overcame this lack of data in a recent study where healthy adult rats received citrulline supplementation (1 g/kg/ day) or an isonitrogenous diet during 4 weeks. Just like in catabolic situations, citrulline increased muscle protein synthesis (133%), but without any effect on muscle mass and muscle protein content. Interestingly, despite this lack of effect on muscle protein content and mass, when citrulline supplementation is associated with exercise, the authors observed an improvement in performance (running time increased by 14%). Importantly, this ability of citrulline to modulate muscle protein synthesis has also been shown in humans. Oral supplementation with citrulline (10 g vs isonitrogenous placebo) improved muscle protein synthesis by 25% in healthy adults under a low-protein diet (8%) for 3 days [14], and this increased muscle protein synthesis appears to be insulinindependent, since insulin levels were not different between groups. More recently, Bouillanne et al. [15] demonstrated for the first time that 21-day oral supplementation with citrulline (10 g/day) in undernourished patients increases lean body mass (15% 10%) and reduces fat mass (29% in women). Finally, the effect of citrulline on protein synthesis seems to be muscle-specific, as three clinical studies have studied the effects of citrulline supplementation on whole body protein synthesis under different conditions, and none of them was able to show a positive effect of citrulline [14,16,17]. While the capacity of citrulline to modulate muscle protein synthesis has been well demonstrated in several situations, the precise mechanisms of action involved remain unclear. However, a new study has partially remedied this deficit. Le Plenier et al. [18] showed, using a model of isolated incubated rat muscle (epitrochlearis) with or without citrulline in the culture medium, that muscle protein synthesis was higher with citrulline, demonstrating a direct action of citrulline on muscle protein synthesis. Moreover, this increase in muscle protein synthesis could be linked to stimulation of the mTORC1 (mammalian target of rapamycin complex 1) pathway—the main pathway for the regulation of protein synthesis. In this same study and in the same model of isolated incubated rat muscle, the addition of rapamycin, an mTORC1 inhibitor, blunted the positive effect of citrulline on muscle protein synthesis. Moreover, the stimulation of mTORC1 by citrulline has been confirmed in vivo in the model of protein energy undernutrition described earlier, since rats refed with citrulline showed higher activation of S6K1 (p70 ribosomal protein S6 III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) in the muscle [18]. These data have been confirmed by Goron et al. [13] who showed that citrulline supplementation in adult healthy rats during 4 weeks increases phosphorylation of S6K1 (157%) and 4EBP1 (146%) in muscle. These results therefore appear to connect the ability of citrulline to increase muscle protein synthesis to a stimulatory action on the mTORC1 pathway, via activation of S6K1 and 4E-BP1 by phosphorylation. In addition, this stimulation of mTORC1 is partially Akt/PI3K (phosphatidylinositol 3-kinase)-independent, as citrulline maintains 4E-BP1 activation by phosphorylation even when the Akt/PI3K pathway, upstream of mTORC1, is inhibited [18]. Finally, although citrulline clearly stimulates overall muscle protein synthesis, it has a more complex action on protein expression. Indeed, citrulline is able to stimulate the expression of specific muscle proteins and inhibit the expression of others. This citrullinemediated modulation of specific proteins was recently listed in a general review by Bourgoin-Voillard et al. [19]. In particular, citrulline more specifically stimulates the expression of myofibrillar proteins and modulates enzymes that drive energy metabolism. Indeed, again in the same model of protein energy malnutrition as described earlier, a differential proteomics approach showed that at muscle level, citrulline leads to overexpression of the enzymes involved in glycogenolysis (i.e., glycogen phosphorylase) and glycolysis (i.e., phosphoglucomutase 1,6-phosphofructokinase, triosephosphate isomerase, β-enolase and pyruvate kinase M1/M2 isozymes) and lower expression of some enzymes of the Krebs cycle (i.e., isocitrate dehydrogenase and succinate dehydrogenase) and the mitochondrial respiratory chain (i.e., NADH dehydrogenase complex, NADH ubiquinone oxidoreductase, and ATP synthase) [20]. Another proteomics study in “healthy” aged rats showed that citrulline supplementation stimulates the expression of mitochondrial biogenesis factor (TFAM: Mitochondrial transcription factor A) as well as mitochondrial complex I activity [11]. A more recent study has confirmed the effect of citrulline supplementation on energy metabolism: Goron et al. [13] showed that citrulline supplementation in adult rats during 4 weeks stimulates several enzymes involved in the pathways generating acetyl-CoA. These properties of citrulline were further confirmed in vitro. In myotubes derived from primary myoblasts, deprived of amino acids and serum during 16 hours, citrulline (5 mM) is able to stimulate protein synthesis and reallocate ATP to the protein synthesis machinery (unpublished data). Thus, the stimulation of protein synthesis could be related to the energy reallocation toward protein synthesis, but the underlying mechanisms remain to be established. It is now well established that citrulline has positive effects on muscle protein synthesis, yet there is still surprisingly little data in the literature on the effects of citrulline on proteolysis. Ham et al. [21] have demonstrated in immobilized mice that citrulline supplementation downregulates Bnip3, a proautophagic gene strongly stimulated by immobilization. In addition, LC3BII:LC3BI protein ratio, a marker of autophagosome number, is increased during immobilization but restored when the mouse is supplemented with citrulline. This result could tie into work by Faure et al. [20] showing that a 5-day citrulline complement decreases proteasome activator complex subunit 1. However, despite interesting but fragmentary data, the regulation of muscle proteolysis by citrulline remains to be explored. Finally, in a very preliminary set of data [22], we focused our attention on a still underexplored aspect of muscle protein metabolism, i.e., muscle secreted proteins. Indeed, a new aspect of muscle protein metabolism has emerged in the past few years. Several III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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authors have pointed out the ability of muscle to secrete specific proteins in response to various stimuli, in particular exercise (see Pedersen et al. [23,24] for reviews). We observed that citrulline is able to modify the patterns of proteins secreted by muscle cells in cultures by decreasing the production of five proteins and increasing the secretion of four proteins compared to amino acid-free media [22]. Four of these proteins (calumenin, cystatin C, fetuin-A, and transcobalamin) are involved in citrulline-modulated functions, i.e., cardiovascular functions (see [3,4] for reviews), brain functions [25,26], and protein synthesis. This proteomics study therefore suggests, for the first time, that the effect of citrulline on general metabolism could hinge on its effect on muscle, opening up an important new field for research. As citrulline has key properties in muscle functions, there is major interest in the effects of citrulline on exercise in humans. Surprisingly though, only a few studies have evaluated the potential benefit of citrulline on performance, and the results seem to be different according to duration of supplementation, type of exercise, and form of citrulline given. Indeed, an acute citrulline supplementation seems to have zero if not deleterious effect on anaerobic or aerobic exercise performance [27,28]. However, the results change with chronic citrulline administration: a 7-day citrulline ingestion leads to increased time to exhaustion and total amount of work completed in high-intensity cycling exercise, and decreased time to complete a 4-km cycling time trial associated with better subjective feelings of muscle fatigue [29,30]. It was proposed that is due to effects of citrulline on vascular function (see [3,4] for reviews). These data are in line with the fact that citrulline preserves splanchnic perfusion in the gut and attenuates intestinal disorders during exercise in athletes [31]. However, the athletes usually consumed citrulline as malate salt. Several studies have been performed with this chemical form to evaluate the properties of citrulline. The effects of citrulline malate were beneficial on performance, recovery, and immune system, which is altered after exercise [32 37]. The effects on performance could be due to the ability of citrulline to increase the use of amino acids during exercise, especially branched-chain amino acids [38]. However, results found with citrulline malate supplementation must be interpreted with caution, as it is impossible to know whether such effects are due to the citrulline itself or to the malate, which is an intermediate of the Krebs cycle [39].

CONCLUSION The beneficial value of citrulline to stimulate protein synthesis to maintain muscle mass, and thus improve muscle functionality, has been incontrovertibly demonstrated, particularly in situations of protein deficiency. An original hypothesis, proposed by Moinard et al. [40], is that citrulline, which is synthesized and released by the intestine mainly in postabsorptive state or in the situation of low protein intake, could maintain a minimal level of protein synthesis to preserve muscle proteins during the postabsorptive state, unlike leucine, a well-known amino acid activator of protein synthesis during the postprandial state. Citrulline and leucine could be the two essential amino acids that control nitrogen balance and muscle protein composition, depending on nutritional state. This elegant hypothesis needs to be confirmed by further studies.

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To conclude, despite the recent surge in data on the effects of citrulline on muscle, further explorations are still needed in order to gain a complete overview of the role of citrulline in muscle metabolism and the mechanisms involved. Moreover, promising data on the effect of citrulline on proteins secreted by muscle cells open up important avenues for research on the indirect action of citrulline, and of nutrients in general, on general metabolism, through its effects on muscle. Conflict of Interest C. Moinard and C. Breuillard are shareholders in Citrage.

References [1] Windmueller HG, Spaeth AE. Source and fate of circulating citrulline. Am J Physiol 1981;241:E473 80. [2] Crenn P, Coudray-Lucas C, Thuillier F, Cynober L, Messing B. Postabsorptive plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal failure in humans. Gastroenterology 2000;119:1496 505. [3] Romero MJ, Platt DH, Caldwell RB, Caldwell RW. Therapeutic use of citrulline in cardiovascular disease. Cardiovasc Drug Rev 2006;24:275 90. [4] Figueroa A, Wong A, Jaime SJ, Gonzales JU. Influence of L-citrulline and watermelon supplementation on vascular function and exercise performance. Curr Opin Clin Nutr Metab Care 2017;20:92 8. [5] Breuillard C, Cynober L, Moinard C. Citrulline and nitrogen homeostasis: an overview. Amino Acids 2015;47:685 91. [6] Osowska S, Moinard C, Neveux N, Loı¨ C, Cynober L. Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection. Gut 2004;53:1781 6. [7] Osowska S, Duchemann T, Walrand S, Paillard A, Boirie Y, Cynober L, et al. Citrulline modulates muscle protein metabolism in old malnourished rats. Am J Physiol Endocrinol Metab 2006;291:E582 6. [8] Le Ple´nier S, Walrand S, Noirt R, Cynober L, Moinard C. Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway? Amino Acids 2012;43:1171 8 [9] Ventura G, Noirez P, Breuille´ D, Godin JP, Pinaud S, Cleroux M, et al. Effect of citrulline on muscle functions during moderate dietary restriction in healthy adult rats. Amino Acids 2013;45:1123 31. [10] Bourdon A, Parnet P, Nowak C, Tran N-T, Winer N, Darmaun D. L-Citrulline supplementation enhances fetal growth and protein synthesis in rats with intrauterine growth restriction. J Nutr 2016;146:532 41. [11] Moinard C, Le Plenier S, Noirez P, Morio B, Bonnefont-Rousselot D, Kharchi C, et al. Citrulline supplementation induces changes in body composition and limits age-related metabolic changes in healthy male rats. J Nutr 2015;14:1429 37. [12] Faure C, Raynaud-Simon A, Ferry A, Dauge´ V, Cynober L, Aussel C, et al. Leucine and citrulline modulate muscle function in malnourished aged rats. Amino Acids 2012;42:1425 33. [13] Goron A, Lamarche F, Cunin V, Dubouchaud H, Hourde´ C, Noirez P, et al. Synergistic effects of citrulline supplementation and exercise on performance in male rats: evidence for implication of protein and energy metabolisms. Clin Sci 2017;131:775 90. [14] Jourdan M, Nair KS, Carter RE, Schimke J, Ford GC, Marc J, et al. Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low protein diet—a pilot study. Clin Nutr 2015;34:449 56. [15] Bouillanne O, Melchior J-C, Faure C, Canouı¨-Poitrine F, Paul M, Boirie Y, et al. Effects of citrulline (CIT) oral supplementation during 21 days on body composition in malnourished elderly patients. Clin Nutr 2015;34: S17 18. [16] Thibault R, Flet L, Vavasseur F, Lemerle M, Ferchaud-Roucher V, Picot D, et al. Oral citrulline does not affect whole body protein metabolism in healthy human volunteers: results of a prospective, randomized, doubleblind, cross-over study. Clin Nutr 2011;30:807 11. [17] Jirka A, Layec S, Picot D, Bernon-Ferreira S, Darmaun D. Effect of citrulline supplementation on protein metabolism in patients with short bowel syndrome: a stable isotope study. Clin Nutr 2016;35:S131.

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[18] Le Ple´nier S, Goron A, Sotiropoulos A, Archambault E, Guihenneuc C, Walrand S, et al. Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies. Am J Physiol Endocrinol Metab 2017;312:E27 36. [19] Bourgoin-Voillard S, Goron A, Seve M, Moinard C. Regulation of the proteome by amino acids. Proteomics 2016;16:831 46. [20] Faure C, Morio B, Chafey P, Le Ple´nier S, Noirez P, Randrianarison-Huetz V, et al. Citrulline enhances myofibrillar constituents expression of skeletal muscle and induces a switch in muscle energy metabolism in malnourished aged rats. Proteomics 2013;13:2191 201. [21] Ham DJ, Kennedy TL, Caldow MK, Chee A, Lynch GS, Koopman R. Citrulline does not prevent skeletal muscle wasting or weakness in limb-casted mice. J Nutr 2015;145:900 6. [22] Breuillard, C., Goron, A., Cunin, V., Bourgoin-Voillard, S., Se`ve, M., Moinard, C. (2017). Modulation of muscle protein synthesis by amino acids: consequences on the secretome—a preliminary in vitro study. In: XXXIXth ESPEN congress, The Hague, The Netherlands. September 2017. Clin Nutr. 36 (S1), S157. [23] Pedersen BK. Muscle as a secretory organ. Compr Physiol. 2013;3:1337 62. [24] Pedersen BK, Febbraio MA. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Nat Rev Endocrinol. 2012;8:457 65. [25] Moinard C, Tliba L, Diaz J, Le Ple´nier S, Nay L, Neveux N, et al. Citrulline stimulates locomotor activity in aged rats: implication of the dopaminergic pathway. Nutrition 2017;38:9 12. [26] Marquet-de Rouge´ P, Clamagirand C, Facchinetti P, Rose C, Sargueil F, Guihenneuc-Jouyaux C, et al. Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus. Age 2013;35:1589 606. [27] Hickner RC, Tanner CJ, Evans CA, Clark PD, Haddock A, Fortune C, et al. L-citrulline reduces time to exhaustion and insulin response to a graded exercise test. Med Sci Sports Exerc 2006;38:660 6. [28] Cutrufello PT, Gadomski SJ, Zavorsky GS. The effect of l-citrulline and watermelon juice supplementation on anaerobic and aerobic exercise performance. J Sports Sci 2015;33:1459 66. [29] Bailey SJ, Blackwell JR, Lord T, Vanhatalo A, Winyard PG, Jones AM. L-Citrulline supplementation improves O2 uptake kinetics and high-intensity exercise performance in humans. J Appl Physiol 2015;119:385 95. [30] Suzuki T, Morita M, Kobayashi Y, Kamimura A. Oral L-citrulline supplementation enhances cycling time trial performance in healthy trained men: double-blind randomized placebo-controlled 2-way crossover study. J Int Soc Sports Nutr 2016;13:6. [31] van Wijck K, Wijnands KAP, Meesters DM, Boonen B, van Loon LJC, Buurman WA, et al. L-Citrulline improves splanchnic perfusion and reduces gut injury during exercise. Med Sci Sports Exerc 2014;46:2039 46. [32] Bendahan D, Mattei JP, Ghattas B, Confort-Gouny S, Le Guern ME, Cozzone PJ. Citrulline/malate promotes aerobic energy production in human exercising muscle. Br J Sports Med 2002;36:282 9. [33] Pe´rez-Guisado J, Jakeman PM. Citrulline malate enhances athletic anaerobic performance and relieves muscle soreness. J Strength Cond Res Natl Strength Cond Assoc. 2010;24:1215 22. [34] Glenn JM, Gray M, Wethington LN, Stone MS, Stewart RW, Moyen NE. Acute citrulline malate supplementation improves upper- and lower-body submaximal weightlifting exercise performance in resistance-trained females. Eur J Nutr 2017;56:775 84. [35] Wax B, Kavazis AN, Weldon K, Sperlak J. Effects of supplemental citrulline malate ingestion during repeated bouts of lower-body exercise in advanced weightlifters. J Strength Cond Res Natl Strength Cond Assoc. 2015;29:786 92. [36] Glenn JM, Gray M, Jensen A, Stone MS, Vincenzo JL. Acute citrulline-malate supplementation improves maximal strength and anaerobic power in female, masters athletes tennis players. Eur J Sport Sci. 2016;16:1095 103. [37] Sureda A, Cordova A, Ferrer MD, Tauler P, Perez G, Tur JA, et al. Effects of L-citrulline oral supplementation on polymorphonuclear neutrophils oxidative burst and nitric oxide production after exercise. Free Radic Res 2009;43:828 35. [38] Sureda A, Co´rdova A, Ferrer MD, Pe´rez G, Tur JA, Pons A. L-citrulline-malate influence over branched chain amino acid utilization during exercise. Eur J Appl Physiol 2010;110:341 51. [39] Wagenmakers AJ. Muscle amino acid metabolism at rest and during exercise: role in human physiology and metabolism. Exerc Sport Sci Rev 1998;26:287 314. [40] Moinard C, Cynober L. Citrulline: a new player in the control of nitrogen homeostasis. J Nutr 2007;137:1621S 5S.

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Sulfur Amino Acids and Skeletal Muscle Isabelle Papet, Didier Re´mond, Dominique Dardevet, Laurent Mosoni, Sergio Polakof, Marie-Agne`s Peyron and Isabelle Savary-Auzeloux Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France

This chapter is aimed to provide an integrated view of the metabolism of sulfur amino acids and related compounds in regards to skeletal muscle metabolism and function. The first part will give the basic biochemical aspects concerning sulfur amino acids. Then, the specificities of muscle regarding sulfur amino acids and related compounds will be described. Some situations in which sulfur amino acid/glutathione homeostasis is impaired in muscle will be described. Finally, potential nutritional strategies developed to provide muscle with the optimal amounts of sulfur amino acids will be addressed

GENERAL BIOCHEMICAL ASPECTS ON SULFUR AMINO ACIDS Methionine and cysteine can be considered special among amino acids based on the following features: intrinsic chemical properties and roles of their related metabolites.

Intrinsic Chemical Properties of Sulfur Amino Acids In addition to carbon, hydrogen, oxygen, and nitrogen, the basal constitutive atoms of all amino acids, only methionine and cysteine contain sulfur. Molecular weight of the two sulfur amino acids differs by 20% (Fig. 20.1). The atom of sulfur is within the main chain of methionine and in the thiol side chain of cysteine. The oxidation of the thiol leads to the homodisulfide cystine (Fig. 20.2) as well as to various heterodisulfides. The cysteine/cystine couple is the largest pool of low molecular weight thiols in plasma [1]. The equilibrium between cysteine and cystine is in favor of cystine in plasma [redox potential of cysteine/cystine couple

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FIGURE 20.1 Chemical structures of methionine and metabolically related compounds. Methionine is basically converted into homocysteine through the TM comprising three steps catalyzed by methionine adenosyltransferase, methyltransferases, and adenosylhomocysteine hydrolase. L-Carnitine derives from the initial trimethylation of lysine residues of proteins, and creatine is the direct product of the methylation of guanidinoacetate (coproduct in the synthesis of ornithine from arginine and glycine). Carnitine and creatine are of primarily interest for skeletal muscle function. Homocysteine is at a metabolic crossroad, being either converted back into methionine through folate- or choline-dependent RM pathways, or used for the synthesis of cysteine through the TS comprising two steps catalyzed by cystathionine β-synthase that uses serine as cosubstrate, and cystathionine γ-lyase. The TR pathway of methionine can occur but is usually quantitatively insignificant. CAS, chemical abstracts service; RM, remethylation; TM, transmethylation; TR, transamination; TS, transsulfuration.

Eh 5 280 mV] and in favor of cysteine within cells (Eh 5 2150 mV) [1]. The formation of disulfide bounds between two cysteine residues plays an important role in the folding of proteins, and the relative ease of reversible oxidation and reduction of both sulfur amino acids serves as regulatory switches of protein bioactivities and signaling pathways [2
4]. On the opposite, the uncontrolled irreversible modification of sulfur amino acid residues can lead to impaired proteins with potential detrimental consequences. Among them, some can be such as the formation of paracetamol-protein adducts that play a key role in the development of liver failure consecutive to intoxication with paracetamol, an analgesic drug widely used [5,6].

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FIGURE 20.2 Chemical structures of cysteine and metabolically related compounds. Cysteine is a building block of glutathione, whose synthesis (1) and degradation (2) are depicted in Fig. 20.3. Cysteine degradation (Fig. 20.4) leads to either taurine, or sulfate anion (3, oxidative pathway) or to hydrogen sulfide (4, desulfuration pathways). Hydrogen sulfide can also be produced by the desulfuration of cystine (5) and homocysteine (not shown). Hydrogen sulfide is metabolized into sulfate (6). Cysteine is a precursor in the synthesis of coenzyme A that occurs in 5 steps (7). The degradation of coenzyme A produces taurine (8). CAS, chemical abstracts service.

Metabolism and Specific Roles of Methionine Transmethylation and Compounds Metabolically Linked to Methionine Aside from its role in protein turnover, methionine also plays other key metabolic roles (Fig. 20.1) [7
9]. In the transmethylation pathway, methionine is irreversibly converted into S-adenosylmethionine, the major methyl donor in the cell. Several enzymes catalyze methyl transfer from S-adenosylmethionine to a wide variety of methyl acceptors leading to the formation of S-adenosylhomocysteine and methylated acceptors. S-Adenosylmethionine is involved in methylation of amino acids residues of proteins, phospholipids, DNA (epigenetic regulations), RNA, and decarboxylated S-adenosylmethionine in the synthesis of polyamines. For example, the trimethylation of lysine residues of proteins (calmodulin, actin, myosin) through transmethylation with S-adenosylmethionine is the first step in the biosynthesis of

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carnitine [10], whose key role in skeletal muscle is described below. In the same line, about 40% of the flux of methionine transmethylation contributes to the synthesis of creatine (Fig. 20.1) [11]—another key compound for muscle, whose role is explained below. S-Adenosylhomocysteine is then converted to homocysteine (Fig. 20.1), a metabolite at crossroads between methionine and cysteine. Homocysteine can be recycled back to methionine through folate/choline-dependent remethylation pathways or used for the synthesis of cysteine and α-ketobutyrate. α-Ketobutyrate contains the carbon skeleton of methionine and is an intermediate of the Krebs cycle [12]. Thus, the synthesis of cysteine determines the use of methionine as an energetic fuel. Transamination A potential alternative pathway for methionine degradation is similar to the catabolism pathway of most amino acids [9]. This pathway involves an initial transamination to 2keto-4-methylthiobutyrate, which is then oxidatively decarboxylated by the branched chain 2-oxo acid dehydrogenase complex to form 3-methylthiopropionate. The latter is metabolized to the highly toxic and volatile molecule methanethiol [13]. The functional significance of methionine transamination pathway in physiological conditions has still not been established [9]. Recent works using experimental models support a role of this pathway in the toxic effect of large excess of dietary methionine (sixfold basal) when the transsulfuration pathway is invalidated [14] and in the suppression of hepatic glucose production after an acute administration of methionine in the fasted state [15]. It is rather unlikely to reach such methionine excess in usual nutritional conditions and the transamination pathway is even very often left out by the reviews dealing with sulfur amino acid metabolism [7,16
18]. Transsulfuration: Endogenous Synthesis of Cysteine Cysteine can be endogenously synthesized, mainly in the liver, from homocysteine issued from the transmethylation of methionine (Fig. 20.1) [19]. The unidirectional transsulfuration pathway producing cysteine consists in two vitamin B6-dependent steps. Homocysteine is first condensed with serine by cystathionine β-synthase (CBS). Cystathionine is then cleaved into cysteine and α-ketobutyrate by cystathionine γ-lyase (CSE, also named cystathionase). According to this transsulfuration process, methionine gives only the atom of sulfur to cysteine, the carbon chain of cysteine coming from serine. Homocysteine, not cysteine, can be converted back to methionine through the remethylation pathway that is dependent on B9 and B12 vitamins. High plasma levels of total homocysteine have been associated with an increased risk of atherosclerotic diseases. However, a recent Cochrane review concluded that supplements of vitamins B6, B9, or B12 alone or in combination, aimed to reduce homocysteine, did not prevent cardiovascular events in patients with or without preexisting cardiovascular disease [20].

Compounds Metabolically Linked to Cysteine In addition to protein turnover, cysteine is used for the synthesis of glutathione, coenzyme A, taurine, reduced and oxidized inorganic sulfurs (Fig. 20.2) [19,21].

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Glutathione ROLES OF GLUTATHIONE

Glutathione [γ-glutamyl-cysteinylglycine (Fig. 20.2)] is the predominant intracellular low-molecular-weight thiol that is normally over 99% reduced glutathione (GSH) [22]. The γ-glutamyl bound makes glutathione resistant to peptidases. GSH is oxidized to glutathione homodisulfide (GSSG) and low molecular weight mixed disulfides (GSSR), then reduced back to GSH by the very effective glutathione reductase using NADPH 1 H1 generated from NADP1 via intracellular metabolism of energetic nutrients (Fig. 20.3) [23].

FIGURE 20.3 Glutathione metabolic pathways. Glutathione is synthetized in two steps catalyzed by glutamate
cysteine ligase (1) then glutathione synthetase (2). The degradation of glutathione through the γ-glutamyl cycle comprises three steps catalyzed by γ-glutamyltranspeptidase located on the external surface of cell membranes (3), γ-glutamyl cyclotransferase (4), and 5-oxoprolinase that releases glutamate (5). Cysteine and glycine are liberated from cysteinlyglycine by a peptidase (not shown). In stressed cells, glutathione can be degraded by a specific γ-glutamyl cyclotransferase (also named Chac 1) (6). Reduced glutathione is converted into glutathione homo- or heterodisulfides through nonenzymatic (oxidation) or enzymatic reaction catalyzed by glutathione peroxidase (7). Other oxidized glutathione derivatives may be S-nitrosoglutathione (GS-NO) and glutathione
metal complexes. Glutathione disulfides are reduced back to reduced glutathione by NADPH-dependent glutathione disulfide reductase (8). The glutathionylation of proteins is a reversible posttranslational modification occurring spontaneously or catalyzed by glutathione-S-transferase or glutaredoxin (7, 8). Electrophiles can be conjugated to glutathione through nonenzymatic or enzymatic reaction catalyzed by glutathione-S-transferase (9). The conjugate is metabolized into the mercapturate (10) with the successive contribution of glutathione-S-transferase, a peptidase and cysteine-S-conjugate N-acetyltransferase. γ-glu-AA, γ-glutamyl-amino acid; γ-glu-cys, γ-glutamyl-cysteine; γ-glu-cystine, γ-glutamyl-cystine; AA, amino acid; Chac1, cation transport regulator-like protein 1; Cys, cysteine; Cys-gly, cysteinylglycine; Glu, glutamate; Gly, glycine; GSH, reduced glutathione; GSSG, glutathione homodisulfide; GSSR, glutathione heterodisulfide; GSX, glutathione thioether conjugate; X, electrophile derived from endogenous compound, xenobiotic or pharmaceutical drug.

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GSH/GSSG couple is a major determinant of intracellular redox potential [24]. In cell culture, the redox potential of GSH/GSSG varies according to growth conditions being more reduced during proliferation (Eh 5 2260 to 2230 mV), intermediate (Eh 5 2220 to 2190 mV) during differentiation and growth arrest, and more oxidized (Eh 5 2170 to 2150 mV) during apoptosis [25]. GSH exerts various antioxidant activities that produce GSSG [24,26,27]. Among these, GSH scavenges electrophiles such as reactive oxygen/ nitrogen species and other free radicals (nonenzymatically or via glutathione peroxidase). Glutathione reductase also quenches H2O2, and converts lipid hydroperoxides into less toxic alcohol derivatives. Moreover, GSH conjugates to electrophiles derived from endogenous (e.g., estrogen, melanins, prostaglandins, and leukotriens) and exogenous/pharmacological (e.g., paracetamol) compounds forming glutathione thioether conjugates glutathione conjugates (GSX) [22]. Theses conjugations are either spontaneous or catalyzed by glutathione-S-transferase, an enzyme of phase II detoxification pathway. GSX conjugates are further metabolized through the mercapturic acid pathway allowing their elimination within bile or urine. Urinary excretion of mercapturates constitutes a net loss of cysteine. At the protein level, glutathione not only limits the oxidative potential of the medium through its antioxidant properties but also can form reversible disulfide bound with cysteine residue located in basic regions of protein architecture [28,29]. This posttranslational modification named glutathionylation occurs either spontaneously or via glutathione S-transferase or glutaredoxin [28]. Deglutathionylation of proteins is mainly catalyzed by glutaredoxin, the most efficient thiol disulfide oxidoreductase [30,31]. Glutathionylation/ deglutathionylation regulates redox state, activity and stability of numerous proteins that are recorded in specific peptidomic databases [32]. Glutathionylation protects proteins against the irreversible oxidation of cysteine residues into sulfinic and sulfonic acid [28]. Glutathionylation/deglutathionylation plays important roles in cell physiology and disturbance of glutathionylated protein homeostasis is emerging as a player in regulation of 20S proteasome activity [33], signaling mechanism in cardiovascular diseases [34], modulation of mitochondrial functions [35], epigenetic mechanisms [36], among many others. GLUTATHIONE METABOLISM THROUGH THE γ-GLUTAMYL CYCLE

Cellular level of glutathione results from the balance between the rate of its intracellular synthesis and the rate of its export by the cells [37]. Intracellular and extracellular reactions of the γ-glutamyl cycle determine the turnover of glutathione (Fig. 20.3) [22]. Glutathione is synthesized intracellularly in two steps and catalyzed by glutamate-cysteine ligase then glutathione synthetase. Glutathione synthesis is regulated by the activity of the first enzyme and the availability of cysteine [38]. High concentration in glutathione exerts a negative feedback on the activity of glutamate-cysteine ligase [22]. GSH, oxidized glutathione derivatives (GSSG, GSSR), and GSX are exported out of cells by active efflux transporters, such as multidrug resistance-associated proteins (MRP) [39
41]; however, identification and characterization of efflux glutathione transporters are not completely established [42]. The extracellular degradation of glutathione and its oxidized derivatives occurs initially via the γ-glutamyl transpeptidase located on the external surface of cell membranes (Fig. 20.3) [22]. This enzyme transfers the γ-glutamyl moiety of glutathione to a

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suitable acceptor, the most active acceptors being cystine and aminoacylglycines [43], and produces γ-glutamyl-acceptor and cysteinylglycine. Cysteine and glycine are liberated by a membrane-bound or cytosolic peptidase and γ-glutamyl-acceptor goes on the intracellular γ-glutamyl cycle owing to regenerate glutamate as well as the acceptor [22,44]. Alternatively, the intracellular reduction of γ-glutamyl-cystine by transhydrogenation involving GSH can liberate cysteine and γ-glutamyl-cysteine, the latter being a substrate of glutathione synthetase [22]. Altogether, γ-glutamyl cycle plays a key role in the cellular import of the three amino acids that are salvaged and reusable for intracellular synthesis of glutathione. It also carries out the transport of other amino acids/peptides, notably cysteine/cystine. In contrast, an irreversible loss of cysteine, but not of the two other glutathione constitutive amino acids, results from GSX metabolism to mercapturates, because mercapturates are excreted along with urine. At the whole body level, there is an interorgan glutathione flow driven by organ-specific activities of glutathione-related enzymes [45]. Indeed, organs and tissues with low γ-glutamyltranspeptidase activity are exporters of glutathione, whereas those with high γ-glutamyltranspeptidase activity utilize plasma glutathione. Liver is recognized as the main organ for the synthesis and the export of glutathione. Consequently, glutathione level in other tissues may decrease when the use of glutathione for detoxification processes dramatically depletes liver glutathione [45]. INTRACELLULAR DEGRADATION OF GLUTATHIONE

In stressed cells, more recent evidences support an intracellular degradation of glutathione. Indeed, a γ-glutamyl cyclotransferase acting specifically toward glutathione but not the other γ-glutamyl peptides leads to 5-oxoproline (a cyclized form of glutamate), and cysteinylglycine (Fig. 20.3) [46,47]. This enzyme actually named Chac1 (cation transport regulator-like protein 1, MGC4504 gene) was first discovered as a member of the PERK-ATF4 (protein kinase RNA-like endoplasmic reticulum kinase—activating transcription factor 4) arm of the unfolded protein response pathway [48], and then characterized as a proapoptotic factor [49]. Chac1 may play an important role in oxidative stress and apoptosis (highly orchestrated and inducible programmed cell death) induced by the activation of the unfolded protein response pathway [47]. The activation of Chac1 decreases intracellular glutathione leading to oxidative damages and the opening of the mitochondrial permeability transition pore, destroying, in turn, the mitochondrial integrity [24]. Permeabilized mitochondria release proapoptotic factors and procaspase into cytoplasm, the determinant mechanism of the mitochondria-mediated intrinsic pathways of apoptosis. The cellular GSH/GSSG redox status also interacts with the death receptormediated extrinsic pathways of apoptosis [50]. Not only Chac1 is an early proapoptotic factor but a swift cellular extrusion of glutathione inducing a prompt decrease of intracellular glutathione concentration also initiates the sequence of events leading to apoptosis [39,51,52]. Other Compounds Metabolically Related to Cysteine Catabolic pathways of cysteine are endogenous sources of taurine, reduced and oxidized inorganic sulfurs, and cysteine is used for the synthesis of coenzyme A (Fig. 20.2).

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CATABOLIC PATHWAYS OF CYSTEINE

Cysteine degradation occurs, mainly in the liver, through cysteine sulfinate-dependent pathways initiated by the cysteine dioxygenase (oxidative pathway) and three cysteine sulfinate-independent pathways (desulfuration pathways) (Fig. 20.4) [19]. Briefly, in the oxidative pathways, cysteine sulfinate is either decarboxylated to produce hypotaurine then taurine or transaminated to form pyruvate and sulfite (SO322). One of the desulfuration pathways involves unspecific aminotransferase then 3-mercaptopyruvate sulfurtransferase and ends up with pyruvate and hydrogen sulfide (H2S). In the two others pathways, the direct desulfuration of cysteine catalyzed by CBS or CSE produces H2S and various molecules depending on cosubstrates. Desulfuration pathways prevail over oxidative pathway when cysteine supply is low, whereas oxidative pathway largely increases when cysteine supply is excessive. The enlargement of cysteine oxidation occurs through increases in both the concentration (up to 45-fold) and the catalytic efficiency (up to 10fold) of cysteine dioxygenase [53,54]. Desulfuration pathway of cystine ends up with cysteine, pyruvate, NH41, and H2S, and the desulfuration of homocysteine by CSE forms homoserine and H2S. The metabolism of H2S to sulfate (SO422) is less defined than H2S biosynthetic pathways [55]. One described pathway is as follows. H2S is converted to thiosulfate (S2SO322)

FIGURE 20.4 Simplified catabolic pathways of cysteine. Cysteine is catabolized through an oxidative pathway initiated by cysteine dioxygenase (1) and three desulfuration pathways (2
4). Cysteine sulfinate is either decarboxylated to produce hypotaurine then taurine, or transaminated to then liberate pyruvate and SO32. The transamination (2) of cysteine leads to β-mercaptopyruvate whose transsulfuration gives pyruvate and H2S. The direct desulfuration of cysteine is catalyzed by CBS (3) or CSE (4) . The oxidation of H2S to SO32 then SO422 provides electrons for the mitochondrial respiratory chain (*). α-KA, α-keto acid; AA, acide amine´; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; H2S, hydrogen sulfide; SO32, sulfite; SO422, sulfate; S2O322, thiosulfate; X, Y, cysteine cosubstrates; X0 , Y0 , H2S coproducts.

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with the cooperation of three enzymes: sulfide quinone oxidoreductase transferring the electrons to ubiquinone an electron carrier in the mitochondrial respiratory chain, sulfur dioxygenase also named persulfide dioxygenase or 2-ethylmalonic encephalopathy protein 1, and thiosulfate sulfurtransferase also named rhodanese [56,57]. This step links sulfur metabolism with the respiratory chain. S2SO322 is a pivotal metabolite that can be either converted back to H2S plus SO322 by the GSH-dependent thiosulfate reductase [21], or further degraded to sole SO322 by the thiosulfate tanssulfurase [54]. SO322 issued from H2S or produced through cysteine sulfinate-dependent pathway (see above) is oxidized to SO422 by sulfite oxidase with ferricytochrome c as the electron acceptor [58]. This step constitutes the second link between sulfur metabolism and the respiratory chain, meaning that sulfur metabolism per se provides energy for adenosine triphosphate (ATP) production. GENERAL ROLES OF TAURINE, HYDROGEN SULFIDE, AND SULFATE

Molecules issued from the degradation of cysteine and a fortiori from methionine are not only by-products but also are metabolic actors. Taurine is an antioxidant, it neutralizes hypochlorous acid, a reactive species produced by neutrophils. It is also involved in conjugation reactions playing a role in elimination of toxins, metabolism of lipids (via bile acid conjugation), activity of glycolipids, to name a few. Moreover, taurine modulates basic processes, such as osmotic pressure, cation homeostasis, enzyme activity, receptor regulation, cell development, and cell signaling [59
62]. H2S has been identified as a “gaseous” signaling molecule such as nitric oxide (NO) and carbon monoxide (CO) [55]. It is also recognized as a cytoprotectant player [63]. At physiological pH, about 80% of total free soluble H2S is present as HS2, 20% as neutral (H2S) and there is essentially no dianionic S22 in biological fluids [64]. There are also two forms of storage with proteins [65]. Intracellular concentrations of total free pool are only 10 nM
3 μM due to rapid degradation under aerobic conditions [65,66]. A recent blooming literature reports the implication of H2S in a variety of physiological functions, notably in vascular function and neurological systems among many others [55]. Mechanisms of action and molecular targets of H2S are incompletely defined, and investigations are complicated by methodological limitations regarding accurate measurement of H2S and metabolic interrelations between various sulfur-containing molecules [55,67
70]. Some of the physiological responses attributed to H2S have recently found to be rather induced by polysulfides (H2Sn) [65]. Free SO422 pools are not only supplied mainly by the catabolism of cysteine [71] but also by the transport-mediated intestinal absorption of SO422, the transport-mediated renal reabsorption of SO422, the degradation of sulfur-containing molecules, and the activity of sulfatases [72]. SO422 is converted to 30 -phosphoadenosine 50 -phosphosulfate (PAPS) [73] that is the universal sulfonate donor implicated in various sulfotransferase reactions [72]. PAPS, the activated form of SO422, is required in (1) the inactivation of thyroid hormones, catecholamines, and steroid hormones; (2) the structure of sulfolipids, glycosaminoglycans, and glycoproteins; (3) the elimination of bile acids; (4) the posttranslational modification of tyrosine-containing secretory proteins; (5) the activation of pharmacological drugs; and (6) the detoxification through phase II conjugation reaction of xenobiotics and pharmacological drugs [74
76]. In the detoxification process of paracetamol, sulfonation (sulfation)

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is a high-affinity but low-capacity reaction that is saturable when increasing dose [77]. This feature contributes to paracetamol toxicity consecutive to overdoses [6]. COENZYME A

Another specific use of cysteine is the synthesis of coenzyme A occurring in five steps [78]. The chemical structure of coenzyme A encompasses three chemical parts: ATP, pantothenic acid (vitamin B5), and cysteine (Fig. 20.2) [79]. Coenzyme A is an ubiquitous cofactor involved in cellular oxidative pathways including fatty acid β-oxidation, carbohydrate and amino acid oxidations, and Krebs cycle; and in lipid synthesis, protein modifications and membrane trafficking [78,79]. Coenzyme A is degraded into cysteamine and pantothenic acid that can be recycled in the synthesis of coenzyme A [80]. The oxidation of cysteamine produces hypotaurine [81] that is oxidized into taurine. That makes a “transversal” metabolic link between coenzyme A and taurine, two compounds issued from cysteine. Cysteamine breaks disulfide bonds within proteins yielding to mixed disulfides and inhibits γ-glutamyl-cysteine ligase, the rate-limiting enzyme in the synthesis of glutathione [80].

SULFUR AMINO ACIDS AND RELATED COMPOUNDS IN SKELETAL MUSCLE Skeletal Muscle Concentrations of Sulfur Amino Acids Contained in Proteins Like for other amino acids, proteins are the main pool of sulfur amino acids in skeletal muscle. Muscle protein mass varies according to various time scales (Fig. 20.5). In a daily scale, muscle protein mass slightly increases in the postprandial period, whereas it decreases during the postabsorptive state [82]. At the scale of life, the major accretion of muscle protein mass occurs during the growth period. Adult muscle protein mass averages 4
8 kg [83,84] and decreases slowly during aging associated or not with chronic diseases, named respectively primary and secondary sarcopenia [82,85]. Moreover, acute health problems are associated with acute muscle wasting followed by more or less efficient recovery lasting few days to months depending on the severity of the problem and the age of patients [86]. Skeletal muscle concentrations of sulfur amino acids contained in FIGURE 20.5

Simplified views of skeletal muscle changes and approximate concentrations of sulfur amino acids and metabolically related compounds. Concentrations are expressed per g of wet weight of skeletal muscle. Cys, cysteine; met, methionine.

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proteins follow the variations of muscle content in proteins, since no major change in amino acid composition of muscle proteins occurs over time. Measurement of sulfur amino acid contents of proteins necessitates performic acid oxidation prior to hydrochloric acid hydrolysis liberating methionine sulfone and cysteic acid that are quantified using an amino acid analyzer with ninhydrin detection, the reference method. Amino acid content of proteins is historical expressed as g of amino acid per 16 g of nitrogen (N), considering that 16 g N corresponds to 100 g of proteins (i.e., protein content 5 6.25 3 N content). Amino acid composition can also be calculated as the percent of the sum of all amino acids. Mammal whole body proteins contain about 2.1 g/16 g N of methionine and 1.4 g/16 g N of cysteine [87]. Consistently, mean sulfur amino acid composition of body proteins from rat, pig, and cattle was reported to be 3.4 g/100 g proteins [88]. Mammal skeletal muscles contain about 2.5 and 1.4 g/16 g N of methionine and cystine, respectively [87]. Similar values have been reported in beef meat: 2.6 and 1.1 g/100 g proteins of methionine and cysteine, respectively [17], and in pig carcass: 2.1 and 0.9 g/ 100 g proteins [89]. For comparison, organ proteins contain 1.6 g/100 g proteins of methionine and 1.2 g/100 g proteins of cysteine at least in pigs [89]. Therefore, methionine content is about twice the cysteine content in muscle proteins and less than 1.5-fold in organ proteins, at least in pig. Muscle containing about 20% of proteins, muscle concentrations of methionine, and cysteine contained in proteins are 4.8 and 2.3 mg/g of muscle i.e., 32 and 19 μmol/g, respectively. Of course, the turnover of sulfur amino acids contained in proteins follows muscle protein renewal, with a fractional rate of protein synthesis of 0.5
2%/day, a positive protein balance in the postprandial period, and a negative protein balance in the postabsorptive period [82,90,91].

Free Sulfur Amino Acids Skeletal Muscle Concentrations of Free Sulfur Amino Acids As all amino acids, free sulfur amino acid concentrations are very much lower than their tissue concentrations as protein constituents (Fig. 20.5). After an overnight fast, skeletal muscle free methionine concentration is about 40
50 nmol/g or nmol/mL of intracellular fluid [92
95] in human as well as in rat [96], i.e., more than 1000-fold lower than muscle concentration of methionine contained in proteins. The quantification of free cysteine is complicated by its unstability in course of the conventional method of amino acid measurement and its presence under various forms: free cysteine per se, free cystine, free small heterodisulfides, and cysteine linked to cysteine residues of proteins. Free total cysteine totalizes all these forms and the sum of free cysteine, cystine and small heterodisulfides is usually named free cyst(e)ine. To quantify free total cysteine with the colorimetric method, disulfides are reduced prior to the precipitation of proteins, whereas free cyst(e) ine is quantified when the reduction is performed after protein precipitation [97,98]. However, this method is usually not used to determine free cysteine concentrations of muscle due to a lack of sensitivity and no reference values are available. However; using HLPC methods, free cyst(e)ine was reported to be 33 nmol/g of muscle for healthy volunteers [99] and 60
70 nmol/g (95% confidence interval) in a reference group of metabolically healthy patients [100].

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FIGURE 20.6 Metabolism of sulfur amino acids in skeletal muscle. Methionine and cysteine along with all other amino acids are implicated in protein turnover. Cysteine is additionally used for the synthesis of glutathione and coenzyme A inside skeletal muscle. Cystine is reduced into cysteine by thioredoxin or glutathione.

In human skeletal muscle, cysteine would not be synthesized from methionine assuming the lack of transsulfuration as in rodents (Fig. 20.6). Indeed, enzyme activities of CBS and CSE are undetectable in rat muscle [101] and corresponding mRNAs are undetectable in rat and mouse muscle, as well [54,102]. Consequently, plasma supply of not only methionine but also cysteine is required to sustain muscle protein metabolism, glutathione, and coenzyme A synthesis. Plasma Concentrations of Sulfur Amino Acids Human plasma free methionine is 20
50 μmol/L after an overnight fast [95,103
105] and the gradient between skeletal muscle and plasma (i.e., muscle concentration/plasma concentration ratio) is 1.6 [95]. Plasma free methionine is about doubled 1 hour after a high protein mixed breakfast meal [105] and can transitorily increase up to 33-fold (from 30 μmol/L to 1 mmol/L) after an experimental oral load of methionine (0.1 g/kg) [104] corresponding to fivefold the population-safe intakes of total sulfur amino acids (21
27 mg/kg/day [106,107]). Plasma free cysteine, cyst(e)ine, and total cysteine are about 10, 120
200, and 350 μmol/L after an overnight fast, respectively [103,108,109]. Plasma concentration of total cysteine is maintained in a much narrow range than methionine, mostly due to the highly regulated and effective liver cysteine dioxygenase avoiding any accumulation of cysteine (see above). For example, rat plasma concentration of total free cysteine was about 75% higher 6 hours after the ingestion of a diet containing 20-fold of cysteine than the 10% casein control diet [110]. To our knowledge, gradients of concentrations between muscle and plasma for the three free forms of cysteine have never been assessed within the same study. Nevertheless, the gradient of concentration of free cyst(e)ine estimated from the above data would be about 0.2; however, this gradient is expected to be higher for cysteine than cystine because cystine is the major form in plasma, whereas cysteine predominates in muscle. Transmembrane Transporters Skeletal muscle has several amino acid transporter systems (1) sodium-dependent amino acid symporters cotransporting sodium down the electrochemical gradient along

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with amino acid against the concentration gradient; (2) uniporters allowing passive transport of amino acids according to the concentration gradients; and (3) antiporters exchanging actively two amino acids [111]. Amino acid transporters share specificities for several amino acids. Symporters are “loaders” because they accumulate amino acids into the cytosol. Antiporters are “harmonizers” because they can bring in indispensable amino acids using abundant intracellular nonindispensable amino acids leading to a harmonized mixture of all amino acids within cells. Moreover, some amino acid transporters are highly regulated depending on the metabolic state and amino acid availability. Methionine is uploaded in myocells with the sodium-coupled neutral amino acid transporter 2 (SNAT2, SLC38A2 gene) (Fig. 20.7) whose translocation to cell membrane is regulated by insulin and mTORC1 (mammalian target of rapamycin complex 1) [111,112]. Moreover, uptake or release of methionine by muscle occurs through 4F2hc-LAT1/LAT2 antiporters (SLC3A2-SLC7A5/SLC7A6 genes) that exchange large neutral amino acids [113]. Cysteine can be loaded in muscle by SNAT2 and taken up or released by the alanine
serine
cysteine (ASC) transporters (ASCT1 and ASCT2, SLC1A4 and SLC1A5 genes, respectively) (Fig. 20.7) [111,113]. SLC7A6 (LAT2), SLC1A4 (ASCT1), and SLC38A2 (SNAT2) mRNA expressions are the highest in skeletal muscle amongst numerous tissues tested [114]. Cystine that is more abundant than cysteine in plasma is exchanged for intracellular glutamate by 4F2hc-xCT (SLC3A2-SLC7A11 genes) antiporter (Fig. 20.7) [113]. Uptaken cystine is immediately reduced into cysteine inside myofibrils because sarcoplasm is a more

FIGURE 20.7 Simplified view of the movements of sulfur amino acids and metabolically related compounds between plasma and skeletal muscle. The uptake and the release of sulfur amino acids are carried out through various amino acid transporters. Glutathione and potentially oligopeptides allow the intake of sulfur amino acids, indirectly. Taurine, creatine and carnitine are taken up via specific transporters. Skeletal muscle may release glutathione via unknown mechanism. AA, amino acid; Cys, cysteine; Glu, glutamate; Gly, glycine; γ-Glu-cys, γ-glutamyl-cysteine; Met, methionine; SLCxAy, gene encoding solute carrier family x member y; SLC1A4, alanine-serine-cysteine transporter 1 (ASCT1); SLC6A6, taurine transporter (TauT); SLC6A8, creatine transporter; SLC7A6, neutral amino acid antiporter (LAT2); SLC7A11, cystine-glutamate exchanger (xCT); SLC15A2, oligopeptide transporter (PepT2); SLC22A5, organic cation transporter 2 (OCTN2); SLC38A2, neutral amino acid symporter (SNAT2).

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reducing environment than plasma [115]. In vitro and in vivo studies, unrelated to muscle, indicate that xCT is highly inducible through the nuclear factors Nrf2 and ATF4 involved in the response to oxidative stress and amino acid starvation [115]. 4F2hc-xCT plays important role not only as a supplier of cysteine for the synthesis of proteins and glutathione but also as a regulator of the extracellular cysteine/cystine couple in favor of cysteine because cysteine formed intracellularly after cystine uptake can be released through ASC transporters [115]. Plasma glutathione and cysteinylglycine are providers of cysteine for muscle. Human plasma concentration of GSH and GSSG (2.8 and 0.14 μmol/L [116]) are lower than that of cysteinylglycine (32 μmol/L [117]), whereas it is the opposite in rat plasma [118]. This species discrepancy resides in the presence of the γ-glutamyltranspeptidase only in human plasma. In overnight-fasted human, an intravenous infusion of glutathione leads to an equivalent increase in plasma cyst(e)ine flux, suggesting that glutathione degradation may amount 50% of plasma cyst(e)ine flux [119]. In rats, liver is recognized as the main producer and exporter of glutathione supplying cysteine for peripheral tissues, notably muscle [120]. Other circulating peptides can also be suppliers of sulfur amino acids for muscle, notably in the postprandial phase (Fig. 20.7). Indeed, peptides can be taken up by peptide transporters (PepT). PepT1 (SLC15A1 gene) is expressed in rat and pig muscles [121,122] and PepT2 (SLC15A2) in human muscle [123]. PepT2 actively transports cysteinylglycine at least in brain cells [124]. Uptaken peptides can be hydrolyzed by cytosolic oligopeptidases. Altogether, methionine uptake depends mainly on its plasma concentration and aminoacid/peptide transporters, whereas cysteine supply occurs through various mechanisms, most of them being independent from the plasma concentration of free cysteine.

Glutathione Homeostasis in Skeletal Muscle Glutathione synthesis occurs in skeletal muscle, where neither glutamate-cysteine ligase nor glutathione synthetase is expressed [125], and glutathione as such is not taken up by muscle from plasma (Figs. 20.6 and 20.7). Muscle synthesis of glutathione is expected to depend on glutamate-cysteine ligase activity and the muscle concentration of cysteine, the two key factors mentioned above. Muscle concentration of free GSH and free total glutathione are about 1.2 and 1.4 μmol/g in young healthy volunteers, respectively [99,126]. In another study muscle concentration of free total glutathione was 2.3 μmol/g [127]. The fractional synthesis rate of muscle glutathione measured in two studies with labeled glycine was 40 and 268%/day [99,127], meaning that in muscle glutathione turns over largely more rapidly than proteins (about 0.5
2%/day, [90,91]). The quantity of cysteine contained in glutathione is 20
40-fold the free cyst(e)ine content. Contrasting with proteins that can liberate cysteine intracellularly, the liberation of cysteine from glutathione occurs only extracellularly in unstressed cells, as described above. Various transporters may be involved in the transmembrane efflux of GSH and GSSG by myocells (Fig. 20.7). However, the expression of such transporters in muscle have been scarcely studied, and MRP1 does not seem to be expressed in muscle at least in rat [128]. Glutathione and GSH-linked enzymes playing various antioxidant roles, the impairment of muscle glutathione status associated with aging [129] could alter protein turnover in muscle contributing to sarcopenia [130].

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Skeletal Muscle Concentration and Plasma Supply of Taurine Taurine is a catabolite of cysteine, whose skeletal muscle concentration ranges from 9 to 19 μmol/g [94,95]; thus, it is almost as abundant as cysteine contained in proteins (Fig. 20.5). In contrast with glutathione, taurine is not synthesized in muscle due to the lack of cysteine dioxygenase, the first enzyme of the catabolic pathway of cysteine leading to taurine [54]. Thus, muscle content of taurine directly depends on its uptake carried out by the chloride sodium dependent taurine transporter (TauT, SLC6A6 gene) (Fig. 20.7) [131]. Human plasma concentration of taurine is about 50 μmol/L [95] and plasma taurine originates not only from its synthesis in the liver but also from the diet, notably meat [132]. Plasma concentration of taurine increases up to 1 mmol/L in young volunteers taking two oral experimental loads of 1.66 g of taurine with a meal, each [133]. Such a daily load represents a pharmacological intake compared to taurine intakes of 60
180 mg/day in omnivorous humans [134
136]. In spite of this, 7-day supplementation with twice 1.66 g of taurine failed to increase muscle concentration of taurine during 2 hours cycling [133]. That means muscle concentration of taurine is highly regulated in humans and unresponsive to large variations in plasma concentration. Nevertheless, transgenic mice with disrupted taurine transporter exhibited an almost complete depletion of taurine in muscle and a reduction of exercise capacity by .80% compared to wild-type controls [137]. That clearly supports the role of physiological level of taurine in muscle physiology, at least in rodents. In muscle, “main roles of taurine are to facilitate calcium-dependent excitation
contraction processes, contribute to the regulation of cellular volume, and aid in antioxidant defense from stress responses” [61]. Moreover, “taurine may control muscle metabolism and gene expression, through yet unclear mechanisms” [62]. In humans, beneficial effect of taurine on muscle may occur through targets outside the muscle [61]. Altogether, the usefulness of taurine supplementation for muscle functioning in human is still discussed, and taurine supplementation might be useful only when muscle content of taurine is dramatically decreased [61,62].

Skeletal Muscle Concentrations, Plasma Supply, and Roles of Other Compounds Metabolically Related to Sulfur Amino Acids Coenzyme A, carnitine, and creatine, all metabolically related to sulfur amino acids, contribute to the production of energy that is essential for skeletal muscle metabolism and contraction (Fig. 20.8). Muscle contraction requires both the production of ATP linked to energy metabolism and the effective regeneration of ATP. Coenzyme A and carnitine play key roles is ATP production, whereas creatine is involved in ATP regeneration. Coenzyme A and Carnitine Human skeletal muscle concentration of coenzyme A ranging from 30 to 70 nmol/g is similar to muscle concentrations of free sulfur amino acids (Fig. 20.5). Coenzyme A synthesis occurs in muscle, and cellular content is well regulated (Fig. 20.6) [79]. At rest, muscle concentration of free carnitine is between those of glutathione and taurine i.e., 3
5 μmol/g (Fig. 20.5) [138,139]. Acetylcarnitine (0.6 μmol/g) and acylcarnitine

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FIGURE 20.8 Roles of coenzyme A, carnitine, and creatine in the provision of energy for metabolic and contractile activities of the skeletal muscle. Sulfur amino acid related compounds are implicated in key steps of ATP production. Coenzyme A is necessary for the conversion of pyruvate and long chain fatty acids into acetyl-CoA and long chain acyl-CoAs, respectively. Long-chain acyl-CoAs use the carnitine shuttle to enter into mitochondria matrix, where they are metabolized through Lynen helix, the cyclic process of β-oxidation generating acetyl-CoA, NADH 1 H1 and FADH2. AcetylCoA is metabolized through Krebs cycle generating NADH 1 H1 and FADH2, too. NADH 1 H1 and FADH2 are consumed in the respiratory chain process fueling the ATP formation at the inner mitochondrial membrane (oxidative phosphorylation). Creatine plays a key role in the regeneration of ATP that is initially produced in sarcoplasm and mitochondria. ATP is highly-rate used in myofibril metabolism notably protein synthesis and proteolysis, and in sarcomere contractile activity. Reaction stoichiometry is not reported to alleviate the figure. ADP, adenosine diphosphate; ATP, adenosine triphosphate; CoA, coenzyme A; FAD/FADH2, oxidized/reduced flavin adenine dinucleotide; GTP, guanosine triphosphate; NAD1/NADH 1 H1, oxidized/reduced nicotinamide adenine dinucleotide.

(0.2 μmol/g) are less abundant than carnitine at rest; however, acetylcarnitine increases under exercise at the expense of free carnitine [139]. Human muscle largely contributes to the initial step of carnitine synthesis but the final step does not occur in muscle [140]. Thus, carnitine is taken up, predominantly through the sodium-dependent organic cation transporter-2 (OCTN2, SLC22A5 gene), and against a large gradient of concentration

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(Fig. 20.7) [141]. Indeed, plasma concentration is about 25
50 μmol/L [142]. Plasma carnitine comes from food, mainly red meat, and the endogenous synthesis described above [10]. Food accounts for 75% of daily carnitine requirement in meat consumers and the absorption of carnitine from food (50%
85%) is much higher than from carnitine supplements (14%
18%) [10]. Muscle concentration of carnitine was independent from the daily carnitine intake that varied from 0.18 to 319 mg/day (mean 38 mg/day) accordingly to the large variation of meat consumption [143]. Coenzyme A and carnitine are involved in the formation of reduced NADH 1 H1 and FADH2 that are consumed in the respiratory chain process fueling the ATP formation at the inner mitochondrial membrane (oxidative phosphorylation) (Fig. 20.8). Coenzyme A is an obligate cofactor of numerous biochemical reactions involved in oxidative metabolism and biosynthetic reactions (intermediary metabolism). Notably, coenzyme A is needed in the reaction that links glycolysis, ending in pyruvate, with the Krebs cycle that requires acetyl-coenzyme A [12]. The cooperation of coenzyme A and carnitine permit the transport of long-chain fatty acids into mitochondrial matrix, where β-oxidation occurs (Fig. 20.8). Muscle concentration of carnitine determines the fuel selection in favor of carbohydrate use when the carnitine content is low, and privileges fatty acids use when the carnitine content is high [144]. Long-term (24 weeks) carnitine supplementations are needed to increase muscle concentration of carnitine, modify fuel selection and improve exercise performance in healthy volunteers [139]. Creatine Skeletal muscle concentration of total creatine (creatine and phosphocreatine) is about 30 μmol/g [145] i.e. as abundant as protein-bound methionine and about 1.5-fold more abundant than protein-bound cysteine (Fig. 20.5). Muscle creatine is 5
10 μmol/g and young men of 70 kg contain 120 g of total creatine [11]. Muscle does not synthetize creatine but contains up to 95% of whole body content [83]. Muscle takes up creatine via the chloride sodium-dependent creatine transporter (SLC6A8 gene) against a huge concentration gradient, plasma concentration of creatine being about 50
100 μmol/L (Fig. 20.7) [146]. Plasma concentration of creatine increases to almost 1 mmol/L after an oral experimental load (4.4 g) in healthy volunteers [147]. That load is 5.5-fold the estimate dietary creatine of 0.8 g/day [146]. Creatine is converted nonenzymatically but irreversibly to creatinine that is lost in the urine. The formation of creatinine is correlated with muscle mass and the urinary excretion is a clinical marker of renal function [145]. Plasma concentration of creatinine regulates the formation of creatine that necessitates the cooperation of kidney and liver [146]. Dietary creatine and endogenous synthesis are about equal parts in omnivorous individuals [11]. Creatine/phosphocreatine couple plays a key role in the rephosphorylation of sarcoplasmic ADP into ATP that is required to sustain muscle metabolism and contraction (Fig. 20.8). Cytosolic creatine crosses the outer membrane of mitochondria, where it is converted to phosphocreatine by mitochondrial creatine kinase using ATP produced by the oxidative phosphorylation within mitochondrial matrix. Then, phosphocreatine is transported into the sarcoplasm, where it allows cytosolic creatine kinase to regenerate ATP from ADP liberated by the contractile activity and metabolic activity [148].

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Creatine supplementation is commonly used as an ergogenic agent by athletes performing exercise of high intensity and short duration [146]. However, potential mechanisms contributing to the anabolic effect of creatine are currently controversial and whether, or not, creatine supplementation could improve muscle mass and function in the elderly is still unknown [149]. Inorganic Sulfurs The degradation of cysteine up to inorganic sulfurs is not expected to occur within human myocells, due to the lack of expression of the key enzymes of cysteine degradation pathways [54]. We are aware of no specific role of SO422 in skeletal muscle. Vessels irrigating muscle may be sensitive to the vasorelaxant effect of H2S, which can be produced by vascular smooth muscle cells [150]. Evidences supporting a vascular effect of H2S in muscle are lacking. That might warrant further studies since (1) aging may be associated with a cysteine deficiency [151] and (2) age-associated alteration of the endothelial function could contribute to sarcopenia [152].

SULFUR AMINO ACIDS REQUIREMENTS AND SUPPLEMENTATIONS Whole Body Requirement and Usual Intakes of Sulfur Amino Acids Whole Body Requirement in Sulfur Amino Acids From a global nutritional perspective, only methionine is an indispensable amino acid i.e., it must absolutely be present in the diet. Cysteine is a dispensable amino acid when 100% of needed cysteine can be synthetized from methionine. The total sulfur amino acid requirement is defined as “the methione intake, in the absence of dietary cysteine, which satisfies all of the physiological requirements for both methionine and cysteine” [153]. The minimum obligatory requirement for methionine is “the intake of methionine that cannot be replaced by cysteine and that will not be reduced by addition of any methyl donor, cofactor or any other metabolite” [153]. A recent study performed on piglets suggests that dietary methyl donors (folate, choline, and betaine) should be considered in the determination of methionine requirement [154]. The ability of cysteine to reduce the total sulfur amino acids requirement, as defined above, is known as the sparing effect of cysteine on the methionine requirement [153]. When all of the requirements for cysteine exceed the endogenous capacity for the cysteine synthesis, a dietary supply of cysteine is necessary. The strict indispensability of cysteine is clear for fetus and preterm infants due to the absence or poorly active CSE, a key enzyme in the transsulfuration pathway [155]. Based on a recent review, the total sulfur amino acid requirement ranges from 12 to 15 mg of methionine/kg/day according to the study, the sparing effect of cysteine is at least 60%, but the minimum methionine requirement is not consensual [7]. Usual Sulfur Amino Acid Intakes Basically, food provides proteins containing methionine and cysteine together, and the meat also provides sulfur amino acid related compounds. Grains and animal proteins are

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rich (3.5%
5%), whereas legume proteins are rather poor (2%
3.5%) in total sulfur amino acids [17]. Protein content of methionine varies from 0.86% in almond to 2.97% in egg and protein content of cysteine from 0.35% in casein to 1.99% in wheat. Cysteine/methionine ratio varies from 0.4 to 1.4 except for α-lactalbumin that contains 6.9% and 1.0% of cysteine and methionine, respectively [17]. Average French intake of (the sole) methionine is 2.1 g/ day [156]. Average American intakes of methionine and cysteine are 2.3 and 1.3 g/day for men and 1.6 and 0.9 g/day for women [19]. Taking into account reaction stoichiometry of transsulfuration and molecular weights of sulfur amino acids, 1 g of cysteine can spare 1.23 g of methionine. Therefore, American intake of total sulfur amino acids, expressed in equivalent methionine, is 3.9 and 2.7 g/day for men and women, respectively. These intakes are largely above the population-safe requirement of total sulfur amino acids set at 1.5 g/day (upper limit of the 95% CI for mean requirement) for a 70-kg human [106,156]. Despite the fact that the average intake of sulfur amino acids appears sufficient, deficiency could occur when diets are based exclusively on plant proteins. Assuming an extreme diet based on proteins poorly digestible (70%) containing no more than 1.5% of methionine equivalent, the minimum quantity of protein necessary to cover the sulfur amino acid requirement would be 110 g/day, i.e., almost twice the RDA of good-quality protein for a 70-kg human. In order to equilibrate dietary pattern of amino acids, especially when protein intake is low, consumers of plant based diet are encouraged to associate legumes, poor in sulfur amino acids and rich in lysine, with grains, rich in sulfur amino acids and poor in lysine.

Skeletal Muscle Requirement in Sulfur Amino Acids and Metabolically Related Compounds Qualitative Requirements and Interorgan Fluxes The metabolic aspects described in the second part of the chapter underlies that skeletal muscle mass and function rely on the ability of arterial plasma to provide appropriate amounts of all the following indispensable (i.e., not synthetized in the muscle) compounds. (1) Methionine to sustain protein metabolism (Fig. 20.6). Basically, methionine is not engaged in any other metabolic pathways within the muscle, except the potential transamination pathway in case of huge methionine supply [157]. (2) Cysteine, and to a larger extent cystine, as well as peptides containing cysteine, because cysteine cannot be produced from methionine within muscle (Fig. 20.6). Cysteine is required for protein metabolism, and the synthesis of glutathione and coenzyme A in muscle. (3) Taurine is indispensable to support muscle physiology and (4) carnitine plus creatine are essential to sustain ATP required for metabolic and contractile activities (Figs. 20.7 and 20.8). The pheripheral availability of these compounds depends not only on nutritional intakes but also on the specific rates at which the organs other than muscle use and/or contribute to their productions. The splanchnic area (chiefly digestive tract and liver), which contributes to a significant part of the whole body amino acid/protein metabolism [158], is a key determinant of the peripheral availability of amino acids [159]. Moreover, it is well known that part of the dietary amino acids/ peptides liberated from the digestive proteases inside the intestinal lumen is directly used for intestine metabolism without reaching portal blood. Indeed, about 60% of the dietary

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cysteine is sequestrated, at first pass, by the intestine in pigs fed with elemental enteral nutrition [160]. Due to its anatomical position, the liver takes up, also at first pass, portal food-derived nutrients as well as intestine metabolites released into the portal vein. Liver as well as kidney play a central role in interorgan movements of amino acids and related compounds [161]. In the postprandial period, the dominant flux is from the intestine to the other tissues notably muscle, which slightly gains proteins, whereas the dominant flux originates from muscle in postabsorptive period and hypercatabolic states, leading to slight and acute loss of muscle proteins, respectively [158,161,162]. Specific Situations and Potential Supplementations CRITICALLY ILL PATIENTS

We are aware of no specific conditions altering free methionine homeostasis in skeletal muscle. In contrast, decreased muscle concentration of glutathione have been largely reported in surgical patients [125,163
165] as well as in critically ill patients with sepsis [100,166,167]. However, neither concentration nor fractional synthesis rate of muscle glutathione was altered under mild experimental endotoxemia [99]. As well, short-term (3 days) fasting does not affect muscle concentration of glutathione nor plasma and muscle concentrations of cyst(e)ine [126]. Moreover, decreased muscle glutathione was rather attributed to a lower activity of the glutathione synthetase [125] or a shortage of glutamate since glutamine infusion attenuates glutathione decrease in trauma patients [167]. These data are contrasting with the general belief that cysteine and glutamate-cysteine ligases are regulators of glutathione synthesis. Nevertheless, altered glutathione homeostasis leading to defective redox regulation in the muscle may contribute to muscle impairment [168]. Using a rat model of sepsis that reproduces the anabolic acute phase response in the liver and the progressive loss of muscle mass [169], we observed major alterations in cysteine and glutathione metabolism in favor of an increased requirement of cysteine in catabolic states, already reviewed [158,170,171]. Then, we showed that supplementation with a mixture of amino acids including cysteine alleviates muscle wasting in a rat model of septic patients [172]. ELDERLY

In 2005, Dro¨ge questioned whether aging could be a “cysteine deficiency syndrome?”, on the basis of studies using the pharmacological precursor of cysteine, i.e., N-acetylcysteine or whey protein isolates naturally rich in cysteine [151]. Supporting this idea, we observed that the proportion of methionine entering the transsulfuration pathway is higher in elderly than young subjects [173]. Of note, an increased requirement of methionine in persons over 50 had already been suggested in 1957 [174]. A recent review underlines that the increased risk of mortality associated with low protein intake in elderly may be linked to an uncovered need of cysteine for glutathione synthesis [175]. In our hands, doubling the dietary intake of cysteine in old rats during 14 weeks increased splanchnic cysteine and glutathione pools and alleviated aging-associated anorexia [176]. Similarly, long-term supplementation with cystathionine, the endogenous precursor of cysteine, increased glutathione pools in the splanchnic area as well as in muscle [177].

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PARACETAMOL TREATMENT

Chronic treatment with paracetamol is another condition where cysteine/glutathione homeostasis is challenged. Indeed, the detoxification of paracetamol requires SO422 and glutathione, both originated from cysteine. Paracetamol-induced decrease of the liver content of glutathione is the initial event leading to hepatotoxicity and N-acetlycysteine is the antidote aimed to rescue glutathione [6]. Requirement of sulfur amino acids may be increased by a paracetamol treatment, considered to be representative of the maximum therapeutic human dose of 4 g/day, since the decreased growth rate of paracetamoltreated is counteracted by methionine or cysteine [178]. According to the idea that sulfurdependent detoxification of paracetamol could occur at the expense of the use of sulfur amino acid for muscle protein synthesis, we recently showed that muscle wasting occurs in adult rats and that sarcopenia is worsened in old rats under nontoxic paracetamol treatment [118,179]. Our results suggest that a dietary supplementation with sulfur amino acids may be helpful to preserve muscle under chronic treatment with paracetamol. Cystathionine is protective against paracetamol-induced hepatotoxicity in rats [180] and could be a good candidate for alleviating paracetamol-induced negative effects on muscle. Safety and Practical Considerations When transsulfuration of methionine is not a limiting factor, sulfur amino acid supplementation can theoretically be administered as methionine alone. However, methionine excess can induce hyperhomocysteinemia, a feature known to be associated with cardiovascular diseases, even if the causal relationship has not been established [20]. Plasma concentration of homocysteine has just been used to determine the no-observed-adverse-effect level (NOAEL) and the lowest-observed-adverse-effect level (LOAEL) of methionine supplement in healthy older adults [181]. NOAEL and LOAEL of supplemented methionine intake were set at 46.3 and 91 mg/kg/day, i.e., three- and sixfold the requirement of total sulfur amino acids (without taking into account of the methionine intake from dietary proteins). We are aware of no NOAEL neither LOAEL of supplemented cysteine. Free cysteine supplementation is possible when animals are fed with dry pellets [172,176]. However, due to the unstability of cysteine in solution, most of the clinical studies used the drug N-acetylcysteine or whey protein isolate as cysteine providers. A preclinical study warns against the fact that the beneficial effect of N-acetylcysteine on normalized measures of muscle function was associated with reduced muscle weight in a rat model of myopathy [182]. To summarize, methionine and cysteine are the two sulfur amino acids supplied by dietary proteins. Apart from the use of sulfur amino acids in protein turnover, these amino acids are metabolically connected with important players in skeletal muscle metabolism and function. Indeed, basal biochemical pathways teach us that methionine gives methyl for the formation of carnitine and creatine among many others, sulfur to cysteine, whereas the carbon skeletal of methionine is orientated toward the Krebs cycle. Cysteine is the precursor of glutathione, the main intracellular antioxidant, and of coenzyme A. Cysteine degradation produces taurine (implicated in muscle physiology) and pyruvate (an energy intermediate), H2S (a physiological mediator), and the ultimate formation of SO422 also provides energy for ATP production. Carnitine, creatine, and coenzyme A play essential roles in the production of ATP from energy substrates and the regeneration of ATP that

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support metabolic and contractile activities of the muscle. Both methionine and cysteine are strictly required by skeletal muscle. Muscle requirements of methionine and cysteine are satisfied according to the availability of not only methionine and cysteine but also cystine, glutathione, and peptides in the arterial plasma, and the presence of specialized transporters. The respective contribution of each precursor to cysteine supply is still unknown. It might differ between human and rats, notably due to species difference regarding γ-glutamyltranspeptidase activity in plasma. Various clinical situations can alter the muscle homeostasis of sulfur amino acids or metabolically related compounds. These alterations may be consecutive to glutathione depletion in the liver. Conceptually, supplementations with sulfur amino acids or specific precursors might be of help to sustain metabolic and contractile activities of muscle. However, there are still numerous gaps to be filled, notably concerning the metabolic and functional consequences of decreased muscle glutathione, and the importance of the interorgan exchanges of sulfur amino acids and metabolically related compounds, in humans.

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Regulation of Skeletal Muscle Metabolism by Saturated and Monounsaturated Fatty Acids 1

Cedric Moro1,2 and Frederic Capel3

INSERM, UMR1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse, France 2 University of Toulouse, UMR1048, Paul Sabatier University, Toulouse, France 3 Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, Clermont-Ferrand, France

INTRODUCTION Dietary Fat and Health Dietary fat quality is a major determinant of several physiological, biochemical, and molecular processes in the body and tissues. Lipids are essential nutrients and a very important source of energy. Lipids are mainly stored in fat cells and within lipid droplets (LDs) in oxidative and steroidogenic tissues as a fuel source, but significant amounts are also found in cell membranes where their structural role is crucial for membrane’s protein functions and the control of cellular functions. Differential effects between the different types of fatty acids (FAs) on inflammatory, metabolic processes during a basal state or in response to physical exercise or a chronic disease are now clearly demonstrated. The most recent dietary guidelines advice that lipids should represent 35% of daily energy intake in order to prevent the deleterious effect of high glycemic index carbohydrates and the deficiency in essential FAs [1]. Furthermore, advices should be focused on the improvement of the quality of fat, more than the reduction of total fat intake [2]. Previous classical guidelines recommended to reduce fat intake, but the evidence is that the prevalence of obesity could rise dramatically despite a fall in total fat intake [3]. FAs are classified according to the presence or not of double bonds in their carbon chain. Dietary fat sources provide a mixture of saturated FA (SFA, no double bond), monounsaturated FA (MUFA, one double bond), and polyunsaturated FA (PUFA, at least 2 double bonds). A complete overview of

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00021-X

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the health benefits/risks balance of the intake of the different types of FA is out of the scope of the present chapter which will be dedicated to SFA and MUFA. The effects of PUFA on muscle metabolism and function or metabolic syndrome are described in review articles [4
6]. Although some controversies exist, most of the institutional dietary guidelines claim that the consumption of SFA should be limited (less than 10% of calories) at the expense of MUFA and PUFA (consumed in replacement of SFA) as a nutritional strategy for the prevention of chronic diseases. The role of dietary SFA and MUFA in cardiometabolic risk remains controversial in the scientific community [7,8]. Furthermore, the translation of scientific results to sustainable modification of consumer choices and understanding of food composition is also challenging [8]. The major SFA in our diet are myristic (or myristate, C14:0), palmitic (or palmitate, C16:0), and stearic (or stearate, C18:0) acids. Milk and coconut oils contain shorter SFA, since we can find SFA from butyric (C4:0) to stearic acid. Animal fat (beef, lamb, lard, skin from poultry), milk fat (cream, butter, cheese, etc.), and some vegetable fats (palm, coconut oils) are the major sources of dietary SFA. Many industrially made products, snacks, and fried food products also usually contain high amounts of SFA. Vegetable oils are frequently used by the industry because of their lower cost and the high stability of the products during cooking. The main dietary MUFA is oleic acid or oleate (C18:1 n-9). MUFAs are nonessential FA since they can be produced endogenously. Oleic acid is abundant in vegetal fat sources, such as olive oil, rapeseed oil, sunflower oil, peanuts, but also in fat of animal origin. Olive oil is a major component of the Mediterranean diet which is frequently described as an ideal dietary model for the general population. Oleic acid intakes should contribute to 15%
20% of daily energy requirements for the French population as proposed by the ANSES [1]. It was reported that only 7% of the population follows this recommendation. The mean intake of oleic acid represents less than 11% of the daily energy requirement. Palmitoleic acid or palmitoleate (C16:1 n-7) is a MUFA, mainly produced from de novo lipogenesis mediated by stearoyl-CoA desaturase 1 (SCD1) the rate-limiting enzyme catalyzing the synthesis of MUFA. High concentrations are found in adipose tissue, liver and skeletal muscle where it represents 3%
7% of FA in triacylglycerols (TAG). Palmitoleic acid is now considered as a lipokine released from adipose tissue which could have systemic metabolic effects on other organs [9]. It was described that fat sources containing high amount of MUFA could be helpful in the prevention of metabolic complications associated with the development of obesity. Postprandial lipid response, characterized by the increase in TAG and lipoprotein levels following a high fat test meal, could be improved if butter was replaced by olive oil. Furthermore, olive oil consumption could normalize the increase in plasma incretin glucagon-like peptide 1 which is affected in type 2 diabetic subjects [10,11]. GLP-1 is a peptide hormone secreted in response to nutrient ingestion by the intestine and inhibits gastric emptying, stimulates insulin secretion and satiety [12]. GLP-1 production occurs especially in response to high fat and carbohydrate intake. Such a response could prevent the deleterious consequences of the high glucose and plasma lipid levels on key metabolic tissues such as liver, pancreas, adipose tissue, and skeletal muscles.

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Fatty Acid Composition and Skeletal Muscle Function Muscle dysfunction is frequently observed in patients with chronic diseases and obesity. The loss in muscle mass and function hinders mobility, exercise capacity, life quality, and expectancy. The potential causes are multiple, involving inflammation, mitochondrial and microvascular dysfunctions, apoptosis and defects in cell’s energy metabolism and response to anabolic hormonal stimuli (notably to insulin). A transition to unhealthy diets with high amounts of calories and SFA has occurred during the last decades. It is commonly considered as a major public health problem because of the multiple physiological and metabolic consequences in insulin sensitive tissues. It has been shown that the FA composition of skeletal muscle phospholipids could be related to insulin sensitivity [13,14]. However, it remains unclear if the reduction in the degree of unsaturation of FA could be a cause or a consequence of insulin resistance. Skeletal muscle is the main insulin sensitive tissue, playing a crucial role in postprandial glucose uptake. Therefore, it is important to preserve muscle phospholipids FA composition for an optimized membrane fluidity and ligand
receptor interactions. The proportion of oleic acid in muscle phospholipids is strongly correlated to the proportion of this FA in muscle TAG, serum phospholipids, and cholesterol esters [15]. Thus, dietary intervention aiming at improving the FA composition of the diet could also be efficient to induce the same modifications at the muscle level. It is also important to mention that the metabolic protection of MUFA could be lost if total fat consumption is excessive and above the recommendation [16].

MECHANISMS OF ACTION OF SFA AND MUFA IN SKELETAL MUSCLE SFA-Mediated Lipotoxicity and Inhibition of Insulin Action The mechanisms of action of SFA and MUFA have been largely studied in skeletal muscle which is a major insulin-responsive tissue, a major site of lipid oxidation and glucose storage, and a major reserve of amino acids through protein metabolism. In vitro studies using cell-based model systems have been helpful to study the mechanism of action of SFA and MUFA. A number of skeletal muscle cell lines such as L6 (from rat) and C2C12 (from mouse) have been used for this purpose. Skeletal myoblasts are seeded into culture plates till 80% confluence in a classical DMEM medium containing 10% fetal bovine sera. This medium is then switched to a differentiation media containing 2% of horse serum. Differentiation into myotubes is typically obtained within 5
6 days. Treatment of myotubes with BSA-bound FA is performed on fully differentiated cells. Primary muscle cells from rodent models and human biopsy samples have also been used. The accumulation of TAG into LDs rich in SFA is now recognized as a key element in the development of insulin resistance in several tissues (Fig. 21.1). Insulin sensitivity has been shown to be inversely related to intramyocellular triacylglycerols (IMTGs) content in sedentary populations [17
19]. Several molecular mechanisms have been suggested to explain this relationship. First, IMTG is associated with emergence of toxic lipids such as

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FIGURE 21.1 SFA-mediated insulin resistance in skeletal muscle Insulin binds to its receptor IR which leads to IRS-1/2 phosphorylation. Akt is activated downstream leading to anabolic response and glucose uptake through GLUT4 translocation to the plasma membrane. A high FA flux to the muscle can be partly buffered into TAG pools stored within lipid droplets, but also give rise to toxic lipid intermediates such as ceramides and DAG which further activate PP2A and PKC, respectively, inhibiting IRS1/2 and Akt. Inflammatory pathways through LPS, SFA, and TNF-α can also activate the transmembrane receptors TLR and TNFR which lead to NFkB and JNK activation that can inhibit IRS1/2 and insulin signaling. IR, insulin receptor; FA, fatty acid; TAG, triacylglycerols; DAG, diacylglycerols; PP2A, protein phosphatase 2A; PKC, protein kinase C; LPS, lipopolysaccharide; SFA, saturated fatty acid; TNF-α, tumor necrosis factor-α; TLR, Toll-like receptor; TNFR, TNF receptor; JNK, c-Jun N-terminal kinase.

DAGs and ceramides which decrease the insulin-stimulated activity of insulin receptor substrate-1 (IRS-1)-associated phosphoinositide 3-kinase (PI3K) [20,21]. This in turn results in reduced glucose transport and glycogen synthesis [22]. Second, in sedentary populations, the IMTG pool is thought to be more prone to oxidative stress, because of reduced depletion/repletion cycles with physical inactivity, therefore generating lipid peroxidation products with the potential to inhibit insulin signaling [23]. An increased peroxidation of IMTG may reduce insulin sensitivity by increasing tumor necrosis factor α (TNF-α), which is known to increase the expression of suppressor of cytokine signaling (SOCS) proteins. The TNF-α/SOCS3 pathway inhibits tyrosine phosphorylation of IRS-1 and subsequently the activation of the downstream signaling cascade [24]. Third, skeletal muscle inflammatory pathways including NFkB and JNK are activated during lipid infusions, mediate intramuscular fat accumulation and promotes serine phosphorylation of IRS-1 and inhibition of insulin signaling [25]. Collectively, a high SFA pressure and LDs accumulation in skeletal muscle activate multiple signaling pathways [protein kinase C (PKC), NFkB, JNK] which further disrupt the insulin signaling pathway and inhibit insulin action (Fig. 21.1). These events lead to an alteration of muscle protein anabolism, a key event in the loss of muscle mass with aging [26] or obesity [27].

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De Novo Production of Diacylglycerols by SFA Several clinical studies demonstrated that total DAGs content is elevated in vastus lateralis of obese individuals without or with type 2 diabetes mellitus (T2DM) compared to lean controls [19,28]. In addition, longitudinal studies show a decrease of skeletal muscle DAG content and improvement of insulin sensitivity after several weeks of endurance training [29]. Lipid infusion studies have helped to gain further insight into the molecular mechanisms of DAG-induced insulin resistance. In this experimental setting, TAG/heparin infusion typically raises plasma FA levels in a supra-physiological range, between 1.5 and 2 mM and promotes a strong reduction of insulin-stimulated glucose disposal within 4 h [22]. This impairment in insulin signaling is associated with an accumulation of DAG in skeletal muscle (Fig. 21.1). Defects were mainly observed at the level of IRS1, which exhibits reduced tyrosine phosphorylation, thus leading to a down-regulation of PI3K activity and glucose uptake in response to insulin [30]. Causal relationships between DAG accumulation and insulin resistance have been also reported in culture of human primary myotubes [31]. DAG are well-known allosteric activators of the conventional members of PKC (α, βI, βII, γ) and novel PKC (δ, ε, η, θ), whereas the atypical PKC (ζ, λ) devoid of a C1 binding site are not DAG-responsive. Activation of PKC by DAG induces serine phosphorylation of IRS1 and inhibits insulin signaling [32]. A few studies have shown that PKCθ activation is strongly associated with skeletal muscle insulin resistance in humans and rodents [33
36]. The role of other PKC isoforms remains unclear. Itani et al. reported an increase of membrane PKCδ and β isoforms in biopsies of vastus lateralis during lipid infusion in healthy individuals [33]. PKCε has also been associated with insulin resistance in skeletal muscle [35,37] and liver [38].

De Novo Production of Ceramides by SFA Ceramides have also been described as potential lipid mediators of skeletal muscle insulin resistance. Inhibition of ceramide synthesis by administration of the pharmacological inhibitor myriocin protects against SFA-induced insulin resistance in rodents [39]. Some studies suggest that a positive association between total ceramide content and insulin resistance exists in human skeletal muscle. Adams et al. found a higher level of ceramides in skeletal muscle of obese insulin resistant patients [40], whereas Straczkowski et al. observed an increased ceramide content in response to lard oil infusion in healthy subjects [41]. Other cross-sectional studies reported an elevated level of muscle ceramides in obese and T2DM subjects [19] and in lean insulin-resistant versus matched insulin-sensitive subjects [41]. Those data were corroborated by interventional studies employing chronic and acute exercise training that showed an increase in insulin sensitivity which was paralleled by a decrease in skeletal muscle ceramide content [29,42]. Myotubes exposed to the SFA palmitic acid (0.4
0.5 mM) exhibit a 2
3-fold increase in several ceramide species [43
45]. This is due to de novo ceramide synthesis by the serine palmitoyl-CoA transferase-I (SPT-I) which transfer one serine molecule to one palmitoyl-CoA. Interestingly, palmitate-mediated ceramide synthesis is totally abrogated by the SPT-I inhibitor myriocin

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in vitro in C2C12 [44] and human primary myotubes [31,45]. The molecular mechanism of ceramide-induced insulin resistance has been unraveled (Fig. 21.1). Ceramides block the translocation of Akt to the plasma membrane and further activation by insulin. Two independent mechanisms have been proposed. On the one hand, ceramides activate PKCζ which phosphorylates Akt on an inhibitory Ser34 residue [46], and on the other, they activate a protein phosphatase 2 A which dephosphorylates Akt on its activating Ser473 residue [44]. These two mechanisms markedly impair Akt activation in response to insulin.

Salvage Effect of MUFA on SFA-Mediated Lipotoxicity The health benefits of MUFA have been extensively studied. In vitro studies based on muscle cell exposure to different combinations and concentrations of FA have suggested a major protective effect of MUFA against insulin resistance in skeletal muscle. Oleic acid is the main MUFA in neutral and phospholipid fractions of the body. Therefore, most studies have used oleic acid to look at the effect of MUFA. Treatment of L6 myotubes with 0.75 mM of oleic or palmitoleic acid could increase basal glucose uptake by inducing glucose transporter localization at the plasma membrane [47]. Beneficial roles of palmitoleic acid were found since an increased concentration significantly reduced hepatosteatosis and promoted glucose transport in skeletal muscle [9]. Several reports have clearly demonstrated that oleic acid could rescue palmitate-mediated Akt inhibition. Equimolar concentrations of oleic acid completely rescue the lipotoxic effect of palmitate when concomitantly incubated on C2C12 [44] or human primary myotubes [45]. A complete normalization of intracellular ceramide levels, insulin-mediated Akt activation and glycogen synthesis was achieved. In other studies, it was found that oleic acid (0.1
0.3 mM) could reduce the accumulation of DAG in C2C12 myotubes exposed to palmitate (0.5 mM), alleviating PKC activation and inflammation [48]. Myotubes exposed to SFA exhibit an increase of NFkB activity and IL-6 production which are blunted by a coincubation with oleic acid. Oleic acid allows a channeling of palmitate to less toxic lipid fractions and stimulate the expression of genes involved in FA β-oxidation [48,49]. Even when used at a 10-fold lower concentration than palmitate, oleic acid could normalize FA oxidation and increase insulin-induced Akt phosphorylation in C2C12 myotubes [49]. MUFA could also inhibit both the accumulation of SFA in DAG and ceramide and the accumulation of these lipotoxic metabolites [49]. A recent study using human myotubes has shown that ER stress was induced concomitantly to Akt inhibition by palmitate [50]. This effect was prevented by oleic acid, independently of any change in glucose uptake, or ROS production. In vivo studies have also demonstrated that the degree of FA unsaturation could modulate transcriptomic and metabolic adaptations of skeletal muscle. Some of these events are strongly related to the control of muscle protein anabolism [51] and metabolic flexibility and involve key modulations of mitochondrial activity. MUFA are able to increase Peroxisome proliferator-activated receptor gamma coactivator (PGC1)-β but not PGC1-α mRNA level in human skeletal muscle without any effect on transcript levels related to glucose transport (GLUT4) or mitochondrial oxidative phosphorylation (COX) [52]. PGC1-α

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and PGC1-β are highly expressed in tissues with high oxidative capacities and control the expression of mitochondrial genes to increase oxygen consumption capacity [53]. Furthermore, overexpression of this transcriptional coactivator could protect from obesity [54]. Although the effects of MUFA on adipose tissue are not in the scope of this chapter, it appears relevant to mention that the regulation of adipose tissue gene expression by MUFA is affected by T2DM, which could strongly contribute the accumulation of lipids within skeletal muscle and thus insulin resistance [55]. Some bioactive lipid mediators produced from SFA and unsaturated FA precursors also contribute to the control of muscle functions. As an example, oleoylethanolamide (OEA) is an endogenous N-acylethanolamine synthesized from membrane glycerophospholipids and derived from the MUFA, oleic acid. The identified biological targets of OEA are fat catabolism and the control of food intake [56]. Metabolic explorations have shown that OEA administration stimulated lipolysis in adipocytes and fat oxidation in soleus muscle strips isolated from mice by a peroxisome proliferator-activated receptors alpha (PPAR-α) dependent mechanism [57]. This mediator could then help to limit the accumulation of toxic lipid mediators. Increasing dietary intake of oleic acid increases OEA level in different tissues and could at least partially explain the beneficial effects of MUFA intake [58]. Furthermore, in one human study, the palmitate:oleate ratio decreased and the oleate:stearate ratio increased in serum and muscle phosphatidylcholine after the consumption of a high-oleic diet [59], allowing a reduction in the FA saturation degree in different tissues and cellular compartments.

Storage Into Lipid Droplets and Lipotoxicity In contrast to oleic acid, palmitic acid is poorly incorporated into TAG pools and induces accumulation of ceramides and apoptosis by activation of caspase-3 [60]. Interestingly however, both palmitic and oleic acids are lipotoxic in DAG O-acyltransferase 1 (DGAT1) null mouse embryonic fibroblasts, used as a model of impaired TAG synthesis. The lipotoxic effect of palmitate can be prevented in Chinese hamster ovary cells by overexpression of SCD1, suggesting a beneficial effect of unsaturated FA [60]. Similar results have been recently observed in rat L6 myotubes in which palmitate exposure increases de novo ceramide synthesis, caspase-3 activation, and apoptosis [61]. In a recent study by Hulver et al. [62], elevated activity of SCD1 was associated with disorders in lipid partitioning (defined as the ratio between TAG synthesis and fat oxidation) in muscle tissue obtained from morbidly obese subjects. The overexpression of SCD1 in muscle cells obtained from lean donors mimicked the obese cellular phenotype, suggesting a role for this enzyme in obesity. One previous report of the potential protective role of SCD1 against lipotoxicity has been confirmed in rat L6 myotubes [63]. Targeted inhibition of SCD1 by short interfering RNA in those cells increases the incorporation of exogenous FA into DAG and ceramides and ultimately impairs insulin-mediated glucose uptake. In the same line, we recently demonstrated that overexpression of the LD protein perilipin-5 (PLIN5) protects skeletal muscle cells against palmitate-mediated lipotoxicity by channeling more palmitate into IMTG [64].

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LIFESTYLE INTERVENTIONS TO ALLEVIATE SFA-MEDIATED LIPOTOXICITY IN SKELETAL MUSCLE Recent human studies have shown that SFA-induced muscle insulin resistance is associated with an increase in DAG content [65]. An increased level of ceramide has been also reported in obesity [40], while 8 weeks of endurance exercise training decreased DAG and ceramide content in an obese population and increased insulin sensitivity but did not alter IMTG content [66]. This is consistent with other studies in rat showing a reduction in DAG and ceramide content in the muscle after 4
8 weeks of exercise training with IMTG content remaining not affected [67,68]. Interestingly, Haugaard et al. [69] demonstrated that the improvement of insulin sensitivity during a very low calorie diet in obese subjects was related to desaturation of both structural (phospholipids) and neutral lipids (IMTG) in muscle. This supports the concept that esterification of exogenous FA into TAG stores of LDs may be protective against lipotoxicity and prevent insulin resistance in muscle. In addition, this confirms the critical role of muscle FA desaturase in regulating insulin sensitivity as previously discussed. Recent studies have shown that TAG accumulation does not seem primarily affected in myotubes from obese and T2DM subjects exposed to palmitic and oleic acids [70], suggesting that there is no genetic or intrinsic increase in TAG accumulation. IMTG accumulation would therefore mainly rely on environmental stimuli involving higher FA availability (as is seen in obesity), FA uptake due to a passive FA transport, and reduced oxidative capacity due to decreased mitochondrial content and/or function in obese and T2DM individuals. IMTG accumulation may be an adaptive mechanism to protect against lipotoxicity and insulin resistance. In support of this concept, overexpression of DGAT1 in mouse skeletal muscle protects mice against HFD-induced insulin resistance by channeling FA into TAG stores and reducing DAG and ceramide content [71]. Similarly, Schenk and Horowitz [42] found that FA (lipid/heparin)-induced insulin resistance was completely prevented by one session of 90 minutes of moderate-intensity exercise. This is again supporting the concept of a higher IMTG turnover being protective against insulin resistance. In conclusion, IMTG accumulation is an adaptive mechanism to buffer excess FA. Besides improving muscle oxidative capacity, regular physical activity increases muscle lipid storage capacity and increases IMTG stores, which in turn reduce the concentrations of toxic lipid intermediates within the muscle cell. Thus, the failure to appropriately sequester SFA into LDs may be a key determinant of insulin resistance.

CONCLUSIONS All clinical, biochemical, and physiological evidences have to be carefully analyzed and combined. Studies in cellular and animal models have been crucial to unravel the molecular mechanisms of action of SFA and MUFA in skeletal muscle. However, the clinical relevance of these observations in humans still remains to be established. This recalls that a huge basic research background is required before translation into dietary guidelines. Experimental evidences clearly highlight that limiting SFA-mediated ectopic fat deposition

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in skeletal muscle is crucial to preserve hormone responsiveness notably to insulin. Therefore, consumption of SFA should be limited for long-term preservation of skeletal muscle mass and function.

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8 Epub 2007/05/19. [43] Coue M, Badin PM, Vila IK, Laurens C, Louche K, Marques MA, et al. Defective natriuretic peptide receptor signaling in skeletal muscle links obesity to type 2 diabetes. Diabetes. 2015;64(12):4033
45 Epub 2015/08/09. [44] Chavez JA, Knotts TA, Wang LP, Li G, Dobrowsky RT, Florant GL, et al. A role for ceramide, but not diacylglycerol, in the antagonism of insulin signal transduction by saturated fatty acids. J Biol Chem. 2003;278 (12):10297
303 Epub 2003/01/15. [45] Pickersgill L, Litherland GJ, Greenberg AS, Walker M, Yeaman SJ. Key role for ceramides in mediating insulin resistance in human muscle cells. J Biol Chem. 2007;282(17):12583
9 Epub 2007/03/06. [46] Fox TE, Houck KL, O’Neill SM, Nagarajan M, Stover TC, Pomianowski PT, et al. Ceramide recruits and activates protein kinase C zeta (PKC zeta) within structured membrane microdomains. J Biol Chem. 2007;282 (17):12450
7 Epub 2007/02/20. [47] Dimopoulos N, Watson M, Sakamoto K, Hundal HS. Differential effects of palmitate and palmitoleate on insulin action and glucose utilization in rat L6 skeletal muscle cells. Biochem J. 2006;399(3):473
81 Epub 2006/07/11. [48] Coll T, Eyre E, Rodriguez-Calvo R, Palomer X, Sanchez RM, Merlos M, et al. Oleate reverses palmitateinduced insulin resistance and inflammation in skeletal muscle cells. J Biol Chem. 2008;283(17):11107
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10 Epub 2016/10/22. [50] Makinen S, Nguyen HY, Skrobuk P, Koistinen HA. Palmitate and oleate exert differential effects on insulin signalling and glucose uptake in human skeletal muscle cells. Endocr Connections 2017;6(5):331
9 Epub 2017/06/07. [51] Tardif N, Salles J, Landrier JF, Mothe-Satney I, Guillet C, Boue-Vaysse C, et al. Oleate-enriched diet improves insulin sensitivity and restores muscle protein synthesis in old rats. Clin Nutr. 2011;30(6):799
806 Epub 2011/06/28. [52] Turco AA, Guescini M, Valtucci V, Colosimo C, De Feo P, Mantuano M, et al. Dietary fat differentially modulate the mRNA expression levels of oxidative mitochondrial genes in skeletal muscle of healthy subjects. Nutr, Metab, Cardiovasc Dis: NMCD. 2014;24(2):198
204 Epub 2013/12/26. [53] St-Pierre J, Lin J, Krauss S, Tarr PT, Yang R, Newgard CB, et al. Bioenergetic analysis of peroxisome proliferator-activated receptor gamma coactivators 1alpha and 1beta (PGC-1alpha and PGC-1beta) in muscle cells. J Biol Chem. 2003;278(29):26597
603 Epub 2003/05/08. [54] Kamei Y, Ohizumi H, Fujitani Y, Nemoto T, Tanaka T, Takahashi N, et al. PPARgamma coactivator 1beta/ ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditure and antagonizes obesity. Proc Natl Acad Sci U S A 2003;100(21):12378
83 Epub 2003/10/08. [55] Pietraszek A, Gregersen S, Pedersen SB, Holst JJ, Hermansen K. Acute effects of monounsaturated fat on postprandial lipemia and gene expression in first-degree relatives of subjects with type 2 diabetes. Eur J Clin Nutr. 2014;68(9):1022
8 Epub 2014/05/16. [56] Lo Verme J, Gaetani S, Fu J, Oveisi F, Burton K, Piomelli D. Regulation of food intake by oleoylethanolamide. Cell Mol Life Sci: CMLS. 2005;62(6):708
16 Epub 2005/03/17. [57] Guzman M, Lo Verme J, Fu J, Oveisi F, Blazquez C, Piomelli D. Oleoylethanolamide stimulates lipolysis by activating the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR-alpha). J Biol Chem. 2004;279(27):27849
54 Epub 2004/05/05. [58] Artmann A, Petersen G, Hellgren LI, Boberg J, Skonberg C, Nellemann C, et al. Influence of dietary fatty acids on endocannabinoid and N-acylethanolamine levels in rat brain, liver and small intestine. Biochim Biophys Acta 2008;1781(4):200
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63 Epub 2012/12/15. [60] Listenberger LL, Han X, Lewis SE, Cases S, Farese Jr RV, Ory DS, et al. Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci U S A. 2003;100(6):3077
82. [61] Turpin SM, Lancaster GI, Darby I, Febbraio MA, Watt MJ. Apoptosis in skeletal muscle myotubes is induced by ceramides and is positively related to insulin resistance. Am J Physiol Endocrinol Metab. 2006;291(6): E1341
50 Epub 2006/07/20. [62] Hulver MW, Berggren JR, Carper MJ, Miyazaki M, Ntambi JM, Hoffman EP, et al. Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans. Cell Metab. 2005;2(4):251
61 Epub 2005/10/11. [63] Pinnamaneni SK, Southgate RJ, Febbraio MA, Watt MJ. Stearoyl CoA desaturase 1 is elevated in obesity but protects against fatty acid-induced skeletal muscle insulin resistance in vitro. Diabetologia. 2006;49 (12):3027
37 Epub 2006/10/13. [64] Laurens C, Bourlier V, Mairal A, Louche K, Badin PM, Mouisel E, et al. Perilipin 5 fine-tunes lipid oxidation to metabolic demand and protects against lipotoxicity in skeletal muscle. Sci Rep. 2016;6:38310 Epub 2016/ 12/07. [65] Montell E, Turini M, Marotta M, Roberts M, Noe V, Ciudad CJ, et al. DAG accumulation from saturated fatty acids desensitizes insulin stimulation of glucose uptake in muscle cells. Am J Physiol Endocrinol Metab. 2001;280(2):E229
37 Epub 2001/02/13. [66] Bruce CR, Thrush AB, Mertz VA, Bezaire V, Chabowski A, Heigenhauser GJ, et al. Endurance training in obese humans improves glucose tolerance and mitochondrial fatty acid oxidation and alters muscle lipid content. Am J Physiol Endocrinol Metab. 2006;291(1):E99
107 Epub 2006/02/09. [67] Lessard SJ, Rivas DA, Chen ZP, Bonen A, Febbraio MA, Reeder DW, et al. Tissue-specific effects of rosiglitazone and exercise in the treatment of lipid-induced insulin resistance. Diabetes 2007;56(7):1856
64 Epub 2007/04/19. [68] Smith AC, Mullen KL, Junkin KA, Nickerson J, Chabowski A, Bonen A, et al. Metformin and exercise reduce muscle FAT/CD36 and lipid accumulation and blunt the progression of high-fat diet-induced hyperglycemia. Am J Physiol Endocrinol Metab 2007;293(1):E172
81 Epub 2007/03/22. [69] Haugaard SB, Vaag A, Hoy CE, Madsbad S. Desaturation of skeletal muscle structural and depot lipids in obese individuals during a very-low-calorie diet intervention. Obesity (Silver Spring) 2007;15(1):117
25 Epub 2007/01/18. [70] Gaster M, Beck-Nielsen H. Triacylglycerol accumulation is not primarily affected in myotubes established from type 2 diabetic subjects. Biochim Biophys Acta 2006;1761(1):100
10 Epub 2006/01/31. [71] Liu L, Zhang Y, Chen N, Shi X, Tsang B, Yu YH. Upregulation of myocellular DGAT1 augments triglyceride synthesis in skeletal muscle and protects against fat-induced insulin resistance. J Clin Invest 2007;117 (6):1679
89 Epub 2007/05/19.

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Polyunsaturated Omega-3 Fatty Acids and Skeletal Muscle Gordon I. Smith Center for Human Nutrition, Division of Geriatrics and Nutritional Science, Washington University, St. Louis, MO, United States

LIST OF ABBREVIATIONS ALA DHA EPA g LCn-3PUFA MPB MPS SDA TNF-α

alpha-linolenic acid docosahexaenoic acid eicosapentaenoic acid gram long-chain omega-3 polyunsaturated fatty acids muscle protein breakdown muscle protein synthesis stearidonic acid tumor necrosis factor alpha

INTRODUCTION There is growing evidence that fish oil derived long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA) modulate muscle protein metabolism, muscle mass, strength, and physical function. This chapter will review current evidence from studies performed in cell cultures, in animal models and in humans and provide recommendations for future studies where gaps in our knowledge exist.

WHAT ARE POLYUNSATURATED OMEGA-3 FATTY ACIDS? There are three distinct classes of fatty acids, namely, (1) saturated fatty acids which do not contain any double bonds; (2) monosaturated fatty acids, which contain a single

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FIGURE 22.1 Chemical structure of the omega-3 polyunsaturated fatty acids, alphalinolenic acid, eicosapentaenoic acid, and docosahexaenoic acid.

Omega-3 CH3

COOH α-Linolenic acid, ALA (C18:3)

CH3

COOH

Eicosapentaenoic acid, EPA (C20:5) CH3 COOH Docosahexaenoic acid, DHA (C22:6)

double-bond; and (3) polyunsaturated fatty acids, which contain two or more doublebonds. Polyunsaturated fatty acids are further classified into two distinct species depending on where the first double bond from the methyl end of the fatty acid chain occurs. As the methyl end is considered the tail of the chain, it is frequently described as the omega tail after the last letter in the Greek alphabet. As the first double bond can occur at the third or sixth carbon from the methyl end, polyunsaturated fatty acids are described as either omega-3 fatty acids (abbreviated as ω-3 or n-3) or omega-6 fatty acids (abbreviated as ω-6 or n-6), respectively. The three omega-3 fatty acids most commonly consumed in the diet is shown in Fig. 22.1. α-Linolenic acid (ALA; C18:3n-3) is found in many plants, nuts, seeds, and vegetable oils with eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) predominantly found in oily fish such as salmon, mackerel, and sardines. As mammals are unable to synthesize omega-3 fatty acids, they are considered essential to the diet. Total omega-3 fatty acid intake in the United States is typically between 1.4 and 1.6 g/day [1 3]; however, the majority (B90%) is consumed as ALA with EPA and DHA typically consumed at very low levels (i.e., B0.1 0.2 g/day) in the United States [1 3]—a level that is substantially lower than  0.5 g/day, which is currently recommended for healthy individuals [4,5], and  1 g/day, which is recommended for individuals with documented cardiovascular disease [6]. As shown in Fig. 22.2, it is possible through a series of enzymatic reactions to elongate and desaturate ALA to produce the longer fatty acids, EPA and DHA. The first step in this cascade [i.e., desaturation of ALA to produce stearidonic acid (SDA)] is rate limiting in humans with ,4% of ALA converted to DHA in men [7 10] and ,10% in women [11]. To circumvent this step, a number of studies have examined whether intake of SDA can increase EPA and DHA levels to a greater extent that an equimolar dose of ALA. As expected EPA content in plasma, red blood cells and peripheral blood mononuclear cells was greater following SDA compared to ALA ingestion [12 15]. Unfortunately, DHA content was minimally affected by SDA supplementation, and as a result to increase EPA and in particular DHA levels to

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α-Linolenic acid ALA (C18:3) Stearidonic acid SDA (C18:4)

Desaturase

361

FIGURE 22.2 Series of enzymatic reactions required to convert alpha-linolenic acid to docosahexaenoic acid.

Elongase

Eicosatetraenoic acid ETA (C20:4) Desaturase Eicosapentaenoic acid EPA (C20:5) Elongase Docosapentaenoic acid DPA (C22:5)

Elongase

Tetracosapentaenoic acid (C24:5) Desaturase Tetracosahexaenoic acid (C24:6) β-Oxidation Docosahexaenoic acid DHA (C22:6)

any significant degree, sources of the longer fatty acids (i.e., fatty fish or dietary supplements) must be consumed. There is considerably heterogeneity in the omega-3 fatty acid content of different tissues and cells, with for example B50% higher LCn-3PUFA content found in erythrocytes than in muscle phospholipids [16]. EPA and DHA content does, however, increase in all cell types in a dose-dependent manner [17,18], but the rate of EPA and DHA incorporation (i.e., time to reach a plateau) is not uniform ranging from weeks in plasma cholesterol esters, months in red blood cells to over a year in adipose tissue [17]. To the author’s knowledge, the duration taken to achieve steady-state levels in skeletal muscle during omega-3 fatty acid supplementation is not known. Nonetheless, a doubling in EPA and DHA content in muscle phospholipids occurs when supplemented at relatively large doses (i.e., 4 g/day) for 8 weeks [19,20] suggesting that significant increases in muscle LCn3PUFA content can be achieved in weeks rather than years.

REGULATION OF MUSCLE MASS Muscle mass is determined by the net balance of the rate of muscle protein synthesis (MPS) and breakdown (MPB) with anabolism only observed when protein synthesis is greater than breakdown. Rates of MPS and MPB vary throughout the day with MPB greater than MPS in the fasting state and positive net protein balance typically limited to following protein ingestion [21]. Resistance exercise also stimulates MPS, but in the fasting state, net protein balance remains negative due to a concomitant increase in MPB [22].

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Positive net balance with resistance exercise is therefore only observed when protein is consumed postexercise, which results in greater increases in MPS and net balance compared to protein ingestion or exercise alone [22 24]. Aging-induced muscle loss is thought to be largely attributable to a reduced response to anabolic stimuli such as exercise and hyperaminoacidemia [25,26] in combination with a blunted suppression of MPB by insulin [27]. A reduced basal rate of MPS in the fasting state has also been reported in older adults by some groups; however, majority of the studies reports no difference in basal rates of MPS or MPB between healthy young and older adults [28]. Anabolic resistance has also been reported following periods of physical inactivity and bed rest and in individuals with colorectal cancer [29 33]. Pharmaceutical and/or nutritional approaches to stimulate muscle protein turnover to prevent the decline in muscle mass during catabolic states and increase muscle hypertrophy in response to anabolic stimuli are highly sought-after. One such approach may be treatment with EPA and/or DHA with growing evidence that these LCn-3PUFAs are potentiators of muscle anabolism under various conditions. The sections below will review results of studies that have examined EPA and DHA-induced changes (or otherwise) in muscle protein turnover, muscle mass, and muscle strength and function. The results will further be broken down to separately discuss findings in response to (1) an overnight fast in humans and control conditions in in vitro studies (i.e., where no anabolic or catabolic stimuli is provided); (2) anabolic (exercise or nutritional) stimuli; and (3) catabolic conditions/states such as sepsis and cancer cachexia.

EFFECT OF OMEGA-3 FATTY ACIDS ON MUSCLE PROTEIN TURNOVER LCn-3PUFA Treatment Under Basal/Fasting Conditions Under fasting conditions in animal models or basal conditions (i.e., without addition of anabolic or catabolic stimuli) in C2C12 myotubes, treatment with EPA and/or DHA has been found to suppress MPB with little to no effect on MPS [34 39]. The first studies to be examine combined EPA and DHA treatment on MPS in people found that taking relatively high doses (3.35 to 4.50 g/day of EPA and DHA) for 8 weeks did not affect fasting rates of mixed-muscle (i.e., the average synthesis rate of all proteins found in muscle) or myofibrillar (contractile proteins only) protein synthesis in either healthy young/middle aged [20,40] or older individuals [19]. These data contrast to results from a recent assessment of MPS in the fasting state [41] where synthesis rates in myofibrillar, mitochondrial and sarcoplasmic (cytosolic) proteins in muscle were fractionated and assessed before and after taking a comparable dose of LCn-3PUFA (3.9 g/day) to earlier studies [19,20,40], but for twice as long (i.e., 16- vs 8-weeks). In this study LCn-3PUFA-induced increases in protein synthesis rates of mitochondrial and sarcoplasmic fractions were reported with a trend for the myofibrillar protein synthesis rate to also be stimulated [41]. Despite the majority of in vitro, animal and human studies reporting no effect of EPA and DHA on fasting rates of MPS the latter findings leave open the possibility that a substantial latency period is required before improvements in fasting rates of MPS are observed. Future studies

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examining responses at multiple time points over a prolonged period (i.e., several months) are therefore required to address this prospect. Moreover, despite convincing data in in vitro and from animal studies that MPB is inhibited in the fasting/basal state by EPA and/or DHA treatment currently no information exists on whether LCn-3PUFA treatment affects rates of MPB in healthy individuals.

Response to Anabolic Stimuli EPA and DHA treatment prior to acute resistance exercise or during resistance training has consistently been found to have no impact on mixed MPS in men and women [40 42]. This response has, however, been found to be extremely heterogenous. For example, in one study a 1 2 fold increase in the mean MPS response to exercise was not found to be statistically significant due to considerable between-subject variability in the magnitude of the exercise-induced change in MPS [41]. While these negative findings may simply be due to inadequate power with a greater these results open up the intriguing possibility of responders and nonresponders, which if true would necessitate the discovery of biomarkers to identify who will best respond to treatment. Whether LCn-3PUFA supplementation affects MPS in response to nutritional stimuli is unclear. For example, greater rates of MPS have been found following mixed meal ingestion in piglets fed a DHA-enriched diet for 34 days [37] but not during continuous infusion (for 240 min) of mixed nutrients in young piglets fed a diet high in LCn-3PUFA for 28 d [43]. Similarly, stimulation of MPS by leucine administration in C2C12 cells is enhanced with treatment with EPA but not DHA for 24 hours [34]. Findings in humans are similarly equivocal. We have reported an improved anabolic response (defined as the increase in MPS above fasting levels) to insulin and B20 g amino acid infused over a 3-hour period. Specifically, the anabolic response was B50% greater in young/middle aged (from 0.027 6 0.005 to 0.042 6 0.005%/h above basal values; P 5 0.01) and B3-fold greater in older adults (from 0.009 6 0.005 to 0.031 6 0.003%/h above basal values; P , 0.01) [19,20]. In contrast, McGlory et al. reported that consumption of a similar dose of LCn-3PUFA also for 8 weeks did not affect myofibrillar MPS following consumption of 30 g whey protein ingestion [40]. Nonetheless, there appeared to be a trend for MPS to be greater in the fed state in the fish oil supplemented group in this study, and it is unclear whether differences would have been found if the subject size was greater. There is a lack of information on how MPB responds to exercise and nutritional stimuli following EPA and DHA treatment. As a result, it is currently unknown whether net protein balance is improved by LCn-3PUFA supplementation following exercise and/or during increased amino acid availability.

Response to Catabolic Stimuli Studies using C2C12 myotubes or myoblasts have under a variety of catabolic conditions [hyperthermia and treatment with tumor necrosis factor alpha (TNF-α; a proinflammatory cytokine), proteolysis-inducing factor, and palmitate] found that the decline in MPS and increase in MPB are attenuated with EPA and DHA treatment [44 47]. Studies

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Treatment effect (%)

4

y = 1.1556x – 1.1763 R² = 0.7892

3 2 1 0 –1 0

1

2 3 Dose (g/day)

4

FIGURE 22.3 Change in lean body mass and muscle volume following supplementation with long-chain omega-3 polyunsaturated fatty acids. Filled symbols represent % change in lean body mass against the response in the respective control group taken from References [41,54 58]. The dotted line represents the line of best fit using the change in lean body mass against the dose of omega-3 fatty acids, which has a correlation coefficient of r 5 0.89. The open circle represents the change in muscle mass against the omega-3 fatty acid dose provided in Reference number [61], which is consistent with the lean body mass findings.

in rodents have not been as comprehensive and have mostly focused on models of cancer cachexia. These studies have found no effect on MPS but unequivocally been shown to improve MPB [48 52]. Despite considerable interest on the effect of EPA and DHA on body composition in humans with cancer (summarized in the next section), it is surprising that no information exists on the effect of EPA and DHA treatment on muscle protein turnover in this population. The only study to date to examine the effect of EPA and DHA on muscle protein turnover under catabolic conditions used arterial and deep vein sampling across the forearm to assess rates of phenylalanine uptake and release (as surrogates for MPS and MPB, respectively) in individuals undergoing hemodialysis [53]. It was reported that after 12 weeks of EPA and DHA treatment (1.93 and 0.97 g/day, respectively), rates of phenylalanine uptake and release were both decreased compared to individuals randomized to the control arm. After multivariate adjustment for baseline values, age, sex, race, C-reactive protein (CRP), diabetes, and fat mass no difference in phenylalanine uptake across the forearm was found, but phenylalanine release from the forearm remained lower in the LCn-3PUFA group.

MUSCLE MASS AND VOLUME Responses in Healthy, Weight Stable Adults There has been recent interest into whether LCn-3PUFA can be included as a treatment option to prevent, lessen, or even reverse the slow decline in muscle mass with aging. Despite results from prospective studies on changes in lean body mass being mixed, when analyzed together they reveal a positive association between the dose provided and the treatment effect defined as the % change in lean mass in the LCn-3PUFA group against the % change in the corresponding placebo/control group (Fig. 22.3). Moreover, in studies

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where the lowest doses were provided (#1.8 g/day), no statistically significant effect of LCn-3PUFA was reported [54 56], whereas those that gave larger doses (2.4 3.4 g/day) all found LCn-3PUFA-induced improvements in lean body mass [41,57,58]. While these results demonstrate promise that LCn-3PUFA supplementation stimulates muscle growth in older adults, one major limitation in all these studies was the focus on whole-body lean mass (determined by dual-energy X-ray absorptiometry, air displacement plethysmography, or bioelectrical impedance analysis) rather than measuring changes in muscle mass directly. Muscle only contributes B50% to total body lean mass [59] and changes in lean body mass do not always correlate with a change in muscle mass [60]. To address whether muscle mass is affected by LCn-3PUFA treatment, we performed a randomized, controlled double-blind study in 60 healthy adults aged between 60 and 81 years with thigh muscle volume measured by magnetic resonance imaging [61]. Subjects were randomized to consume pills containing 1.86 g/day EPA and 1.50 g/day DHA or an equivalent dose of corn oil (control arm) for 6 months. In the control group, no change in muscle volume was found. In contrast, a 2.2% (95% confidence interval: 0.3% 4.2%) increase in muscle volume above baseline with a treatment effect (i.e., compared to responses in the control group) of 3.6% (95% confidence interval: 0.2% 7.0%) was found following LCn-3PUFA supplementation. The change in muscle volume was almost exactly as predicted based on lean body mass responses from earlier studies (Fig. 22.3) adding further weight to the notion that a relationship exists between LCn-3PUFA dose and improvement in lean body/muscle mass in healthy adults.

Response to Exercise Training and Catabolic Stimuli Only a few studies have examined the effect of LCn-3PUFA treatment on body composition during exercise training with no additional effect on lean body mass reported [42,56]; a finding similar to the negligible effect of resistance exercise on MPS [40,41]. This contrasts to the considerable number of studies to examine the effect of LCn-3PUFA treatment on muscle or lean mass in cell lines, animal models, and in humans during highly catabolic conditions. With few exceptions, addition of EPA and/or DHA to C2C12 cells and their supplementation in mainly rodent models has been found to markedly attenuate muscle loss [49,62 65]. For example, in C2C12 cells, addition of EPA at physiological levels (50 μM) prevents TNF-α induced decreases in myotube diameter [62,63]. In addition, most [49,64 67], but not all [68], studies in rodents and pigs examining proinflammatory states (cancer cachexia, burns, or lipopolysaccharide injection) have reported attenuated loss of muscle mass following predominantly EPA treatment. This is echoed by results in individuals with cancer where LCn-3PUFA treatment has been shown to attenuate loss of lean or muscle mass in the majority, but again not all, studies [69 74]. While loss of lean mass is less extensive during calorie restriction-induced weight loss compared to cancer cachexia, it is still typical for a B2-kg reduction in lean mass to be observed following B10% weight loss [75], which could increase the risk of sarcopenia (defined as low muscle mass and function [76,77]) especially in older adults. Despite LCn-3PUFA treatment unequivocally attenuating loss of muscle mass in rodent models [78 80] in humans across a wide range of ages (18 60 years), LCn-3PUFA treatment has been

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found to have little effect on lean body mass loss during calorie restriction [81 86]. It is unclear why no effect on lean body mass is observed during calorie restriction in contrast to the weight stable state or during cancer cachexia where LCn-3PUFA appears to have positive effects.

STRENGTH AND PHYSICAL FUNCTION The maximum force produced by individual muscle fibers is largely determined by the fiber type (i.e., type II . type I) and the cross-sectional area [87]. While aging is well known to reduce muscle mass, the decline in muscle function occurs at a B2 3-fold greater rate (declines of B2% 3% per year for muscle function have been reported vs B0.5% 1.0% per year for muscle mass) [88,89] suggesting that the “quality” of the muscle (defined as the amount of force that can be generated per unit of muscle) declines with aging. Muscle quality is thought to be determined by the muscle fiber type, architecture, ability to innervate, and the amount of fat and connective tissue within the muscle [90]. As a result, differences in muscle mass with LCn-3PUFA intake or treatment may not always translate to improved physical function and strength and likewise improvements in strength may be apparent without a corresponding change in muscle mass. One such example of this disconnect are results from cross-sectional studies where greater LCn3PUFA intake is not associated with increases in lean body/muscle mass [91,92] but is consistently associated with greater hand-grip, 1-repetition maximum leg press and isokinetic knee extension strength, and faster chair rise times [91,93 95]. For example, it has been reported in a large cohort of 2983 men and women aged between 59 and 73 years that for every additional portion of fatty fish consumed per week, grip strength increased by 0.43 kg (B1.0% increase) in men and 0.48 kg (B1.8% increase) in women [93]. These findings were confirmed by a randomized, controlled, double blind study we recently performed, which examined the effect of LCn-3PUFA or corn oil (control) supplementation for 6 months on grip strength, 1-repetition maximum strength for several upper and lower exercises, and isometric power in older adults [61]. At the end of the treatment period, hand grip and 1-RM muscle strength were 2.3 (B6.5%) and 9.1 kg (B4.0%) greater than the control arm, respectively, with a trend for isometric power to also be improved. Moreover, the extent of these improvements in strength was greater than the increase in muscle volume we observed. Taken together, these findings suggest that muscle quality is improved in older adults following LCn-3PUFA treatment. The mechanisms responsible for this putative improvement are, however, unclear but may be related to improved neuromuscular function [96,97] and/or reduce intermuscular fat infiltration [61]. Resistance exercise training is well known to increase strength and physical function, and there is evidence that LCn-3PUFA supplementation augments this improvement [42,96]. For example, in older women, LCn-3PUFA supplementation during a 12-week exercise training regimen increased peak isometric torque to a greater extent that strength training alone with the rate of force development also improved [96], findings that were recently confirmed by a separate group of investigators [42]. Interestingly, in the later study, improvements were observed in women only with no additional benefit of taking

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LCn-3PUFA in men. Due to the small number of studies performed to date, it is premature to recommend women only take fish oil during exercise training to augment its effect on strength and physical function, but these data do suggest that further studies are needed to confirm these results and elucidate the mechanisms responsible for any potential sexually dimorphic response.

CONCLUSIONS There is growing evidence that the long-chain omega-3 polyunsaturated fatty acids, EPA and DHA, have anabolic properties, and their addition to the diet, either a consumption of fatty fish or as fish oil supplements, leads to clinically significant improvements in muscle protein metabolism, lean body/muscle mass, and strength and muscle function under a variety of conditions. In particular, increases in lean body and muscle mass have consistently been reported in weight stable older adults and in individuals with cancer cachexia. LCn-3PUFA-induced improvements in physical function and strength have also been reported, although at present, this effect may be limited to in women with the response in men less clear. Future studies are required to examine the effect of EPA and DHA on rates of MPB in humans, focus more on changes in muscle mass rather than total lean body mass and determine whether responses to exercise training differ between men and women.

Acknowledgments Gordon Smith was supported by the Washington University Institute of Clinical and Translational Sciences grant UL1TR000448, subaward KL2TR000450, from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH) during the writing of this chapter. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH.

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Vitamin D Signaling and Skeletal Muscle Cells Carla Domingues-Faria and Ste´phane Walrand Universite´ Clermont Auvergne, INRA, UNH, Unite´ de Nutrition Humaine, CRNH Auvergne, F-63000 Clermont-Ferrand, France

INTRODUCTION Vitamin D is a fat-soluble vitamin discovered in 1919 by Sir Edward Mellanby. It is not strictly speaking a vitamin: an organic precursor was identified in the skin in the 1920s that leads to vitamin D formation when irradiated by sunlight or ultraviolet radiation (UVB). Vitamin D was subsequently synthesized for the first time in 1952 by Woodward, who was awarded the Nobel Prize in chemistry in 1965. In the late 1920s, it was clearly established that rickets could be prevented and cured by direct exposure to sunlight, UVB irradiation, and by the consumption of some irradiated foods or cod liver oil. With the systematic supplementation of children with vitamin D in the mid-20th century, rickets disappeared in economically developed countries. Hypovitaminosis D promotes osteomalacia in the young adult, and osteopenia or even osteoporosis in older persons. Vitamin D is not only essential for the prevention of rickets and bone loss: research has progressed considerably since the 1980s, and receptors of active derivatives of vitamin D have been discovered in the cells of most body tissues, explaining the pleiotropic effects of this nutrient. Epidemiological and experimental data thus support a role of vitamin D in the prevention of many diseases (cancers and autoimmune diseases, cardiovascular events and hypertension, sarcopenia, etc.). Maintenance of vitamin D status is currently a global public health issue, as it is estimated that at least one billion people in the world have a proven vitamin D deficiency [1]. More than 50% of healthy young adults are vitamin D deficient [2], and more than 90% of patients consulting primary care for nonspecific musculoskeletal pain have demonstrated vitamin D deficiency [3]. Of note, the prevalence of vitamin D deficiency in patients aged over 65 years ranges from 40% to 100% [1].

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00023-3

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Besides its established role in maintaining bone mass and mineral homeostasis, a significant amount of emerging evidence reveals that vitamin D exerts a wide range of effects on skeletal muscle by driving several key intracellular pathways.

VITAMIN D AND SKELETAL MUSCLE FUNCTION The first associations between vitamin D and muscle function stemmed from clinical observations on muscle weakness and stiffness in chronic vitamin D deficiency, particularly during osteomalacia [2]. In these situations, a high dose of vitamin D was found to limit muscle abnormalities [2,4]. In infants, vitamin D deficiency-associated myopathy is typically characterized by reduced muscle tone and hypotonia [5]. In adults, this syndrome is predominantly characterized by proximal muscle weakness, with difficulties climbing stairs, getting up from a sitting or crouching position, and lifting heavy objects. Added to these clinical observations, Birge and Haddad [6] showed in the mid-1970s that vitamin D directly affected phosphocalcic metabolism in the diaphragm of vitamin D deficient rats. From these early observations, our basic knowledge about the roles of vitamin D on muscle health has continued to grow, and vitamin D is still being studied today for its muscle actions, notably in older persons frequently affected by sarcopenia. Given the benefits of vitamin D on muscle function in young or athletic persons, studies have investigated its effects on the mobility of older persons. Vitamin D deficiency is particularly prevalent in the elderly owing to reduced dietary intake and a concomitant decrease in sun exposure and in the ability of the skin to synthesize it [7,8]. Some chronic diseases in the elderly, such as renal failure, also appear to be a further contributing factor in hypovitaminosis D. A significant relationship was found between vitamin D status and the extension force deployed by the lower limb muscles in a group of older individuals [9]. This finding is consistent with the study of Mowe et al. [10] on the association between serum vitamin D concentration and muscle function. This study, carried out in 349 people aged 70 on average, showed that serum vitamin D concentrations were significantly lower in those persons with the lowest grip strength, inability to climb stairs, with no outdoor activity, and who had fallen in the previous month [10]. In addition, a low serum level of vitamin D was positively associated with a reduction in handgrip strength and in walking distance in community-dwelling old persons [11,12]. These results have been confirmed very recently. In a prospective study, it was evidenced that a low serum vitamin D concentration was associated with a 1-year decline in muscle strength in community-dwelling older persons [13]. Similarly, a reduction in distal lower limb strength has also been observed in older people with impaired glucose tolerance and suffering from vitamin D deficiency [14]. Interestingly, the expression of vitamin D receptor (VDR) in muscle tissue decreased progressively with aging [15]. This observation points to a possible loss of muscle sensitivity to vitamin D with age. As a result, muscle weaknesses related to vitamin D deficiency may occur in older adults at higher blood levels than in younger subjects. All these observations imply that vitamin D requirements to maintain muscle mass and function may be higher in the elderly than in younger persons.

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In agreement with observational analyses, interventional studies have demonstrated the value of vitamin D for muscle health. Vitamin D supplementation improves muscle quality and fiber type morphology, indicating a vitamin D targeted action on skeletal muscle remodeling [16]. Additionally, several studies have established that the supplementation of healthy but vitamin D deficient athletes improves their muscle strength [17]. Similar results were observed in older subjects. Muscle strength and mobility were measured in vitamin D deficient older women (mean age 76 years) after a treatment for 6 month with 0.5 μg/day of α-calcidol [18]. In this study, both knee extension strength and walking distance were significantly improved in women receiving vitamin D, while no improvement was seen in the placebo group. Additionally, in frail older patients, vitamin D supplementation significantly improved the time required for dressing and functional abilities as measured by a functional assessment questionnaire [19]. A protocol of sustained vitamin D supplementation significantly improved the strength deployed by the lower and upper limbs in a group of vitamin D deficient women [20]. Another investigation also revealed that an intake of 1000 IU/day of vitamin D for 1 year by people aged 70
90 restored vitamin D status and increased the strength deployed by lower limb muscles [21]. In this study, improved mobility capacities as evaluated by the Timed Up and Go Test were observed in the subjects receiving vitamin D. The same results were reported in a trial using lower intakes of vitamin D [22]. In this latter study, a daily intake of 400 IU of vitamin D for 9 months resulted in the restoration of vitamin D status, and in an improved walking speed and balance in 70 year-old subjects initially with vitamin D deficits. We note that similar effects (increased muscle strength in the lower limbs) were observed in younger subjects, i.e., from 60 years of age, regardless of physical activity level [23]. According to several authors, vitamin D deficiency primarily affects the muscles of the lower limbs responsible for postural balance and walking [20]. A significant negative correlation between vitamin D status and falls has been reported in elderly patients [10,24]. The improvement of lower limb muscle strength and balance with vitamin D intake may explain fewer fall-related fractures in the supplemented elderly [25]. Recent work confirmed these previous studies. Muscle strength was enhanced in women suffering from a vitamin D deficiency after 6 months of vitamin D supplementation with either 1000 IU/day of cholecalciferol or 1 μg/day of α-calcidol [26]. Unfortunately, other recent interventional studies failed to demonstrate any positive effects of vitamin D supplementation. In a clinical trial, postmenopausal women aged 60 with vitamin D insufficiency were supplemented for 1 year with a daily low dose (800 IU) or a twice-monthly dose (50,000 IU) [27]. This protocol failed to produce any benefit for muscle mass and function [27]. In home-dwelling older women, muscle strength was unchanged following a vitamin D supplementation (800 IU/day) for 2 years, whether or not it was combined with physical exercise [28]. Of note, the participants of this last study were characterized by only a mild vitamin D insufficiency. Similar results were found in other clinical trials where no additive effect of vitamin D intake (1920 IU/day) in the course of 12 weeks of resistance training was detected on muscle strength in young and older men [16]. Such variability in vitamin D supplementation efficiency is likely due to several parameters (characteristics of participants, baseline vitamin D status, treatment time period, form and/or dose of vitamin D used), all of which should be considered in interventional protocols and when interpreting results.

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Taken together, these studies suggest that vitamin D supplementation is effective only in subjects suffering from a vitamin D deficiency. In a metaanalysis, Beaudart et al. evidenced that vitamin D supplementation had a small but positive impact on global muscle strength, more specifically in the lower limbs [29]. They also found a possibly greater effect of vitamin D supplementation in subjects with a baseline vitamin D level below 30 nmol/L. However, Rosendahl-Riise et al. concluded from a recent metaanalysis that supplementation of vitamin D did not improve muscle strength [30]. We note that this analysis was based on fewer studies and participants than the previous one by Beaudart et al. Observational studies provide evidence that vitamin D is an important nutritional substance for muscle health, but it is still difficult to assert consensually that vitamin D supplementation can improve muscle mass and/or strength. Vitamin D supplementation appears to be effective in deficient patients, as observed in older subjects or athletes. The impact of vitamin D treatment may thus depend on parameters such as sex and age of the participants, lifestyle, quality of life, baseline vitamin D status, type of vitamin D intake, gene polymorphisms or ethnicity. A vitamin D treatment plan to improve muscle health would thus be more effective if individualized [31].

VITAMIN D AND SKELETAL MUSCLE MORPHOLOGY It is noteworthy that commonalities concerning the effects of vitamin D deficiency and aging per se on skeletal muscle (sarcopenia) have been described in the literature. Muscle weakness due to vitamin D deficiency in young people manifests mainly as a feeling of heaviness in the legs, general muscle fatigue and difficulty in carrying out everyday activities associated with muscle function such as climbing stairs or rising from a chair [32,33]. The same functional aspects are systematically observed in older subjects experiencing the impact of aging on the functional properties of skeletal muscle. In addition, muscle biopsies in young subjects with severe vitamin D deficiency showed atrophy mainly of type II muscle fibers: type II muscle fibers are those that are specifically lost during aging. Interestingly, type II muscle fibers are rapidly contracted and are therefore the first to be recruited to maintain equilibrium and avert falls. Thus, the fact that type II fibers are affected by vitamin D deficiency and aging per se may help explain the more frequent falls in persons with deficiencies, notably among older persons [34,35]. In addition, studies have shown that vitamin D supplementation increases the relative number and size of type II muscle fibers in elderly women [36
38]. A positive correlation between serum vitamin D level and the diameter of the type II muscle fibers was noted in these studies. All authors working in this scientific area agree that muscle atrophy associated with aging (sarcopenia) arises from a selective loss of type II fibers [39,40]. It has therefore been proposed that the specific atrophy of type II fibers in the elderly may be related to a reduction in the status and muscle action of vitamin D, itself explained by decreased muscle vitamin D receptor (VDR) density. Muscle histological sections from young vitamin D deficient individuals also revealed enlarged intermyofibrillar spaces, a sign of fibrosis, and increased lipid infiltration, as conventionally described in the elderly [41,42]. In accordance with these earlier results, our team demonstrated that age associated with vitamin D

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deficiency led to triglyceride and ceramide accumulation in rat skeletal muscle [43]. Interestingly, vitamin D supplementation tended to reduce skeletal muscle lipid accumulation [43]. Several aspects of the morphological characteristics of myopathy associated with aging are therefore identical to those observed in vitamin D deficient subjects [40,44,45]. Taken as a whole, descriptive data from epidemiological studies and clinical investigations have clearly shown that a low vitamin D status is closely associated with reduced muscle function, strength and altered morphology. In addition, vitamin D supplementation can improve muscle strength in vitamin D deficient patients with myopathy, muscle weakness and sarcopenia [31].

VITAMIN D STATUS AND SKELETAL MUSCLE DEVELOPMENT AND REGENERATION Twenty years ago, Yoshizawa et al. first demonstrated the importance of vitamin D and its receptor in muscle development, when VDR-null mice showed drastic growth retardation and early mortality [46]. Using the same model, Girgis et al. observed a decrease in grip strength and muscle fiber number and size, likely leading to skeletal muscle atrophy in VDR knock-out mice compared with their wild-type counterparts [47]. These early results demonstrate that vitamin D and its receptor are involved in skeletal muscle development. Dietary vitamin D restriction also influences skeletal muscle development. In mice, Girgis et al. demonstrated that vitamin D restriction led to a reduction in grip strength, but with no impact on muscle fiber diameter or mass [47]. Conversely, Oku et al. observed that muscle mass tended to decrease in vitamin D restricted young rats compared with controls [48]. This is in line with our previous work showing a significant decrease in tibialis anterior muscle mass in vitamin D restricted old rats [49]. The discrepancies between these two studies are likely related to the different periods of vitamin D restriction and/or the age of the animals, i.e., 9 months versus 1 month [49]. Vitamin D is also essential for skeletal muscle regeneration. Srikuea et al. recently demonstrated that key markers of vitamin D metabolism were up-regulated during skeletal muscle regeneration after a barium chloride induced lesion in mice [50,51]. However, treatment with a supraphysiological dose of vitamin D delayed the formation of the regenerative muscle fiber and increased muscle fibrosis [50]. In this study, the expression of the enzyme controlling vitamin D deactivation, i.e., CYP24A1, was increased following vitamin D infusion [50]. This suggests that vitamin D is deactivated at the site of infusion, and eliminated, a mechanism locally controlling vitamin D concentration. This phenomenon could explain why a local high dose infusion of vitamin D in skeletal muscle failed to improve muscle regeneration. Conversely, in another animal model, the infusion of vitamin D led to an increase in proliferation and a decrease in apoptosis in injured muscle, which could promote skeletal muscle repair [52]. These discrepancies in vitamin D effects between these two studies could be due to the fact that the animal used, the nature of the injury and also the vitamin D form used were different. Resistance training also induces a deterioration of muscle fibers, requiring a subsequent phase of regeneration. Thus, up-regulation of markers of vitamin D metabolism in skeletal

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muscle was also observed in rats following resistance training, consistent with the study of Srikuea et al. [50,51,53,54]. Vitamin D seems therefore to be essential in resistance training, with a role of sustaining the regeneration process, and also preventing skeletal muscle damage [54]. A reduced concentration of blood markers of skeletal muscle damage following exhaustive exercise was noted in rats receiving a vitamin D infusion [54]. Benefits of vitamin D on muscle regeneration are also confirmed in humans. Owens et al. have demonstrated that muscle cell migration dynamics, myotube fusion, differentiation, and hypertrophy of myoblasts derived from biopsies of young volunteers presenting vitamin D insufficiency were improved following vitamin D treatment [55]. In a clinical trial, the same authors highlighted that a supplementation with vitamin D in young men presenting vitamin D insufficiency led to the improvement of peak torque recovery following damaging eccentric contractions of the knee extensors [55]. Taken together, these results demonstrate the direct effect of vitamin D on muscle cells, leading to the improvement of the skeletal muscle recovery process. Muscle regeneration involves not only the intervention of the muscle cells themselves, but also other cell types such as immune cells [56]. The latter interact closely with myoblasts leading to the formation of a new muscle fiber or to the repair of an injured fiber [56]. Interestingly, immune cells are natural targets of vitamin D action. Vitamin D could thus promote muscle regeneration not only by directly targeting muscle cells themselves [37], but also indirectly by acting on immune cells [57]. It is therefore of prime importance to investigate the action of vitamin D on specific immune response during muscle regeneration. In an animal model of skeletal muscle regeneration, the number of infiltrated immune cells on the injury site was not modified by vitamin D infusion [52]. Of note, in this study, the overall immune cell population was investigated without distinction of immune cell subsets. Interestingly, in another rodent model, vitamin D infusion interfered with the increase in proinflammatory cytokine expressions in muscle following intensive physical exercise [58]. It has also been demonstrated that vitamin D deficient subjects display a decrease in blood concentration of antiinflammatory cytokines, i.e., interleukin (IL)-10 and IL-13, following physical training, compared with nondeficient volunteers. Furthermore, muscle strength was more rapidly recovered in nondeficient men [59,60]. Taken together, these results highlight the key action of vitamin D in modulating circulating and muscle-specific cytokine expression, and possibly immune and muscle cell cross-talk during muscle repair. Finally, our research group has demonstrated in old rats that vitamin D deficiency down-regulates the Notch pathway, known to play a leading role in muscle regeneration [49]. In this context, vitamin D deficiency could aggravate age-related decrease in muscle regeneration capacity. These results show that the effect of vitamin D on skeletal muscle regeneration efficacy is a topic of interest. Unfortunately, investigations on vitamin D and muscle repair in the context of vitamin D deficiency are very scarce. More research is needed in this scientific area to define strategies that can prevent ineffective and/or incomplete muscle repair in different pathophysiological situations. This would be of great interest particularly in older persons who experience ineffective muscle repair, contributing to muscle mass loss and sarcopenia [40]. Nutritional strategies need to be considered not only during aging but in all situations where muscle repair may be needed: in pathologies associated with muscle damage, or among athletes, who regularly undergo muscle regeneration processes [56].

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EFFECT OF VITAMIN D ON SKELETAL MUSCLE CELL SIGNALING Many studies have demonstrated the effect of vitamin D on skeletal muscle function, development, morphology or regeneration. This is not surprising, since several studies have shown that the active metabolite of vitamin D (1α,25(OH)2vitamin D) affects muscle cell metabolism by regulating the expression of many genes and various intracellular pathways. The VDR has been found in muscle tissue in both animal models and humans [51,61
63]. Further, other authors have shown that VDR expressed in skeletal muscle cells specifically binds 1α,25(OH)2vitamin D. VDR is a nuclear receptor acting as a transcriptional factor; other authors have postulated that VDR can also be expressed at the plasma membrane level. At the nucleus, the ligand
receptor interaction is modulated by various other transcription factors, resulting in a final transcription complex responsible for the genomic effects of vitamin D [64]. However, 1α,25(OH)2vitamin D
VDR complex is also able to modulate signaling pathways [65]. Clearly, vitamin D drives many muscle cell functions by distinct general mechanisms: (1) by a positive or negative regulation of the transcription of target genes, i.e., the genomic effect of vitamin D and (2) by the modulation of intracellular signaling pathways independently of a transcriptional mechanism, i.e., the nongenomic effect of vitamin D. This is particularly the case for the effects of vitamin D on muscle cell proliferation and differentiation, or on muscle cell metabolism.

Effect of Vitamin D on Muscle Cell Proliferation and Differentiation Vitamin D helps control the proliferation and differentiation of muscle cells through its genomic action, by controlling central genes directly involved in these processes [63,66
68]. In addition, a vitamin D genomic regulation of muscle cell proliferation and differentiation has been shown through its positive effect on the expression of insulin growth factor, a positive inducer of skeletal muscle hypertrophy [69]. Vitamin D controls muscle cell proliferation and differentiation not only through a genomic action, but also through nongenomic effects. Recent data clearly indicate that the nongenomic action is dependent on a rapid activation of the mitogen-activated protein kinase (MAPK) signaling pathway [5,37,51,67]. Overall, this pathway transmits extracellular signals to their intracellular targets, which eventually leads also to the initiation of myogenesis, proliferation, differentiation or even apoptosis [9,65,67,68]. In mammalian cells, the conventional MAPK family is represented by several regulatory proteins: extracellular signal-regulated kinases (ERK-1/2), c-Jun N-terminal kinases, ERK5, and p38 MAPK [70]. When activated, these intermediates regulate cellular processes through the phosphorylation of other kinases and transcription factors. Among them, ERKs are key factors of the signaling pathway modulating cell growth and differentiation mechanisms [11,18]. In proliferating myoblasts, 1α,25(OH)2vitamin D activates rapidly (within 1 minute) ERK-1/2, phospholipase C and c-myc: it causes the translocation of an active phosphorylated form of the ERK-1/2 protein from the cytoplasm to the nucleus and triggers the synthesis of the growth-related protein (c-myc) [65,67,68,71]. This whole signal cascade leads to the stimulation of muscle cell proliferation [67,68]. Additionally, it has been demonstrated recently that vitamin D improves murine and human skeletal muscle cell

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differentiation [55]. Conversely, recent work concluded that vitamin D inhibited human skeletal muscle cell proliferation and differentiation [72]. The discrepancy in the differentiation process was likely due to the culture medium used and cell plating condition. Taken together, these results demonstrate that vitamin D regulates muscle cell proliferation and differentiation positively or negatively, probably according to the cell type used, vitamin D dose, or culture conditions.

Effect of Vitamin D on Muscle Cell Metabolism The genomic action of vitamin D is also responsible for a strong controller of calcium absorption and fluxes in cultured myotubes. In particular, it has been shown that vitamin D acts on the postcontraction capture of calcium by sarcoplasmic reticulum and the transport of phosphate through the cell membrane. We note that the vitamin D induced changes in intracellular calcium levels appear to modulate myofibrillar contraction and relaxation, affecting the contractile function of this tissue [66,73]. Further, vitamin D can cause instantaneous changes in calcium flux and metabolism within muscle cells, which cannot be explained by a slow genetic mechanism. In addition, the use of specific inhibitors of gene transcription did not inhibit all vitamin D induced muscle effects. These data indicate that 1α,25(OH)2vitamin D can act directly on muscle cell membrane, in lipid rafts, in particular the calveolae, without impacting gene regulation and expression [65,67,68,71,74,75]. It is hypothesized that 1α,25(OH)2vitamin D causes the activation of second messengers involved in different cell signaling pathways and inducing increased calcium absorption (within a few minutes) through the voltage-dependent calcium channels [76
78]. Apart from its action on calcium pools, 1α,25(OH)2vitamin D promotes protein synthesis and affects cellular growth in skeletal muscle [36,79]. According to recent results, vitamin D may have a sarcoplasmic action on the intracellular pathways regulating protein metabolism [79]. Vitamin D is able not only to stimulate protein synthesis in muscle cells but also to increase the amount of the insulin receptor by causing an overexpression of its gene and its phosphorylation state. Clearly, the signaling pathway (Akt/mTOR/p70S6k/ 4EBP1 in particular) participating in the positive regulation of muscle protein synthesis is activated in myotubes treated with vitamin D. Mechanistically, vitamin D seems to enhance the stimulating effect of insulin and leucine on the regulation pathway of protein translation initiation (Akt/mTOR/p70S6k/4EBP1), resulting in additional activation of protein synthesis in myotubes. Vitamin D therefore potentiates the effect of insulin and leucine on protein anabolism in muscle cells. The effect of vitamin D on the genes coding for the insulin receptor and for VDR may partly explain this action [79]. Conversely, Van Der Meijden et al. [80] have recently argued that vitamin D has no effect on the Akt/ mTOR signaling pathway in differentiating C2C12 myotubes [80]. However, Van Der Meijden evaluated the effect of vitamin D treatment on the Akt/mTOR signaling activation without acute protein synthesis stimulation during myotube formation [80]. These results suggest that vitamin D activates protein synthesis mostly by amplifying the action of factors stimulating the mTOR signaling pathway. The effect of vitamin D on muscle protein synthesis was confirmed in vivo. The rate of muscle protein synthesis was reduced

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in old rats suffering from a vitamin D depletion [43]. This process was restored by vitamin D supplementation [43]. Finally, a study has evidenced that vitamin D increases mitochondrial oxygen consumption, and seems to enhance mitochondrial biogenesis in human skeletal muscle cells [81]. Overall, this work reveals that vitamin D may regulate mitochondrial function, dynamics, and enzyme activities in human skeletal muscle cells, which likely influence muscle strength [81]. Taken as a whole and despite some discrepancies, these studies clearly demonstrate that vitamin D modulates muscle proliferation, differentiation, and metabolism by driving key intracellular pathways.

GENETIC CONTRIBUTION TO VITAMIN D EFFECTS Vitamin D modulates muscle cell function and metabolism by several mechanisms, depending on its genomic or nongenomic action. In addition, vitamin D acts as a key controller of muscle cell function also through the existence of allelic variants of the VDR. Research has shown that skeletal muscle strength and mass are inheritable traits, and some genes have been identified as contributors to skeletal muscle mass or strength [82]. Several single-nucleotide polymorphisms in the gene encoding the VDR have been associated with muscle strength, in particular FokI and BsmI polymorphisms [4]. FokI polymorphism is a T/C transition in the exon 2 of the VDR gene, resulting in a shorter protein with enhanced transactivation capacity [4]. BsmI polymorphism is located in the 30 region of the VDR gene, known to play an important role in the regulation of gene expression [4]. One study cohort recently demonstrated that FokI and BsmI polymorphisms were associated with skeletal muscle strength in men and women aged 20 2 90 years [82]. Furthermore, allele FF of FokI was associated with an increased risk of sarcopenia in older women [82]. This and other earlier work demonstrate that the “genetics of vitamin D” plays a real part in the control of muscle function and that VDR genetic variations could aggravate the risk of muscle disease such as sarcopenia in numerous clinical situations. However, further studies are needed to evaluate the associations between genetic variations and muscle mass or strength, or to highlight other polymorphisms and their outcome on skeletal muscle trophicity [31].

THE COMPLEX ACTION OF VITAMIN D Vitamin D signaling modulates muscle development, functions and repair by at least three general mechanisms: (1) by its genomic effect, (2) by its nongenomic effect, and (3) through the existence of allelic variants of VDR. However, it is important to emphasize that these different mechanisms act in coordination to maintain muscle metabolic and contractile function. Hence, the action of vitamin D is probably due to a complex combination of genomic and nongenomic dependent regulations, themselves controlled by the VDR polymorphisms. Furthermore, vitamin D can exert indirect genomic effects, such as by

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modulating signaling pathways, leading to altered genomic expressions. Furthermore, the sometimes divergent effects of vitamin D on skeletal muscle observed in reported studies can depend on the pathophysiological situation, the model used, the vitamin D type or dose, and/or the treatment time. This suggests that the strategies of vitamin D studies used until now may not all be well adapted, notably to distinguish between its genomic and nongenomic action and to consider the importance of the polymorphism of the VDR and enzymes controlling the metabolism of vitamin D. A more holistic approach would be desirable to advance further in the study of muscle vitamin D and elucidate its action in muscle cell signaling and functions.

CONCLUSION Besides its established role in maintaining bone mass and mineral homeostasis, a significant amount of emerging evidence reveals that vitamin D exerts a wide range of effects on skeletal muscle. Early studies reported changes in muscle morphology and contractile weakness in subjects with vitamin D deficiency. These investigations were completed by numerous trials evaluating the impact of vitamin D on muscle mass and strength. Animal models have confirmed that vitamin D deficiency and the presence of congenital aberrations in the systems of vitamin D production and/or activation lead to muscle weakness. Furthermore, vitamin D plays a key role in muscle repair. To explain these effects, some molecular mechanisms by which vitamin D affects the differentiation of muscle cells, the regulation of calcium flow and the expression of key genes, have been partially elucidated. Vitamin D deficiency is widespread in the general population, particularly in older persons, with multiple health consequences. In this context, several epidemiological studies reveal that a low vitamin D status is always associated with a decrease in skeletal muscle mass, strength and repair and reduced contractile capacity. The age-related vitamin D deficiency leads to an accelerated muscle loss, i.e., a vitamin D related sarcopenia, and consequently a reduced physical capacity and an increased risk of falls and fractures. Conversely, an additional intake of vitamin D significantly improves muscle function in the elderly. Nevertheless, although it has been shown that nuclear VDR expression decreases in muscle with age, further work is still needed to gain a better understanding of the cellular, metabolic and molecular roles of this micronutrient and its impact in aging. The study of the muscle effect of vitamin D is therefore currently a dynamic area of research. New findings will soon push back the boundaries of knowledge on the broad functional repertoire of vitamin D, in particular on age-related musculoskeletal loss.

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405. [52] Stratos I, Li Z, Herlyn P, et al. Vitamin D increases cellular turnover and functionally restores the skeletal muscle after crush injury in rats. Am J Pathol. 2013;182(3):895
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Vitamin D Deficiency and Myopathy 1

2

Christian M. Girgis1,2,3

Department of Endocrinology, Royal North Shore Hospital, Sydney, NSW, Australia Department of Endocrinology, Westmead Hospital, Sydney, NSW, Australia 3University of Sydney, Sydney, NSW, Australia

INTRODUCTION The link between vitamin D deficiency and myopathy is well established. Three centuries ago, Daniel Whistler, the English physician, described the presence of weak “toneless muscles” in children with rickets [1]. For years, muscle weakness, falls, and type II muscle fiber atrophy have been reported in adults with vitamin D deficiency [2]. These defects in muscle function improve following vitamin D supplementation, indicating a direct role for vitamin D in muscle contraction and strength. This chapter will discuss effects of vitamin D on skeletal muscle from a range of human studies, including observational studies, human clinical trials on vitamin D and muscle strength, falls, and physical performance.

VITAMIN D The term “vitamin” suggests that vitamin D is a nutrient. Whilst this is partly true as vitamin D is indeed present in certain food types (e.g., oily fish, mushrooms, eggs), vitamin D is more accurately considered a hormone due to its autocrine regulation, secosteroid structure and interaction with a nuclear receptor [3]. The first step in vitamin D’s synthetic pathway requires sun exposure and the photochemical conversion of 7dehydrocholesterol to vitamin D3 under the influence of UV-B radiation. Vitamin D then circulates bound to the D-binding protein and undergoes two hydroxylation steps, first in the liver to form 25(OH)Vitamin D (25OHD) and subsequently in the kidney to form 1,25(OH)2Vitamin D (1,25(OH)2D). 1,25(OH)2D is the biologically active hormone that binds to the vitamin D receptor (VDR) to regulate gene expression and exerts predominant effects in calcium and mineral homeostasis [3]. The VDR is expressed in skeletal muscle, but at very low levels, its expression is greatest at early stages of muscle

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development and during muscle repair and VDR modulates the uptake of vitamin D into skeletal muscle fibers [4 10]. Vitamin D deficiency is recognized to be very common. In Australia, one-third of participants in a study of 11,000 adults were vitamin D deficient (25OHD , 50 nmol/L) [11]. In the United States, an incidence of B40% was reported in a study of 4495 adults [12]. This high incidence of vitamin D deficiency is not limited to adults who are sedentary or simply avoid sun exposure. Athletes who would be expected to have regular access to sunlight also have surprisingly high rates of vitamin D deficiency. Recent studies report the following rates of vitamin D in groups of athletes from Australia (33% deficient) [13], the Middle East (58%) [14], United Kingdom (57%) [15], and USA (13.3%) [16]. With increased awareness of this issue, a broad array of studies, including observational and cohort studies, have associated vitamin D deficiency with several conditions including autoimmune diseases, cancer, diabetes, and myopathy [17]. Direct associations between myopathy and vitamin D deficiency were initially reported in the 1960s. Adults with osteomalacia (i.e., reduced bone mineralization due to vitamin D deficiency) displayed muscle weakness which improved following vitamin D supplementation [18]. Whether these effects represented direct functions of vitamin D in skeletal muscle function or indirectly, a result of vitamin D’s systemic effects on phosphate and calcium handling was uncertain. In the years since this paper, we have formed a greater understanding of this association and clinical studies have elucidated effects of vitamin D on muscle performance, function, and falls [19,20].

MUSCLE WEAKNESS AND VITAMIN D DEFICIENCY Weakness involving proximal muscle groups (i.e., large muscles in the pelvic girdle and quadriceps) was first reported in a group of adults with osteomalacia in the 1960s [18]. Specific proximal muscle defects are commonly described including difficulty standing, squatting or climbing a flight of stairs [18,21,22]. Changes in gait, often described as “waddling,” are widely reported and possibly related to proximal muscle weakness and pain [18,23]. It is unclear why large, proximal muscle groups are preferentially involved, but this pattern is not specific for vitamin D deficiency. Proximal myopathy is seen in a range of other endocrine and hormonal imbalances such as Cushing’s syndrome (cortisol excess), hyperthyroidism and in patients receiving long-term glucocorticoid therapy [24,25]. The reversible pattern of muscle weakness following vitamin D supplementation has been reported in a large number of case series [21,26,27]. In one report, progressive muscle weakness was described in veiled women from Saudi Arabia who were vitamin D deficient [90% had 25D , 20 nmol/L (8 ng/mL)], with a proportion requiring a wheel-chair [28]. Dramatic improvements followed 3 months of vitamin D and calcium supplementation were seen. Wheelchair-bound patients were able to walk without assistance by the end of the study. In another series, five patients with myopathy leading to wheelchair-use received vitamin D2 (50,000 IU weekly) that resulted in substantial improvements in muscle pain, strength, and mobility within a month. Electromyography (EMG) in vitamin D deficient subjects with weakness demonstrates myopathy without specific features [22]. In a paper from the 1970s, 15 women with

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nutritional osteomalacia demonstrated EMG changes including significantly shorter motor unit action potentials and larger proportion of polyphasic potentials as compared to controls [22]. Muscle biopsies demonstrated nonspecific muscle fiber atrophy. Complete resolution in these changes over a 5-week period of vitamin D supplementation was reported. In another study, two patients with osteomalacia demonstrated type 2 muscle fiber atrophy, as well as patchy necrosis and an abnormal intermyofibrillar network on muscle biopsy [29]. In 12 patients with osteomalacia, Young reported significant improvement in quadriceps strength and increases in the proportion of type II muscle fibers on biopsy after 3 months of vitamin D supplementation. More recently, Ceglia reported an increase in muscle fiber diameter and intramyonuclear VDR levels in response to vitamin D supplementation in a group of older, mobility-limited women [30]. In summary, these reports provide evidence that vitamin D deficiency results in a nonspecific proximal myopathy that can be severe, requiring a wheel-chair in most dramatic cases, but is reversible with supplementation. Biopsy studies suggest changes in muscle fiber proportion and fiber atrophy in vitamin D deficient subjects.

MUSCLE PAIN AND VITAMIN D DEFICIENCY Apart from proximal myopathy, vitamin D deficiency may also result in muscle pain (i.e., myalgia) (135 137). Whilst some studies report diffuse muscle pain in subjects with vitamin D deficiency [31 33], other studies do not support this and rather support proximal muscle symptoms [34 36]. The pain distribution is complicated by the possible presence of osteomalacia in subjects and associated bone pain due to microfractures. In a study of around 3000 men living in Europe, the description of chronic widespread pain (8.6%) was more likely in those with low vitamin D [ , 37.5 nmol/L (15 ng/mL)] [37]. However, upon adjusting for season, age, activity, and other factors, the association was weakened. Amongst 153 subjects living in USA who reported persistent musculoskeletal pain, the vast majority (94%) was vitamin D deficient (mean level 30 nmol/L [12 ng/mL]) [31]. Very high rates of vitamin D deficiency were reported amongst specific ethnic groups, namely, African-American, East-African, Hispanic, and American-Indian patients. In a study from the UAE, 86% of the patients who were initially diagnosed with fibromyalgia were vitamin D deficient. The majority of subjects reported improvement in muscle pain following vitamin D supplementation [38]. A case-control study from Australia compared eight urban Aboriginal patients who reported muscle pain with eight matched Aboriginal controls without pain [39]. There were significantly lower vitamin D levels amongst those with pain [41 versus 58 nmol/L (17 versus 23 ng/mL), P 5 0.017]. Wide cultural and gender differences in the reporting of pain, subjective features in the diagnosis of fibromyalgia, and the presence of other features known to affect vitamin D status amongst patients with persistent pain syndromes confound the assessment of the role of vitamin D deficiency in muscle pain. Also, the observational nature of these studies does not equip them to address the question of causality.

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In a randomized controlled trial (RCT) of 50 subjects with diffuse muscle pain [34], there was no benefit of vitamin D supplementation (vitamin D2 50,000 IU weekly) compared to placebo. However, 50% of the control group achieved normal vitamin D levels during the study, and on stratification by achieved vitamin D levels, improvement in pain scores were not different between groups. In another study of 138 patients with fibromyalgia, which is not a purely muscle disorder, patients with mild-to-moderate vitamin D deficiency [levels 25 62.5 nmol/L (0 25 ng/mL)] who received vitamin D3 supplementation (50,000 IU weekly) showed significant improvement over 8 weeks compared to controls. However, this effect did not persist at 12 months and those with severe vitamin D deficiency who received vitamin D in an unblinded fashion did not report any improvement at either 8 weeks or 1 year [40]. In summary, vitamin D deficiency with subsequent osteomalacia is associated with muscle pain that is reversible. The pain is mainly located in large proximal muscle groups rather than diffuse [18,41 43] and is often associated with bone pain and other features of myopathy including weakness [27,28].

VITAMIN D AND PHYSICAL PERFORMANCE MEASURES A variety of studies have assessed specific effects of vitamin D on physical performance measures and muscle function. Cross-sectional studies have demonstrated associations between vitamin D levels and handgrip and lower limb strength, balance and gait speed [44 46], but following multivariate adjustment, other studies have found no association [47,48]. Amongst B4000 adults, vitamin D levels correlated with lower-limb function assessed by 8-ft walk and the sit-to-stand test [49]. In people with vitamin D deficiency (level , 25 nmol/L), significantly poorer physical performance was demonstrated at baseline and higher rate of decline in muscle function compared to those with vitamin D . 75 nmol/L [50]. In a case-control study comparing 55 veiled Arab women with severe vitamin D deficiency and 22 Danish women with higher vitamin D levels, muscle function was significantly reduced in the Arab women at baseline [27]. Following repletion of vitamin D, the Arab women demonstrated dramatic improvements in muscle function and at 6 months, there was no difference in electrically stimulated muscle function versus controls. Subjective improvements in muscle pain were also reported following vitamin D treatment. Over a 3-month period, older subjects on vitamin D and calcium supplementation displayed significant improvements in knee flexor and extensor strength, grip strength, and timed-up-and-go (TUAG) test versus those randomized to calcium supplementation alone [51]. Significant improvements were noted in TUAG and quadriceps strength in another study with reduced falls following 1 year of vitamin D and calcium compared to calcium supplementation alone [51,52]. In 139 older subjects with falls, a single dose of vitamin D2 at 600,000 IU IM improved functional performance but did not affect falls or quadriceps strength at 6 months compared to controls [53]. Community-dwelling women in the lowest tertile of serum

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vitamin D who received vitamin D and calcium supplementation displayed the most pronounced improvements in lower limb muscle function over 1 year compared to those on calcium alone [54]. Several studies have failed to demonstrate improvements in muscle function with vitamin D regardless of baseline vitamin D status [55 59]. Amongst 243 frail, older subjects, there was no difference in physical performance in those receiving a single dose of 300,000 IU of vitamin D despite demonstrated increases in serum levels [55]. A study of 179 vitamin D deficient adolescent females in Lebanon found no effect on grip strength in those receiving vitamin D3 (doses 1400 or 14,000 IU/week), but there were improvements in lean mass after 1 year [59]. The addition of vitamin D supplementation to high resistance training resulted in improved TUAG but not quadriceps strength over 9 months [60]. Metaanalyses on effects of vitamin D have been hampered by great variability in the parameters of muscle function amongst studies, use of measures without established validity and lack of blinded assessment. A metaanalysis of 16 RCTs reported a greater number of studies showing lack of beneficial effect and the lack of obvious characteristics to differentiate studies with positive versus negative findings [61]. A metaanalysis of 13 RCTs involving elderly, vitamin D deficient subjects, reported improvements in lower limb strength and balance [62]. In summary, some studies suggest a beneficial effect of vitamin D supplementation on muscle function, especially in vulnerable populations and those with vitamin D deficiency [27,54]. However, the evidence-base is limited by heterogeneous studies that assess muscle strength by different techniques.

FALLS AND VITAMIN D DEFICIENCY Falls are a manifestation of vitamin D deficiency in older people who may be less likely to self-report muscle weakness and pain. Falls are common and troubling events in the lives of older people, leading to fracture, hospitalization and frailty. Falls are multifactorial, caused by muscle weakness, sarcopenia, postural instability, visual impairment, and a range of chronic conditions. Older people are also at risk of vitamin D deficiency as their skin becomes less efficient in the UV-mediated production of vitamin D sand muscle levels of VDR decline [63]. Observational studies have associated low vitamin D with the risk of falls in older people. A prospective study of B1500 institutionalized, older females reported 20% fewer falls with a doubling in vitamin D levels over 50 months [64]. Other studies have reported increases in the long-term risk of recurrent falls and decline in physical performance and muscle mass in older subjects with vitamin D deficiency [50,65,66]. However, these data are observational and vitamin D deficiency may be a surrogate marker for frailty as frail older subjects spend less time outdoors, receive less sun exposure, and may have malnutrition. In a RCT of 625 residents of assisted-living facilities, a significant reduction in falls over 2 years was reported in those receiving vitamin D2 and calcium (vitamin D: initially 10,000 IU weekly, then 1000 IU daily; calcium: 600 mg day) versus calcium alone [67]. In

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another study, vitamin D3 (800 IU/day) and calcium (1200 mg/day) reduced falls by 50% during the 3-month treatment period versus the preceding 6 weeks, as opposed to calcium alone [51]. Studies indicate that subjects who experience the greatest reduction in falls following vitamin D supplementation are the most vulnerable, older groups: residents of aged-care institutions, recurrent fallers [51], less mobile older women [68], and stroke sufferers [69]. However, the effects of vitamin D on falls incidence are not unanimous [70,71]. In a large study of B5000 older subjects, there was no effect of vitamin D3 on the rate of falls over 26 62 months [71]. Patients in this study had recently suffered a minimal-trauma fracture or were at high risk of fracture, suggesting that their baseline risk of falls and impaired mobility could not be overcome by a moderate daily dose of vitamin D (800 IU day) [70,71]. However, administration of vitamin D in infrequent “mega-doses” may actually increase falls risk. An oral mega-dose of vitamin D3 (500,000 IU day) increased the rate of falls in over 2000 community-dwelling older women [72]. The falls rate was highest in the 3 months following the dose, suggesting a temporal relationship. In a well-cited metaanalysis, vitamin D supplementation reduced the risk of falls in a dose-dependent fashion with a threshold dose of at least 700 IU daily [73,74]. Subjects with serum vitamin D levels above 60 nmol/L had B20% fewer falls. However, this metaanalysis relied on two studies to produce its positive dose-dependent effect and one of these studies was reportedly not sufficiently-powered [74]. In another large metaanalysis including . 45,000 people, the rate of falls was B20% lower amongst those randomized to receive vitamin D, but this was not dose-dependent and not difference in effect was seen in people living in the community versus age-care facilities [19]. The greatest benefit was seen however in those with baseline vitamin D deficiency and with calcium coadministration. In a metaanalysis of the US Preventative Task Force, a 17% reduction in the incidence of falls was seen in response to vitamin D and its analogues (calcitriol and alfacalcidol) [75], whilst another recent metaanalysis of 20 RCTs demonstrated an insignificant 5% reduction in falls in older individuals [76]. In summary, vitamin D deficiency leads to falls in older subjects and supplementation generally reduces the risk of falls in the most vulnerable older groups with the exception of a significantly higher risk of falls in subjects receiving single mega-doses.

CONCLUSIONS This chapter has discussed the myopathy of vitamin D deficiency, focusing on vitamin D’s effects on muscle strength, muscle pain, physical performance and falls. Severe vitamin D deficiency is clearly detrimental for muscle function, results in proximal muscle weakness, myalgia and a greater risk of falls. These features are reversible with vitamin D supplementation. However, interventional trials and metaanalyses in subjects with less severe vitamin D deficiency have found mixed results. Validation of physical performance parameters and the definition of sarcopenia remain unclear making standardization of outcome measures difficult [77,78]. Regarding falls, vitamin D supplementation is effective in vulnerable older subjects, such as those living in aged-care facilities but not excessive,

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single mega-doses which appear harmful [79]. Biological mechanisms indicate the presence of VDR in muscle, a role in intramuscular calcium handling and the modulation of muscle fiber size. Further research will help to clarify optimal vitamin D supplemental doses for muscle health. In the meantime, people with severe vitamin D deficiency [ , 25 nmol/L (10 ng/mL)] are at risk of muscle dysfunction and would certainly benefit from supplementation.

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[20] Stockton KA, Mengersen K, Paratz JD, Kandiah D, Bennell KL. Effect of vitamin D supplementation on muscle strength: a systematic review and meta-analysis. Osteoporos Int. 2011;22(3):859 71. [21] Russell JA. Osteomalacic myopathy. Muscle Nerve 1994;17(6):578 80. [22] Irani PF. Electromyography in nutritional osteomalacic myopathy. J Neurol Neurosurg Psychiatry 1976;39 (7):686 93. [23] Schott GD, Wills MR. Muscle weakness in osteomalacia. Lancet 1976;1(7960):626 9. [24] Floyd M, Ayyar DR, Barwick DD, Hudgson P, Weightman D. Myopathy in chronic renal failure. Q J Med 1974;43(172):509 24. [25] Amstrup AK, Rejnmark L, Vestergaard P, Sikjaer T, Rolighed L, Heickendorff L, et al. Vitamin D status, physical performance and body mass in patients surgically cured for primary hyperparathyroidism compared with healthy controls—a cross-sectional study. Clin Endocrinol (Oxf) 2011;74(1):130 6. [26] Ziambaras K, Dagogo-Jack S. Reversible muscle weakness in patients with vitamin D deficiency. West J Med 1997;167(6):435 9. [27] Glerup H, Mikkelsen K, Poulsen L, Hass E, Overbeck S, Andersen H, et al. Hypovitaminosis D myopathy without biochemical signs of osteomalacic bone involvement. Calcif Tissue Int 2000;66(6):419 24. [28] Al-Said YA, Al-Rached HS, Al-Qahtani HA, Jan MM. Severe proximal myopathy with remarkable recovery after vitamin D treatment. Can J Neurol Sci 2009;36(3):336 9. [29] Yoshikawa S, Nakamura T, Tanabe H, Imamura T. Osteomalacic myopathy. Endocrinol Jpn 1979;26 (Suppl):65 72. [30] Ceglia L, Niramitmahapanya S, Morais MD, Rivas DA, Harris SS, Bischoff-Ferrari H, et al. A randomized study on the effect of vitamin D3 supplementation on skeletal muscle morphology and vitamin D receptor concentration in older women. J Clin Endocrinol Metab 2013;98(12):E1927 35. [31] Plotnikoff GA, Quigley JM. Prevalence of severe hypovitaminosis D in patients with persistent, nonspecific musculoskeletal pain. Mayo Clinic Proc 2003;78(12):1463 70. [32] Macfarlane G, Palmer B, Roy D, Afzal C, Silman A, O’Neill T. An excess of widespread pain among South Asians: are low levels of vitamin D implicated? Ann Rheum Dis 2005;64(8):1217 19. [33] Mouyis M, Ostor AJK, Crisp AJ, Ginawi A, Halsall DJ, Shenker N, et al. Hypovitaminosis D among rheumatology outpatients in clinical practice. Rheumatology (Oxford) 2008;47(9):1348 51. [34] Warner AE, Arnspiger SA. Diffuse musculoskeletal pain is not associated with low vitamin D levels or improved by treatment with vitamin D. J Clin Rheumatol 2008;14(1):12 16. [35] Block SR, editor. Vitamin D deficiency is not associated with nonspecific musculoskeletal pain syndromes including fibromyalgia. Minnesota: Mayo Clinic; 2004. [36] Myers KJ, editor. Vitamin D deficiency and chronic pain: cause and effect or epiphenomenon? Minnesota: Mayo Clinic; 2004. [37] McBeth J, Pye SR, O’Neill TW, Macfarlane GJ, Tajar A, Bartfai G, et al. Musculoskeletal pain is associated with very low levels of vitamin D in men: results from the European Male Ageing Study. Ann Rheum Dis 2010;69(8):1448 52. [38] Badsha H, Daher M, Ooi Kong K. Myalgias or non-specific muscle pain in Arab or Indo-Pakistani patients may indicate vitamin D deficiency. Clin Rheumatol 2009;28(8):971 3. [39] Benson J, Wilson A, Stocks N, Moulding N. Muscle pain as an indicator of vitamin D deficiency in an urban Australian Aboriginal population. Med J Aust 2006;185(2):76 7. [40] Arvold DS, Odean MJ, Dornfeld MP, Regal RR, Arvold JG, Karwoski GC, et al. Correlation of symptoms with vitamin D deficiency and symptom response to cholecalciferol treatment: a randomized controlled trial. Endocr Pract. 2009;15(3):203 12. [41] Reginato AJ, Coquia JA. Musculoskeletal manifestations of osteomalacia and rickets. Best Pract Res Clin Rheumatol 2003;17(6):1063 80. [42] Gloth III FM, Lindsay JM, Zelesnick LB, Greenough III WB. Can vitamin D deficiency produce an unusual pain syndrome? Arch Intern Med 1991;151(8):1662. [43] Nellen JF, Smulders YM, Jos Frissen PH, Slaats EH, Silberbusch J. Hypovitaminosis D in immigrant women: slow to be diagnosed. BMJ. 1996;312(7030):570 2. [44] Boxer RS, Dauser DA, Walsh SJ, Hager WD, Kenny AM. The association between vitamin D and inflammation with the 6-minute walk and frailty in patients with heart failure. J Am Geriatr Soc 2008;56 (3):454 61.

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[67] Flicker L, MacInnis RJ, Stein MS, Scherer SC, Mead KE, Nowson CA, et al. Should older people in residential care receive vitamin D to prevent falls? Results of a randomized trial. J Am Geriatr Soc 2005;53(11):1881 8. [68] Bischoff-Ferrari HA, Orav EJ, Dawson-Hughes B. Effect of cholecalciferol plus calcium on falling in ambulatory older men and women: a 3-year randomized controlled trial. Arch Intern Med 2006;166(4):424 30. [69] Sato Y, Iwamoto J, Kanoko T, Satoh K. Low-dose vitamin D prevents muscular atrophy and reduces falls and hip fractures in women after stroke: a randomized controlled trial. Cerebrovasc Dis. 2005;20(3):187 92. [70] Porthouse J, Cockayne S, King C, Saxon L, Steele E, Aspray T, et al. Randomised controlled trial of calcium and supplementation with cholecalciferol (vitamin D3) for prevention of fractures in primary care. Br Med J 2005;330(7498):1003. [71] Grant AM, Avenell A, Campbell MK, McDonald AM, MacLennan GS, McPherson GC, et al. Oral vitamin D3 and calcium for secondary prevention of low-trauma fractures in elderly people (Randomised Evaluation of Calcium Or vitamin D, RECORD): a randomised placebo-controlled trial. Lancet. 2005;365(9471):1621 8. [72] Sanders KM, Stuart AL, Williamson EJ, Simpson JA, Kotowicz MA, Young D, et al. Annual high-dose oral vitamin D and falls and fractures in older women: a randomized controlled trial. J Am Med Assoc 2010;303 (18):1815 22. [73] Bischoff-Ferrari HA, Dawson-Hughes B, Staehelin HB, Orav JE, Stuck AE, Theiler R, et al. Fall prevention with supplemental and active forms of vitamin D: a meta-analysis of randomised controlled trials. Br Med J 2009;339:b3692. [74] Ross A, Taylor C, Yaktine A, Del Valle H. Dietary reference intakes for calcium and vitamin D. Institute of Medicine report. Institute of Medicine (US) Committee to Review Dietary Reference Intakes for Vitamin D and Calcium. Washington DC. The National Academies Press; 2011. [75] Michael YL, Whitlock EP, Lin JS, Fu R, O’Connor EA, Gold R, et al. Primary care-relevant interventions to prevent falling in older adults: a systematic evidence review for the U.S. Preventive Services Task Force. Ann Intern Med 2010;153(12):815 25. [76] Bolland MJ, Grey A, Gamble GD, Reid IR. Vitamin D supplementation and falls: a trial sequential metaanalysis. Lancet Diabetes Endocrinol 2014;2(7):573 80. [77] Girgis CM. Integrated therapies for osteoporosis and sarcopenia: from signaling pathways to clinical trials. Calcif Tissue Int 2015;96(3):243 55. [78] Girgis CM, Mokbel N, Digirolamo DJ. Therapies for musculoskeletal disease: can we treat two birds with one stone? Curr Osteoporos Rep 2014;12(2):142 53. [79] Girgis CM. Vitamin D and muscle function in the elderly: the elixir of youth? Curr Opin Clin Nutr Metab Care 2014;17(6):546 50.

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Phytochemicals, Their Intestinal Metabolites, and Skeletal Muscle Function 1

Kazumi Yagasaki1,2 Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya, Tochigi, Japan 2Department of Applied Biological Chemistry, Tokyo University of Agriculture and Technology, Tokyo, Japan

LIST OF ABBREVIATIONS AMPK GLUT4 PEPCK G6Pase GS LGP ACC FAS SCD GIN ENL HOMA-IR IPGTT GRE

50 -adenosine monophosphate-activated protein kinase glucose transporter 4 phosphoenolpyruvate carboxykinase glucose-6-phosphatase glycogen synthase liver glycogen phosphorylase acetyl-CoA carboxylase fatty acid synthase stearoyl-CoA desaturase 1 [6]-gingerol enterolactone homeostasis model assessment of insulin resistance intraperitoneal glucose tolerance test green rooibos extract

INTRODUCTION Skeletal muscles are the largest tissue in our body. The tissue has two important roles. The first one is food materials as important sources of proteins and hence amino acids, while the other one is contributors involved in metabolic regulation of main nutrients such as proteins/ amino acids, lipids, and glucose. As to glucose metabolism, the skeletal muscles account for

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the majority (B75%) of insulin-mediated glucose uptake in the postprandial state, indicating that the skeletal muscles play an important role in maintaining glucose homeostasis [1]. The number of diabetic patients is increasing globally due to population growth, aging, urbanization, and increasing physical inactivity and prevalence of obesity [2]. According to the recent estimation by International Diabetes Federation, the number of people with diabetes worldwide is 415 million in 2015 and 642 million in 2040, and in high-income countries, up to 91% of adults with the disease have type 2 diabetes (T2D) [3]. In skeletal muscles, insulin stimulates glucose uptake through activation of phosphatidylinositol-3-kinase and Akt (protein kinase B, commonly known as Akt), this leading to increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane [4]. Another GLUT4 translocation promoter is 50 -adenosine monophosphate-activated protein kinase (AMPK) [5]. In mammalian cells, AMPK activated by an increase in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio plays a role as an energy sensor [5]. AMPK is activated by exercise/muscle contraction [6] and compounds such as metformin [7], leading to promotion of GLUT4 translocation to plasma membrane and hence glucose uptake in skeletal muscle [8]. As was assumed earlier [9,10], novel compounds that activate skeletal muscle glucose uptake and AMPK would be of use for the development of new treatment of insulin resistance and T2D. Defective insulin signaling in insulin-resistant states could be bypassed by actions of AMPK to promote skeletal muscle glucose uptake and suppress hepatic glucose production via insulin-independent mechanisms [10]. In this chapter, we will briefly describe the important role of AMPK activation by edible phytochemicals and their intestinal metabolites in glucose uptake of skeletal muscle cells and tissues.

CULTURED SKELETAL MUSCLE CELLS AND INCUBATED MUSCLE TISSUES AS USEFUL BIOASSAY SYSTEMS TO SEARCH FOR NOVEL BIOACTIVE FACTORS In the early stages of our studies on muscles, effects of edible plant components on the proliferation, DNA, and protein syntheses were estimated in L6 myoblasts, a rat-skeletal musclederived cell line [11]. In the course of experiments, isopentenyladenine, a member of one class of “plant” hormones, i.e., cytokinins, was found to simulate the syntheses of DNA and protein, and hence, the proliferation of L6 myoblasts that come from the “animal” kingdom at 5 μM [12] in a serum-free medium [muscle medium-1 (MM-1)] [13]. This interesting and valuable experience prompted us later to search for novel phytochemicals that would stimulate glucose uptake in L6 myocytes (myoblasts and myotubes) [14]. L6 myoblasts are known to differentiate to L6 myotubes through spontaneous cell fusion, and L6 myotubes, like C2C12 myotubes, are often employed for various metabolic studies instead of muscle tissues [14]. In the meanwhile, we studied stimulatory actions of some amino acids on protein syntheses and their mechanisms in L6 myotubes, focusing our attention on basic amino acids, especially L-histidine [15], and branched-chain amino acids, especially L-leucine [16,17]. Besides these studies, effect of L-leucine on glucose uptake in incubated skeletal muscles of rats (soleus muscles) was studied, employing [1-14C] 2-deoxy-D-glucose and [1-3H] L-glucose [18] by the procedure of Stenbit et al. [19] with slight modifications. In this assay,

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2-deoxy-D-glucose binds to GLUT4 and enters muscle cells without further metabolism. In contrast, L-glucose neither binds to transporter nor enters the cells but causes nonspecific adhesion to cell or tissue surface. At the end of the incubation period, the muscles were rinsed with radioisotope-free buffer, hydrolyzed in NaOH, neutralized with HCl, and centrifuged. Aliquots of the supernatant were transferred to mini vials, mixed with a scintillator, and 14C and 3H were counted simultaneously. A separate aliquot of the supernatant was used for protein determination. Amounts of 2-deoxy-D-glucose and L-glucose (nmol/mg protein) in soleus muscles were calculated by dividing radioactivities of 14C and 3H by specific radioactivities of [1-14C] 2-deoxy-D-glucose and [1-3H] L-glucose, respectively. Net glucose uptake was determined by subtracting the amount of L-glucose from that of 2-deoxy-D-glucose [18].

GLUCOSE UPTAKE ASSAY IN CULTURED L6 MYOTUBES FOR INITIAL SCREENING OF NOVEL PHYTOCHEMICALS Although the abovementioned procedure is precise and reliable, we have to prepare soleus muscles from animals every time when conducting experiments. Thus, we have contrived a simple, rapid, and relatively inexpensive glucose uptake assay system without using radioisotopes for first screening of food and natural substances that promote glucose uptake in muscles, by adopting cultured L6 myotubes [20,21] and, in some cases, L6 myoblasts [22] as illustrated in Fig. 25.1. Briefly, the prefused cells (5myoblasts,

FIGURE 25.1 Outline of an example of glucose uptake assay in cultured L6 myocytes. L6 myoblasts were cultured and grown for 11 days to form myotubes in 10% or 10% then 2% FBS/DMEM. The 11-day-old myotubes were kept for 2 h in KHH buffer, and then they were cultured in KHH buffer containing 11 mM glucose without or with samples for appropriate time intervals. FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle’s Medium; KHH buffer, KrebsHenseleitHepes buffer. Source: Originally drawn by taking Ref. [14] into account. III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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5 3 104 cells/well) were subcultured into 24-place multiwell plates and grown for 11 days to form myotubes in 0.4 mL of 10% or 10% then to 2% fetal bovine serum/ Dulbecco’s Modified Eagle’s Medium. The medium was renewed every 3 days. Later, the 11-day-old myotubes were kept for 2 h in filter-sterilized KrebsHenseleit buffer (pH 7.4, 141 mg/L MgSO 4, 160 mg/L KH2PO4, 350 mg/L KCl, 6900 mg/L NaCl, 373 mg/L CaCl2  2H2O, and 2100 mg/L NaHCO3) containing 0.1% bovine serum albumin, 10 mM Hepes and 2 mM sodium pyruvate [KrebsHenseleitHepes (KHH) buffer]. Then, the myotubes were cultured in KHH buffer containing 11 mM glucose without or with test compound (usually 0100 μM) for another 4 h in the absence of insulin. Differences in glucose concentrations between before and after culture were determined with a microplate reader and a glucose detecting kit, and the amounts of glucose consumed were calculated. The myotubes and myoblasts in this system are helpful to clarify mechanisms in vitro by which phytochemicals promote glucose uptake. Abovementioned in vitro assay has been carried out in the absence of insulin. If a phytochemical tested activates AMPK and promotes GLUT4 translocation to plasma membrane and hence glucose uptake in muscle cells without insulin, then the phytochemical has a potential to overcome insulin resistance in vivo. Natural compounds that activate AMPK in skeletal muscles have a possibility to suppress rises in blood glucose levels even in the diabetic state with insulin resistance (Fig. 25.2).

FIGURE 25.2 Schematic representation of signaling pathways leading to GLUT4 translocation through Akt and/or AMPK activation in muscle cells. GLUT4, glucose transporter 4; AMPK, 50 -adenosine monophosphateactivated protein kinase. Modified by taking Huang S, Czech MP. The GLUT4 glucose transporter. Cell Metab 2007;5:23752; Yagasaki K. Anti-diabetic phytochemicals that promote GLUT4 translocation via AMPK signaling in muscle cells. Nutr Aging 2014;2:3544; and Park SJ, Ahmad F, Philp A, Baar K, Williams T, Luo H, et al. Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell 2012;148:42133 into account.

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PHYTOCHEMICALS, GLUCOSE METABOLISM IN SKELETAL MUSCLE CELLS AND THEIR EFFECTS ON T2D MODEL ANIMALS Stilbenoids, Muscle Cell Glucose Uptake, and Glucose Metabolism in T2D Model Mice Of stilbenoids, resveratrol and piceatannol are well known (Fig. 25.3). Especially, resveratrol has been reported to possess important health beneficial effects such as anticancer and antiinflammatory [23], lifespan extension [24], and antidepressant effects [25]. Piceatannol, a naturally occurring hydroxylated analog of resveratrol, is less studied than resveratrol but displays a wide spectrum of biological activity [26]. In the earlier studies, we could find hypolipidemic effect of resveratrol in nephritic [27] and hepatoma-bearing rats [28], antiinvasive effect of resveratrol on hepatoma cells [2931], antiproliferative effect of pterostilbene, a stilbenoid, on hepatoma cells [32], and antiproliferative as well as antiinvasive effect of piceatannol against hepatoma AH109A cells [33]. Resveratrol was applied to the abovementioned glucose uptake assay and found to dose-dependently promote glucose uptake in L6 myotubes at doses 1100 μM [21]. It stimulated GLUT4 translocation by activating both insulin signaling and AMPK signaling, i.e., increases in phosphorylated Akt (p-Akt)/total Akt ratio (p-Akt/Akt) and phosphorylated AMPK (p-AMPK)/total AMPK ratio (p-AMPK/AMPK). Resveratrol could protect cultured pancreatic β-cells (RIN-5F) from advanced glycation end products (AGEs)-induced oxidative stress and apoptosis. Resveratrol significantly suppressed the elevation in the fasting blood glucose (FBG) level and the serum triglyceride and lipid peroxide measured as thiobarbituric acid reactive substances (TBARS) levels in T2D model db/db mice. Resveratrol is suggested to show antidiabetic effect by stimulating both insulin-dependent and -independent glucose uptake in muscles and by protecting pancreatic β-cells from AGEs-induced oxidative stress and apoptosis.

FIGURE 25.3 Structure of resveratrol and piceatannol.

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Piceatannol, a resveratrol derivative, was found to promote glucose uptake (25100 μM), AMPK phosphorylation, and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence [22]. Promotion by piceatannol of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a glut4 cDNA-coding vector (designated as pFN21A-rat glut4). In this study, L6 myoblasts were used for the transfection of pFN21A-rat glut4 and detection of rat GLUT4, because the transfection efficiency into L6 myotubes was very low. This transfection method made GLUT4 translocation to plasma membrane visible by immunocytochemical staining (Fig. 25.4). Piceatannol suppressed the rises in blood glucose levels at early stages and improved the impaired glucose tolerance at late stages in db/db mice. These in vitro and in vivo findings suggest that piceatannol may be preventive and remedial for T2D and useful as an antidiabetic phytochemical.

Aspalathin, Muscle Cell Glucose Uptake and Glucose Metabolism in T2D Model Mice Aspalathin is a major C-glucosyl flavonoids of rooibos, the endemic South African legume, Aspalathus linearis (Fig. 25.5). Diverse flavonoids have been identified and quantified in fermented rooibos tea [34,35] and unfermented rooibos tea [36]. The main flavonoid in unfermented rooibos aqueous extract was aspalathin (49.92 mg/g), which occupied 84.5% of the total flavonoid contents (59.08 mg/g), while aspalathin content in fermented rooibos aqueous extract (1.243 mg/g) was 22.4% of the total flavonoid contents (5.521 mg/g) [35,36]. The total antioxidant activity of unfermented rooibos was twice as high as that of fermented rooibos [36]. Aspalathin dose-dependently increased glucose uptake in L6 myotubes at doses 1100 μM (Fig. 25.5A) and improved glucose intolerance in db/db mice (Fig. 25.5B). Aspalathin promoted insulin secretion in cultured pancreatic β-cells (RIN-5F) at 100 μM and suppressed the increases in FBG levels in db/db mice [20]. Aspalathin promoted AMPK phosphorylation but not Akt phosphorylation in L6 myotubes and it was demonstrated to enhance GLUT4 translocation to plasma membrane in L6 myoblasts transfected with a glut4 cDNA-coding vector, pFN21A-rat glut4 [37]. In RIN5F cells, aspalathin suppressed AGE-induced rises in reactive oxygen species (ROS) [37]. In ob/ob mice, aspalathin significantly suppressed the increase in FBG levels and improved glucose intolerance. Furthermore, the phytochemical suppressed the increases in the levels of serum TBARS, serum and liver triglyceride, and serum tumor necrosis factor-α (TNF-α) in ob/ob mice as compared with normal mice, while aspalathin normalized the serum adiponectin level reduced in the diabetic state [37]. In addition, aspalathin reduced the gene expression of hepatic enzymes related to glucose production (phosphoenolpyruvate carboxykinase, PEPCK; liver glycogen phosphorylase, LGP) and increased that of glucose storage (glycogen synthase, GS). Aspalathin decreased the expression of hepatic genes related to lipogenesis (acetyl-coenzyme A carboxylase 1; fatty acid synthase; stearoyl-coenzyme A desaturase 1) in ob/ob mice [37].

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FIGURE 25.4 Effect of piceatannol on glucose uptake (A) and cellular GLUT4 localization by bioimaging (B) in L6 myoblasts transfected with pFN21A-mock vector or pFN21A-glut4 vector. (A) Glucose uptake for 4 h was measured without or with piceatannol (0, 100 μM) in L6 myoblasts 48 h after transfection of pFN21A-mock vector or pFN21A-glut4 vector. Each value and bar represents the mean 6 SEM of four wells. Values not sharing a common letter are significantly different at P , 0.05 by TukeyKramer multiple comparisons test. (B) L6 myoblasts stably expressing proteins of HaloTag (Mock PIC 2 and Mock PIC1 ) or Halo-GLUT4 (GLUT4 PIC 2 and GLUT4 PIC 1 ) were processed for immunocytochemistry using anti-HaloTag antibody and anti-caveolin-3 antibody. Cellular localization of HaloTag and HaloTag-GLUT4 is shown in red fluorescence (left) and that of caveolin-3 is shown in green fluorescence (center). Merged image is also shown (right). L6 myoblasts (Mock PIC 1 and GLUT4 PIC 1 ) were exposed to piceatannol (100 μM) for 30 min. SEM, standard error of mean; PIC, piceatannol. Cited from Minakawa M, Miura Y, Yagasaki K. Piceatannol, a resveratrol derivative, promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and suppresses blood glucose levels in type 2 diabetic model db/db mice. Biochem Biophys Res Commun 2012;422:46975 with permission.

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FIGURE 25.5 Effect of aspalathin on glucose uptake in L6 myotubes (A) and glucose intolerance in db/db mice (B). (A) Stimulatory effect of aspalathin on glucose uptake by cultured L6 myotubes was conducted as indicated in Fig. 25.1. Each value represents the mean 6 SEM of six wells. Values not sharing a common letter are significantly different at P , 0.05 by TukeyKramer multiple comparisons test. (B) IPGTT was performed after the final determination of fasting blood glucose level. Diabetic db/db mice of 11 weeks of age were fasted for 15 h and given orally aspalathin at a dose of 20 mg/mL/100 g body weight (ASP) or water (1 mL/100 g body weight) (CNT). Two hours later, blood was collected from the tail vein of db/db mice (0 min). Immediately after blood collection, diabetic mice received an intraperitoneal injection of glucose at a dose of 0.2 g/mL/100 g body weight. Blood samples were successively collected at 30, 60, 90, and 120 min after glucose injection, and blood glucose levels were determined. Each value represents the mean 6 SEM of six (CNT) or four (ASP) mice. Values not sharing a common letter are significantly different at P , 0.05 by Student’s t-test. IPGTT, Intraperitoneal glucose tolerance test. SEM, standard error of mean; CNT, control group; ASP, aspalathin group. Cited from Kawano A, Nakamura H, Hata S, Minakawa M, Miura Y, Yagasaki K. Hypoglycemic effect of aspalathin, a rooibos tea component from Aspalathus linearis, in type 2 diabetic model db/db mice. Phytomedicine 2009;16:43743 with permission.

In summary, hypoglycemic effect of aspalathin is related to increased GLUT4 translocation to plasma membrane via AMPK activation. In addition, aspalathin reduces the gene expression of hepatic enzymes related to glucose production and lipogenesis. These results strongly suggest that aspalathin has an antidiabetic potential. As already noted, green (“unfermented”) rooibos has been shown to contain more aspalathin than “fermented” rooibos tea. We investigated the antidiabetic effect of green rooibos extract (GRE) [38]. GRE increased glucose uptake under insulin-absent condition and induced phosphorylation of AMPK in L6 myotubes as previously demonstrated for

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aspalathin. In addition to AMPK, GRE also promoted phosphorylation of Akt in L6 myotubes unlike aspalathin, suggesting an involvement of GRE component(s) other than aspalathin in Akt phosphorylation. Promotion of GLUT4 translocation to the plasma membrane by GRE in L6 myotubes was demonstrated by Western blotting analysis. GRE suppressed AGEs-induced increase in ROS levels in RIN-5F pancreatic β-cells. Subchronic feeding of GRE suppressed the rise in FBG levels in type 2 diabetic model KK-Ay mice. These in vitro and in vivo results strongly suggest that GRE has antidiabetic potential through multiple modes of action. These results suggest that aspalathin may possess both preventive and/or remedial actions on hyperglycemia in db/db, ob/ob, and KK-Ay mice. Potential of rooibos in prevention of metabolic syndrome has been reviewed recently [39].

Nepodin, Gingerol, Muscle Cell Glucose Uptake, and Glucose Metabolism in T2D Model Mice Nepodin, acetyl-1,8-dihydroxy-3-methylnaphthalene, is a component found from the root of Rumex japonicus [40]. R. japonicus are very common perennial herbs growing mainly in East Asia, including China, Japan, and Korea. Young leaves of R. japonicus have been eaten as an edible wild plant and its infusion has been drunk as a tea. It has traditionally been used for the treatment of acute and chronic cutaneous diseases, constipation, jaundice, uterine hemorrhage, and hematemesis [41,42]. Antioxidant activity and antibacterial activity of R. japonicus have also been reported [43]. However, antidiabetic action of nepodin is poorly understood. We could demonstrate that nepodin stimulated glucose uptake dose-dependently in L6 myotubes. The stimulatory effect of nepodin on glucose uptake was abrogated by an AMPK inhibitor, compound C, suggesting a possible involvement of AMPK in glucose uptake in L6 myotubes. In fact, nepodin stimulated the phosphorylation of AMPK. Nepodin stimulated the translocation of GLUT4 to the plasma membrane in L6 myoblasts transfected with a glut4 cDNA-coding vector and in differentiated L6 myotubes (Western blotting analysis) [44]. In in vivo study, nepodin suppressed the increases in FBG levels and improved the glucose intolerance of T2D model db/db mice. Nepodin rescued the impaired phosphorylation of AMPK in the skeletal muscle (gastrocnemius muscle) of db/db mice. These results suggest that nepodin has an antidiabetic potential [44]. Ginger, the rhizome of the plant Zingiber officinale Roscoe, is widely used in several food products and also in many types of pharmaceutical formulations [45]. Among the various derivatives of ginger, [6]-gingerol (GIN) has been identified as, for example, antioxidant, antiinflammatory, and anticancer principle [46,47]. The antidiabetic effect of GIN and its mechanisms was investigated through studies on glucose uptake in L6 myocytes and on pancreatic β-cell protective ability from ROS in RIN-5F cells. The in vivo effect of GIN was also examined in obese T2D db/db model mice [48]. GIN promoted dosedependently (25100 μM) glucose uptake in L6 myotubes and the rise in glucose uptake at 50 μM of GIN was entirely canceled by compound C, this linking to AMPK activation (5phosphorylation) in the myotubes and hence promotion of GLUT4 translocation to plasma membrane of L6 myoblasts that was transfected with a glut4 cDNA-coding vector. GIN protected RIN-5F pancreatic β-cells from oxidative stress by suppressing AGEsinduced ROS production. In db/db mice, GIN treatment suppressed the elevation of FBG

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levels and improved glucose intolerance in the diabetic state. It also suppressed the increases in serum triglyceride, cholesterol, TBARS, TNF-α levels. In addition, GIN regulated hepatic gene expression of enzymes related to glucose metabolism toward decreases in gluconeogenesis (PEPCK), glucogenesis (glucose-6-phosphatase), and glycogenolysis (LGP) as well as an increase in glycogenesis (GS), thereby contributing to reductions in hepatic glucose production and hence blood glucose concentrations. TNF-α is involved in insulin resistance in skeletal muscle cells [49,50]. Thus, its reduction in the serum of obese diabetic db/db mice by GIN may be associated, at least partly, with the reduction of insulin resistance and blood/serum glucose levels in db/db mice. These in vitro and in vivo findings strongly suggest that GIN has antidiabetic potential through multiple mechanisms. Beneficial effects of ginger on metabolic syndrome have been reviewed recently [51].

Phytochemicals, Their Microbial Metabolites, Muscle Cell Glucose Uptake, and Glucose Metabolism in T2D Model Mice Soy isoflavones, daidzin and daidzein, are known to converted to equol, which was first isolated from equine urine in 1932 and later in human urine as a metabolite of the isoflavones [52]. Equol is produced by intestinal bacteria in some, but not all, adults. This observation led to the term equol-producers and equol-nonproducers. Another isoflavone, genistein, is converted to 5-hydroxy-equol by human intestinal Slackia isoflavoniconvertens [53,54]. Lignans are also converted to enterolactone (ENL) by intestinal microbiota [55]. The mammalian lignans, ENL and enterodiol, are commonly found in blood and urine of pigs [56] and humans [57]. They are formed by the conversion of dietary precursor lignans from, for instance, strawberry [58] and rye [56]. Interestingly, lignins have been also demonstrated to be precursors of mammalian lignans in rats [59]. Hydroxymatairesinol, one of the lignans, from Norway spruce was reported to be metabolized to ENL and found in urine as the major metabolite in rats after oral administration [60]. Hydroxymatairesinol decreased the number of growing tumors and increased the proportion of regressing and stabilized tumors in the rat dimethylbenz[a]anthracene-induced mammary tumor model [60]. ENL also has been shown to inhibit or delay the growth of experimental mammary cancer [61]. Hydroxymatairesinol and ENL have been found to possess antiproliferative and antiinvasive activities against a rat ascites hepatoma cell line of AH109A in culture and hepatoma-bearing rats [62]. Recently, ellagitannins are shown to be metabolized to small molecules by gut microbiota in humans [63]. Of these phytochemicals and metabolites, antidiabetic effects of isoflavones, its metabolite equol and lignan metabolite ENL are described below (Fig. 25.6). Genistein promoted dose-dependently glucose uptake, phosphorylation of AMPK, and GLUT4 translocation to plasma membrane independently of insulin in cultured L6 myotubes [64]. This isoflavone might regulate glucose uptake by increasing the phosphorylation and decreasing the O-GlcNAcylation of proteins related to glucose homeostasis in L6 myotubes. It also increased insulin secretion from RIN-5F pancreatic β-cells. Genistein suppressed the rises in blood glucose levels and urinary glucose excretion in T2D model KK-Ay mice, leading to its antidiabetic effects.

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FIGURE 25.6 Structure of metabolites of isoflavones and lignans.

Daidzein promoted glucose uptake, AMPK phosphorylation, and GLUT4 translocation to plasma membrane by Western blotting analyses in L6 myotubes under a condition of insulin absence [65]. Promotion by daidzein of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a glut4 cDNA-coding vector. Daidzein (0.1% in the diet) suppressed the increases in the FBG levels (Fig. 25.7A), serum total cholesterol levels, and homeostasis model assessment of insulin resistance (HOMA-IR) of db/db mice. In addition, daidzein supplementation markedly improved the AMPK phosphorylation in gastrocnemius muscle of db/db mice (Fig. 25.7B), indicating that the isoflavone activated AMPK in vivo as well as in vitro. Daidzein also suppressed increases in blood glucose levels (Fig. 25.8A) and urinary glucose excretion (Fig. 25.8B) in KK-Ay mice, another type 2 diabetic animal model [65]. These in vitro and in vivo findings suggest that daidzein is preventive for T2D and possesses antidiabetic potential. Equol promoted significantly glucose uptake at very low doses (0.251 μM) in L6 myotubes. It also promoted AMPK phosphorylation and GLUT4 translocation detected by Western blotting analyses in L6 myotubes at 1 μM under a condition of insulin absence [66]. Equol at a low content of 0.05% in diet suppressed the rise in serum glucose, cholesterol, triglyceride, and lipid peroxide concentrations and the hepatic triglyceride level in T2D model ob/ob mice as compared with those in the control mice. Moreover, equol treatment suppressed the rises in FBG level and improved the glucose intolerance in ob/ob mice. Furthermore, equol treatment improved expression of hepatic gluconeogenesis- and

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FIGURE 25.7 Effect of daidzein on (A) fasting blood glucose levels and (B) AMPK phosphorylation in gastrocnemius muscle of db/db mice. (A) The db/db mice were fasted for 3 h before the collection of blood. Blood samples were collected from tail vein. Each value represents the mean 6 SEM of six mice. Values not sharing a common letter are significantly different at P , 05 by TukeyKramer multiple comparisons test. (B) Homogenates of gastrocnemius muscle were subjected to SDSPAGE and Western blotting analysis using antiphospho-AMPK and anti-AMPK antibodies. Each value represents the mean 6 SEM of three mice. Values not sharing a common letter are significantly different at P , 0.05 by TukeyKramer multiple comparisons test. SEM, standard error of mean; SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis; NOR, normal group; CNT, control group; DAI, daidzein group. Cited from Cheong SH, Furuhashi K, Ito K, Nagaoka M, Yonezawa T, Miura Y, et al. Daidzein promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and improves glucose homeostasis in Type 2 diabetic model mice. J Nutr Biochem 2014;25:13643 with permission.

lipogenesis-related genes in terms of glucose and lipid metabolism. Thus, equol is antidiabetic at lower doses than is daidzein [66]. Urinary concentrations of lignan metabolites are reported to be significantly associated with a lower risk of T2D [67]. ENL dose-dependently increased glucose uptake in L6 myotubes at doses 25100 μM under insulin-absent condition [68]. Activation (5phosphorylation) by ENL of AMPK and translocation of GLUT4 to plasma membrane in L6 myotubes were demonstrated by Western blotting analyses. Promotion by ENL of GLUT4 translocation to plasma membrane was also visually demonstrated by immunocytochemistry in L6 myoblasts that were transfected with a glut4 cDNA-coding vector. T2D model db/db mice were fed a basal diet or the basal diet supplemented with ENL (0.001% or 0.01%) for 6 weeks. FBG levels were measured every week and intraperitoneal glucose tolerance test (IPGTT) was conducted. ENL at a higher dose (0.01% in the basal diet) suppressed the increases in FBG levels. ENL was also demonstrated to improve the index of insulin resistance (HOMA-IR) and glucose intolerance by IPGTT in db/db mice [68]. From these results, ENL is suggested to be an antidiabetic chemical entity converted from dietary lignans by gut microbiota.

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FIGURE 25.8 Effect of daidzein on nonfasting blood glucose levels and urinary glucose excretion in KK-Ay/Ta Jcl mice. (A) KK-Ay/Ta Jcl mice were kept on diets during blood collection from tail vein. Each value represents the mean 6 SEM of six mice. Values not sharing a common letter are significantly different at P , 0.05 by TukeyKramer multiple comparisons test. (B) Urinary glucose excretion in mice was determined using urine test paper. Each group consisted of six mice. Abbreviations: SEM, standard error of mean; NOR, normal group; CON or CNT, control group; DAI, daidzein group. Cited from Cheong SH, Furuhashi K, Ito K, Nagaoka M, Yonezawa T, Miura Y, et al. Daidzein promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and improves glucose homeostasis in Type 2 diabetic model mice. J Nutr Biochem 2014;25:13643 with permission.

PLANT HORMONES AND MUSCLE FUNCTION As was already mentioned, isopentenyladenine, a member of one class of “plant” hormones, i.e., cytokinins, was found to simulate the syntheses of DNA and protein, and hence the proliferation of L6 myoblasts [12]. Abscisic acid, a plant hormone playing an important role in the regulation of strawberry fruit ripening [69,70], has been reported to increase in human plasma after hyperglycemia and to stimulate glucose consumption by 3T3-L1 adipocytes and L6 myoblasts at the nanomolar level [71]. Interestingly, abscisic acid is produced and released by human and murine pancreatic β-cells in response to

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glucose, and nanomolar abscisic acid stimulates glucose-dependent and -independent insulin release [72]. Abscisic acid seems to be involved both in the regulation of glycemia and in the pathogenesis of the metabolic syndrome [72].

DIRECT MOLECULAR TARGET OF PHYTOCHEMICALS THAT ACTIVATE AMPK As abovementioned, several phytochemicals activated AMPK and promoted GLUT4 translocation to plasma membrane, thereby increasing glucose uptake in L6 myotubes. However, the direct molecular target of phytochemicals has been elusive [73]. Park et al. have shown that resveratrol directly inhibits cyclic adenosine monophosphate (cAMP)dependent phosphodiesterases (PDEs) [74]. They have reported that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading PDE, leading to elevated cAMP levels. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including glucose tolerance in mice. Authors therefore suggest that administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging [74]. It is likely that other phytochemicals also activate AMPK by inhibiting PDEs, because many of them have been shown to be PDE inhibitors [75] as already pointed out [14].

BROMACOLOGY: PHARMACOLOGY OF FOOD AND THEIR COMPONENTS Nutrients have been shown to possess pharmacological actions in addition to serving their nutritional roles. The research field studying nutritional roles and metabolism of nutrients is called “nutritional science.” The research field studying mutual interaction between nonnutrients and living organisms has been called “functional food science.” However, the research field investigating pharmacological aspects of nutrients has not been definitely designated. Nutrients as well as nonnutrients possess various pharmacological actions (food pharmacology). We have proposed to designate food pharmacology as “bromacology”; “broma” means “food” [76]. Bromacology advocates preventing the progression of prediseases to diagnosed diseases.

Acknowledgments Author is deeply grateful to Dr. Ryuhei Funabiki, Dr. Yutaka Miura, and research colleagues at Tokyo University of Agriculture and Technology and Utsunomiya University for their cooperation and kind help. Part of works described in this chapter was supported in part by the Regional Innovation Strategy Support Program, MEXT, Japan, and in part by the Japan Society for the Promotion of Science [JSPS Grant 15K07424].

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[71] Bruzzone S, Ameri P, Briatore L, Mannino E, Basile G, Andraghetti G, et al. The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts. FASEB J. 2012;26:125160. [72] Bruzzone S, Bodrato N, Usai C, Guida L, Moreschi I, Nano R, et al. Abscisic acid is an endogenous stimulator of insulin release from human pancreatic islets with cyclic ADP ribose as second messenger. J Biol Chem. 2008;283:3218897. [73] Tennen RI, Michishita-Kioi E, Chua KF. Finding a target for resveratrol. Cell 2012;148:3879. [74] Park SJ, Ahmad F, Philp A, Baar K, Williams T, Luo H, et al. Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell 2012;148:42133. [75] Kuppusamy UR, Das NP. Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes. Biochem Pharmacol 1992;44:130715. [76] Yagasaki K, Yamazaki M. Bromacology: pharmacology of food and their components. Trivandrum: Research Signpost; 2008.

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Antioxidants and Polyphenols Mediate Mitochondrial Mediated Muscle Death Signaling in Sarcopenia 1

Stephen E. Alway1,2

Department of Physical Therapy, College of Health Professions, Memphis, TN, United States 2 Department of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN, United States

LIST OF ABBREVIATIONS 4EBP1 8-iso-PGF2α AIF AKT AMPK BAK1 Bcl-2 CR FADD FAS FOXO3 GAS GSH GSSG HLS HMB HNE IGF1 IL15 MAFbx/ Atrogin-1 mTOR

eukaryotic initiation factor 4E binding protein 1 MnSOD-iso-prostaglandin F(2α) apoptosis inducing factor protein kinase B adenosine monophosphate (AMP)-activated protein kinase BCL2 antagonist/killer 1 Bcl-2-associated X protein caloric restriction fas-associated protein with death domain fatty acid synthesis forkhead box protein O3 gastrocnemius reduced glutathione oxidized glutathione hindlimb suspension β, hydroxy-β-methylbutyrate 4-hydroxynonenal (lipid marker for oxidative stress) insulin growth factor-1 interleukin-15 muscle atrophy F-box mammalian target of rapamycin

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MTORC1 MuRF-1 SOD SOD1 SOD2 TA TFAM TUNEL Vo XIAP XJB

mammalian target of rapamycin complex 1 muscle RING-finger protein-1 total superoxide dismutase activity manganese superoxide dismutase 1 Copper-zinc superoxide dismutase (CuZn-SOD) tibialis anterior transcription factor A, mitochondrial terminal deoxynucleotidyl transferase dUTP nick end labeling (a marker for DNA fragmentation in nuclear apoptosis) maximal unloaded shortening velocity X-linked inhibitor of apoptosis protein. mitochondrial targeted electron scavenger XJB-5-131

INTRODUCTION Skeletal muscle comprises about 40% of the total body mass of a young healthy person, but with aging, muscle mass and function decline. The age-associated loss of muscle mass and function has been termed sarcopenia [1]. Sarcopenia is mildly progressive after age 30 but is particularly severe with losses approaching 2% per year after the seventh decade of life [2]. Sarcopenia increases the susceptibility for obesity and diabetes [3], independently lowers mobility [4], and increases mortality [5]. It is estimated that 20% of the population of the United States (B72,000,000 people), will be 65 years of age or older by 2030 [6]. Similar distributions have been predicted for other countries. Several age-associated changes have been proposed as potential mechanisms that mediate sarcopenia. However, it is difficult to determine which of these changes trigger sarcopenia or rather, if they are a consequence of signaling for muscle atrophy. Examples of changes that might contribute to sarcopenia, include an increase in low grade but constant systemic inflammation [7], elevated production or reduced buffering of reactive oxygen species (ROS) [8
10], altered or impaired innervation [11
14], loss of motor units and alpha motor neurons [15], reduced regenerative capability [16,17], and decreased mitochondrial function [18,19]. Low physical activity can exacerbate sarcopenia, whereas exercise training can at least partially attenuate some of the aging-associated alterations in mitochondrial function in aging [9,10,20,21]. Emerging evidence suggests that dysfunctional mitochondria play an important role in regulating the loss of muscle function that is associated with sarcopenia [22
24]). This is due in part because optimal mitochondrial function is critical for energy delivery and expenditure, but mitochondrial function is impaired in aging [25,26]. In this chapter, we will provide evidence that the loss of mitochondrial function is central to the dysregulation of apoptosis, autophagy, proteasome, and lysosomal pathways. These same pathways are key to muscle and motor neuronal loss that contributes to sarcopenia. The manner in which nutrition could be expected to alter the signaling from these pathways to attenuate sarcopenia will be discussed.

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MITOCHONDRIAL FUNCTION IN AGING MUSCLES AND MOTOR NEURONS The general pathways that are initiated by signaling from dysfunctional mitochondria are summarized in Fig. 26.1. These pathways assume that stress to mitochondria in aged cells results in irreparably damaged mitochondria, but aging attenuates removal of these damaged mitochondria. The accumulation of dysfunctional mitochondria initiate the apoptotic death signaling pathway in muscle cells and motor neurons, and communication between these cell types accelerates muscle wasting and denervation, and leads to sarcopenia. Evidence for the components of this pathway are given below.

Aging Attenuates Mitochondrial Function Aging decreases muscle mitochondrial function [27], mitochondrial content, and enzyme activity [28
32], lowers respiration [33] and reduces mitochondrial biogenesis [34]. FIGURE 26.1 Mitochondrial initiated sarcopenia. Initiation of mitochondrial stress that includes ROS, inflammation signals, cytokines, etc. (lightning bolt) can result in dysfunctional mitochondria. Damaged mitochondria are removed by mitophagy signaling and proteasome/lysosomal activation in healthy young muscle and motor neurons. However, in aging, the increased ROS and other mitochondrial stresses, cause mPTP opening (red dashed arrow). The resulting release of the mitochondrial contents (e.g., cytochrome c) to the cell cytosol leads to an apoptotic signaling cascade that ends with DNA fragmentation and removal of nuclei (apoptosis). Sufficient nuclear death in muscle cells will result in the death and removal of the entire muscle cell. Motor neuron death occurs when apoptosis removal of the alpha motor neuron nucleus occurs in the same series of events that occurs in skeletal muscle. The interdependence of muscle cells and motor neurons suggests a potential feedback loop (anterograde and retrograde) communication between the muscle and the motor neuronal compartments, which exacerbates death in both compartments. Death in these cell compartments leads to loss of muscle mass and function in aging. Thus, dysfunctional mitochondria provide the signal to initiate the signaling pathways that lead to sarcopenia.

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Part of the decline in mitochondrial content in aging may be the result of an imbalance between mitochondrial removal and mitochondrial biogenesis. Mitochondrial biogenesis is a complex process consisting of both synthesis, assembly, growth by fusion, and division of preexisting mitochondria by fission dynamics. The mitochondrial DNA (mtDNA) gene encodes for only 37 genes. This includes 13 mRNAs encoding for protein subunits of the enzymes involved in oxidative phosphorylation (cytochrome oxidase subunits I, II, III, IV, and V), 2 ribosomal RNAs, and 22 transfer RNAs that are needed for the translation of the encodes proteins [35]. Thus, the majority of mitochondrial proteins are nuclear encoded, synthesized in the cytoplasm and then must be imported into mitochondria. Because mitochondrial biogenesis can be affected at many different levels including mitochondrial transport, one needs to exercise caution when interpreting mitochondrial markers as outcomes for biogenesis, because some markers may or may not fully reflect new mitochondria assembly. Rather, assessment of mitochondrial protein synthesis provides the gold standard for measuring mitochondrial biogenesis [36]. Maintenance of the balance of mitochondrial biogenesis capacity along with the removal of dysfunctional mitochondrial are key factors in preventing the progression of aging-related diseases including sarcopenia. It should be noted that the aging-associated loss of mitochondrial respiration and function has recently been challenged in other studies [37,38], and reductions in mitochondrial function have been attributed more to lack of use than aging per se [39]. Furthermore, improvements in mitochondrial function in aging can also be achieved by nutritional intervention without changes in mitochondrial biogenesis [29]. Moreover, it has been suggested that changes (or lack of changes) in mitochondrial respiratory function with age, may be influenced by sex [40], muscle fiber type, or motor unit recruitment patterns [41,42].

Mitochondrial Regulation of Neural Dysfunction in Aging Loss of motor function in sarcopenia is partially dependent upon the reduction of muscle cross sectional area, and partly related to reduced neuronal function and innervation. Neural conduction and resetting membrane potentials after each action potential are an energy-consuming event, so that mitochondrial health would be expected to be important for optimizing function in motor neurons. Indeed, decreases in motor neuron mitochondrial enzyme content [31] and the loss of mitochondrial content [31] may precede neuronal death in aging. Given the importance of mitochondrial in the motor neuron, it is not surprising that mitochondria have been implicated in the decline of neuron function and number in aging [31]. Nevertheless, while the role of mitochondria in aging has been well characterized in muscle cells, their importance in regulating motor neuron aging deficits or motor neuron death is not well understood. Mitochondria morphological abnormalities and reduced respiratory activity of several enzymes occur in motor neuron degeneration models such as the wobbler mouse [43], but it is less clear if this is a common aging-associated phenomenon that is independent of disease. Thus, we have good reason to think that mitochondrial dysfunction occurs in motor neurons with aging. Furthermore, with an aging-induced increase in ROS production and perhaps a reduced antioxidant activity, ROS accumulation could impair neural mitochondria in a similar mechanism as proposed for skeletal muscle mitochondria. Denervation III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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has also been suggested to be an important regulator of mitochondrial dysfunction in neural tissue [44] but it is less clear if denervation is the initiator or the contributor to mitochondrial dysfunction.

POTENTIAL SOURCES OF MITOCHONDRIAL DYSFUNCTION IN AGING Reactive Oxygen Species (ROS)-Induced Damage Aging tends to increase ROS production, and this is magnified during reduced muscle activity that typically accompanies aging. There is a large database indicating that mitochondrial ROS production results in widespread oxidative damage to cells [45]. Although the origin of ROS is not fully known, there is generally proposed to be an increase in cytokines that occur with aging. Furthermore, there are data to support a role for myostatin as a prooxidant, which generates ROS in muscle cells through tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor κB (NF-κB) and NADPH oxidase [46]. This is supported by observations that myostatin null mice have lower ROS and sarcopenia [46]. Reducing ROS production would be expected to attenuate oxidant damage to proteins, lipids, and DNA and reduce mitochondrial insult. At least as important as ROS production in determining the accumulation of ROS for potential cell damage is the level of antioxidants present in the mitochondrial and the cytosol of a muscle cell. In fact, most measurements may underestimate ROS production because there is a large antioxidant potential in these compartments [47]. The importance of antioxidants in sarcopenia are highlighted by observations that both neural and muscle losses of the cytosolic antioxidant CuZn-superoxide dismutase (CuZnSOD) recapitulated sarcopenic muscle loss in a mouse model [48]. It his however, noteworthy that the loss of CuZnSOD in only neural or muscle cells did not manifest full sarcopenic muscle loss [48], but this does emphasize the cross talk that probably occurs between these two tissues in aging. Nevertheless, as aging muscles and neurons are associated with an increased ROS accumulation and a reduction of mRNA and protein for several antioxidant enzymes [49
53], aging likely increases the potential for greater ROS-induced damage to mitochondrial components in motor neurons and muscle cells. The interactions between aging and inactivity with ROS production, along with lower antioxidant levels [49,52
54], increase the potential for mitochondrial damage in aged muscles as compared to young skeletal muscle cells. It has also been recently suggested that ROS production might be secondary to denervation that occurs in aged muscles [44], again highlighting the potential communication between neural and muscle cells in aging. Whatever the initial source(s) of ROS production, it is clear that accumulation of excessive ROS leads to damaged mitochondria in muscle and neural cells, which in turn can result in mitochondria that are more dysfunctional.

Mitochondrial DNA Damage and Aging mtDNA deletions or DNA mutations have been proposed as contributors to mitochondrial dysfunction that leads to aging-related muscle fiber loss and atrophy [55,56]. Indeed, III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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there is evidence of increased mtDNA mutations in areas of muscle oxidative damage [55,57]. Similarly, in neurons, DNA damage precedes neuronal apoptosis [58], whereas forced repair of DNA damage rescues neurons from elimination by apoptosis [59]. Although not all increases in ROS production are the result of mtDNA deletions or lead to mtDNA mutations [60], it is clear that such deletions provide another strong contribution to mitochondrial ROS production in aging muscles and neurons [59,61,62]. Furthermore, aging-induced mtDNA deletions are linked closely to the loss of mitochondrial function in motor neurons [31] and neuronal malfunction in neural diseases such as Parkinson’s disease [63] and in muscle loss with aging [56,61]. Together, these observations highlight the important role of mitochondria in maintaining neural and muscle function in aging.

Altered Mitochondrial Dynamics with Aging Mitochondria are not static structures, as they can form individual organelles or an extensive reticulum. Mitochondrial morphology is regulated by interactions between fusion and fission regulatory proteins. Fusion proteins such as mitofusins 1 and 2 (Mfn1 and 2) and optic atrophy 1 (Opa1) can join mitochondrial membranes together to form larger mitochondria or increase the size of the mitochondrial reticular network [64,65]. Fission proteins like dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1) promote mitochondrial fission which can result in smaller, individual or fragmented mitochondria [64,65]. These processes of fission and fusion are important in healthy mitochondria because they allow for exchange of the matrix proteins between individual mitochondrial [66]. However, abnormal mitochondrial dynamics may negatively influence mitochondrial health. For example, both the mRNA level and the protein abundance of important fusion and fission proteins have been reported to be lower in old as compared to young adult skeletal muscle [67], and this would suggest that the potential for mitochondria to respond to changing environments might be reduced as compared to young mitochondria. Indeed, this appears to be the case, because electron microscopy and biochemical analyses have shown distinct mitochondrial profiles in aging muscle. Small, more fragmented mitochondria have been identified in muscles of old animals as compared with mitochondria from muscles in young adult hosts [68,69], although very large mitochondria have also been noted in muscles of old animals [64,70]. Consistent with the fragmented mitochondrial phenotype in muscles from old hosts, there is evidence to suggest that muscles of aged rodents and humans have a greater overall rate of fission vs fusion [67,71] and lower levels of the fusion protein Opa1 [71] as, compared with younger muscles. Fragmented mitochondria tend to have a lower respiratory capacity, and an increased production of ROS, which increases the susceptibility of mitochondria to release its contents and activate the intrinsic caspase apoptotic pathway. Thus, it is not surprising that aging and disuse, which both have excessively fragmented mitochondria, are accompanied by muscle loss [68,69]. It is interesting that a knockout of Mfn1/2 in skeletal muscle, which prevents mitochondrial fusion, increases the accumulation of mtDNA defects and results in muscle atrophy [155]. Together, these observations support the idea that muscle

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mitochondria are important regulators of muscle size. However, to provide a balanced perspective, it is important to point out that other studies have found higher fusion profiles in muscles of humans [72], prematurely aged mice [18], and larger mitochondria in muscles of old mice [64]. Nevertheless, it is interesting that even in studies reporting a higher fusion index in aged muscles as shown by ratios of Mfn1/Mfn2 [18] or Mfn2/Drp1 [64], the protein contents of Mfn1, Mfn2, Opa1, or Drp1 did not change. This means that even when higher fusion indexes are recorded, there still may be more fragmented mitochondria in aged muscles The impact of age-associated changes in mitochondrial dynamics in motor neurons and their potential role in sarcopenia have not been studied. However, mitochondrial dynamic dysfunction has been reported as an early event in Amyotrophic Lateral Sclerosis (ALS) [73,74] which is a common motor neuron disease. Furthermore, increased mitochondrial fragmentation has been found to precede glutamate-induced death of motor neurons [75]. Thus, similar to mitochondrial dynamic changes in muscle with aging, motor neuron dysfunction and death may converge upon mitochondria, and mitochondrial dynamics may play an important role in regulation of neuronal function that contributes to accelerated sarcopenia. The changes in fission and fusion protein mediated functions may be the result of damage to mitochondria such as accumulation of mtDNA defects, increased ROS production, and/or inappropriate import or assembly of electron transport proteins. Indeed, high intensity exercise induces mitochondrial damage and dysregulation of mitochondrial fusion and fission proteins thereby altering mitochondrial structure [76]. Such oxidant damage can lead to increased mitochondria permeability (producing leaky mitochondria) and the release of mitochondria specific proteins, including apoptosis inducing factor (AIF) and cytochrome c, into the cytosol through the mitochondrial transition pore, which triggers death-signaling pathways. mtDNA deletions or DNA mutations have been proposed contributors to aging-related muscle fiber loss and atrophy [55,77]. Indeed, there is evidence of increased mtDNA mutations in areas of muscle oxidative damage [55,57]. Although not all increases in ROS production are the result of mtDNA deletions [60] and/ or lead to mtDNA mutations [60] it is clear that such deletions provide another strong contribution to mitochondrial ROS in aging muscles [61,62]. Furthermore, mtDNA deletions are linked closely to muscle loss with aging [61], again highlighting the important role of mitochondria in maintaining muscle mass in aging. Improving mitochondria structure and increasing mitochondrial biogenesis by supplementing old mice with growth differentiation factor 11 [78] or caloric restriction [79,80] further supports the idea that healthy mitochondria have a pivotal role in maintaining muscle mass and function and that unhealthy mitochondrial can eventually contribute to cell destruction.

PGC-1α REGULATION OF MITOCHONDRIA IN SARCOPENIA PGC-1α Regulation of Mitochondria Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), is thought to be a master regulator of mitochondrial biogenesis and it has a role in regulating antioxidant

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proteins. Aging decreases PGC-1α levels along with muscle mass [28,81]. In contrast, transgenic mice that had a muscle specific overexpression of PGC-1α had elevated mitochondrial biogenesis, oxidative capacity, greater resistance to muscle fatigue, and improved aerobic performance [82
84]. PGC-1α also plays a key role in preventing deterioration of mitochondria function in response to immobilization [85] and conserves mitochondria [86] and skeletal muscle mass in response to catabolic stimuli [87,88]. Overexpression of PGC-1α can block the expression of atrophy associated genes (atrogenes) and minimize muscle loss that is induced by TNF-like weak inducer of apoptosis (TWEAK)-Fn14 [89]. Together, these data support the idea that PGC-1α regulated mitochondrial biogenesis is important for maintaining muscle mass and function and in reducing sarcopenia. However, increasing mitochondrial biogenesis without also removing dysfunctional mitochondria would provide a muscle milieu with a mixture of healthy and unhealthy mitochondria. Thus, not only is the abundance of mitochondria important but the health of the mitochondria are also equally important for maintaining muscle mass.

PGC-1α in Mitochondria in Aged Muscles There is also evidence to support an important role for PGC-1α to regulate antioxidant proteins in aging muscle [90]. Reduced mRNA levels CuZnSOD, MnSOD, and/or GPX1 [90,91] have been reported in PGC-1α knockout mice as compared to age-matched wildtype mice. Furthermore, mice that overexpress PGC-1α have greater MnSOD in skeletal muscle and this SOD is primarily housed in the mitochondria [92]. Indeed, the effect of PGC-1α overexpression is profound because it has been shown to reduce sarcopenia, by inhibiting mitochondrial-associated apoptosis, autophagy, and proteasome degradation. In addition, a PGC-1α-mediated increase in antioxidant production with a corresponding reduction of inflammation has been reported in skeletal muscles of old mice [92]. The central nature of mitochondrial health to muscle mass in aging is highlighted by the dramatic regulation of mitochondria by PGC-1α. Thus, nutritional investigations that target PGC-1α regulation might be important for improving mitochondrial function to offset or eliminate sarcopenia.

PGC-1α Regulation of Mitochondria in Motor Neurons It is likely that there is cross talk between neural and muscle compartments. This is shown in a study where muscle-specific expression of PGC-1α in mice resulted in a remodeling of the postsynaptic and presynaptic components of the neuromuscular junction, in the absence of greater physical activity [93]. Furthermore, PGC-1α overexpression increased mitochondrial enzymes, improved motor neuron function, and reduced cell death in a mouse model of ALS [94]. Although we do not know if the same result would be found in aged motor neurons, it is reasonable to speculate that a motor neuronal overexpression of PGC-1α would be expected to have a protective effect against age-associated mitochondrial dysfunction and motor neuronal death.

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MITOCHONDRIA ARE INITIATORS OF CELL DEATH SIGNALING IN AGING MUSCLES Two independent pathways are involved in regulating signaling for cell death. These are nuclear apoptosis [81,95
98] and autophagy [99
103], and both pathways can be activated in response to dysfunctional or damaged mitochondria in aging. While proper balance between these signaling pathways is important for optimizing, the health of the muscle fiber, altered signaling, and dysregulation of one or both pathways is common in sarcopenia [95,98,100]. Low physical activity can exacerbate sarcopenia, whereas exercise training can at least partially attenuate some of the aging-associated alterations including improvements of mitochondrial function in aging [10,20]. While healthy mitochondria provide proper cellular metabolism and production of adenosine triphosphate (ATP), dysfunctional or damaged mitochondria can initiate intrinsic (mitochondrial) death pathways that result in removal of nuclei via nuclear apoptosis [95,96,98]. Although autophagy can be upregulated during periods of muscle wasting, the overall pattern to dismantle a dysfunctional mitochondria by autophagy (mitophagy) is to eliminate the source of the death signaling and ultimately to save the muscle cell from complete removal by the apoptotic death pathway [100,102]. While proper balance between these signaling pathways is important for optimizing, the health of the muscle fiber, altered signaling, and dysregulation of apoptotic and/or autophagy pathways is common in sarcopenia [95,98,100].

Mitochondrial-Induced Nuclear Apoptosis in Aging Muscle The decline of mitochondrial function with aging is associated with sarcopenia. Losses of mitochondrial function may limit the synthesis of sufficient ATP for homeostasis, and this could be a contributing factor to apoptosis signaling [104]. There are many similarities in mediation of death in single cells that contain only one nucleus and multinucleated skeletal muscle cells. However, an important difference is that loss of a single nucleus (e.g., via apoptosis) does not result in complete cell death in multinucleated muscle cells, whereas the single nucleated cell will die once its nucleus is eliminated. In skeletal muscle, targeting one nucleus in a muscle region but not another suggests that signaling for cell death is not controlled systemically, but rather it is controlled by local targeted signaling networks. There are three primary pathways for apoptosis, but the intrinsically mediated mitochondrial signaling pathway is attractive for explaining much of the apoptotic signaling associated with sarcopenia. Nuclear apoptosis is characterized by increases in DNA fragmentation and by increases in proapoptotic proteins such as Bax, caspase-3, apoptosis protease activating factor-1 (Apaf-1), and AIF [22,23,105]. There are multiple levels of evidence from our lab and other research groups, which document that apoptosis has an important role in regulating muscle mass in aging [106
109] and denervation [110
112]. Furthermore, there is evidence that downregulating apoptotic signaling can accelerate the loss of muscle mass and function in aging animals or after aging-associated conditions

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such as denervation [11,99,113]. In addition, an upregulation of apoptotic signaling has been identified in premature aging models that exhibit accelerated sarcopenia [114]. Increased levels of caspase-3 and DNA fragmentation also have been reported in aged rat muscles. Fig. 26.2 summarizes the main elements of mitochondrial associated (intrinsic) apoptotic signaling, which contributes to nuclear apoptosis in skeletal muscle. Increased mitochondria permeability is an important regulator of intrinsic apoptosis signaling in skeletal muscle. This is initiated by Bax:Bax (or Bax:Bak) dimerization, which creates a pore in the outer mitochondrial membrane. Alternatively, mitochondrial permeability can occur via a greater sensitization of the muscle mitochondrial permeability transition pore opening

FIGURE 26.2 Mitochondrial induced nuclear apoptosis. Induction of mitochondrial stress (lightning bolt) results in dissociation of the anti-apoptotic Bcl-2 with Bax and forming a Bax:Bax pore in the outer membrane and a mitochondrial pore (not shown) in the inner mitochondrial membrane. This opens the mPTP pore and allows mitochondrial housed contents to leak into the cytosol, forming an apoptosome, which activates caspase-9 and subsequently activation of the effector caspase-3. Activated caspase-3 enters the nucleus that is in proximity to the dysfunctional mitochondria, activates enzyme PARP, which cleaves DNA. DNA fragmentation can also be caused by direct activation of mitochondrial housed components such as AIF. Mitochondrial housed second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/ DIABLO) can promote caspase-9 activation via inhibition of the anti-apoptotic XIAP. Although not all DNA damage that occurs in this fashion will result in nuclear removal, sufficient damage will result in the elimination of the nucleus that is targeted by apoptosis. The intersections of extrinsic and endoplasmic reticulum pathways that exist for inducing apoptosis are not shown. PARP, poly ADP ribose polymerase; Bax, Bcl-2-associated X protein; Bcl, B-cell lymphoma; AIF, apoptosis inducing factor; XIAP, X-linked inhibitor of apoptosis protein.

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(mPTP) in response to ROS or calcium. When these channels open, they increase mitochondrial permeability, which allows for the release of mitochondrial housed proteins such as cytochrome c, to the cytosol in muscles of old animals [115]. In the cytoplasm, cytochrome c acts as a proapoptotic protein by binding dATP and apoptosis protease activating factor-1 (Apaf-1), forming an apoptosome that cleaves caspase-9. An agingassociated increase in the proapoptotic Apaf-1 protein and in the level of the proapoptotic cleaved caspase-9 protein, along with increased DNA fragmentation, has been reported in the in the gastrocnemius muscles of old rats. This is not surprising because cleaved caspase-9 will activate caspase-3, the final effector caspase in this apoptotic pathway. Mitochondrial-associated caspase signaling is clearly important in muscle loss, but it is not the only source of mitochondrial-associated apoptotic signaling. Caspaseindependent signaling associated with mitochondrial dysfunction and permeability has been shown to occur in aging-associated muscle loss. The release of endonuclease G (EndoG), AIF, second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO), and X-linked inhibitor of apoptosis protein (XIAP) from the mitochondria to the cytosol begins the initiation of apoptotic signaling in a noncaspase dependent signaling manner. Thus, it is clear that mitochondria are intimately involved in the initiation of the signaling cascades leading to apoptosis of a nucleus in aging skeletal muscle. However, it is interesting that nuclear apoptosis in skeletal muscle involves cell signaling that is so precise that specific individual myonuclei can be targeted for elimination in multinucleated skeletal myofiber without targeting other nuclei. While apoptosis signaling can be somewhat general in a single-nucleated nonmuscle cell as there is only one target in that cell, in multinucleated skeletal muscle, the apoptotic signaling requires amazingly precise targeting of some nuclei but not others. We suggest that local signaling from individual dysfunctional mitochondria will only target nuclei within its vicinity.

Mitochondrial Permeability Transition Accelerates Apoptosis in Aging Muscles It has been well established that apoptosis occurs in aging skeletal muscle [62,81,95,98,106,116
122] including human muscles [123,124]. Much of the greater susceptibility of aged skeletal muscle to apoptosis is related to the elevated mitochondrial sensitivity to open the mPTP [124], thereby releasing the mitochondrial contents to the cytosol to initiate apoptotic signaling [81,108,119]. It could be argued that inactivity that typically accompanies aging might explain some of the increased mPTP susceptibility for opening. However, mPTP opening in aging cannot be solely the function of inactivity, because increased mitochondrial susceptibility to permeability transition opening has also been observed in muscles from active humans [124]. Nevertheless, exercise, which should improve mitochondria number and function, can at least partially reverse aging-associated apoptosis as exercise training has been reported to decrease catabolic and apoptotic signaling in muscles of aged rodents [125]. It is also important to note that the susceptibility for mPTP opening is exacerbated by an imbalance of Ca2 1 homeostasis that likely results from leaky ryanodine receptors that occur in aged skeletal muscle [126]. Sensitization of the mPTP in aging skeletal muscle

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may be an important contributor to the initiation of apoptosis and muscle loss leading to sarcopenia. Aging-associated muscle denervation may also contribute to increased mPTP that in turn induces muscle apoptosis and muscle loss [44,127].

What Role Can Mitochondria Have in Removal of Type II Fibers in Muscles of Older Individuals? Type II fibers have few mitochondria as compared to type I fibers, and therefore, it is not unreasonable to think that the signal for mitochondrial-apoptosis should then be low in type II fibers. Yet, type II fibers are lost in aging, at least at the same rate as type I fibers and may even be preferentially atrophied in older human muscles [128], at least if we ignore hybrid fibers that might contain multiple myosin heavy chain (MHC) isoforms. Furthermore, the age-associated decline in fiber size and function in rodents is generally most prominent in fibers that contain type II MHC [129,130]. If mPTP opening is a critical component of initiating apoptotic signaling from dysfunctional mitochondria, one might wonder why is that that the glycolytic fibers which contain less than 3% of the fiber volume as mitochondria [131,132] also die in aging. While it is true that type II fibers contain a low mitochondrial volume as compared to type I fibers, type II fibers are more susceptible to ROS damage [133]. This means that although there are fewer mitochondria in type II fibers, the greater mitochondrial damage quickly leads to mPTP opening [134], further mtDNA damage [135], and mitochondrial associated apoptotic signaling [81,111,116,134,136] as compared to type I fibers. We do not know if removal of dysfunctional mitochondria by mitophagy signaling is different between type I and type II fibers. Irrespective of fiber type, the levels of death signaling in apoptosis are intimately linked to the health of mitochondria in skeletal muscle [62,100,102,103,134,137,138]. In future studies, it will be intriguing to determine if mitophagy is suppressed more in type II than in type I fibers in aging and if the type II to type I fiber switch may be somewhat protective against sarcopenia.

AUTOPHAGY Autophagy is a network of cellular recycling that involves identifying and tagging a damaged organelle, capturing and surrounding damaged organelles with doubled wall membrane vesicles known as autophagosomes, and degrading the organelle within the lysosome (Fig. 26.3). Accelerated and extensive autophagy has been identified in many diseases and excessive autophagy in muscle can contribute to muscle wasting. However, more modest and appropriately controlled autophagy signaling and particularly autophagic removal of dysfunctional mitochondria (mitophagy) is an important suppressor the overall apoptotic death-signaling program. Thus, dismantling and removal of dysfunctional mitochondria by mitophagy removes the source for apoptotic death signaling, thereby promoting cell survival.

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FIGURE 26.3 Induction of mitophagy. Pink1 is imported to mitochondria where it is localized to the intermembrane and intermembrane spaces or degraded under in healthy mitochondria under basal conditions. Excessive ROS or other stress cause a reduction in the mitochondrial membrane potential. Upon mitochondrial depolarization, Pink1 is recruited to the outer mitochondrial membrane, and transport inside the mitochondria is prevented, resulting in an accumulation of Pink along the outer mitochondrial membrane (depicted by the pink rink around the outer mitochondrial membrane). The E3 ligase Parkin (green) is recruited to insert into the outer

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Removal of Dysfunctional Mitochondria by Mitophagy Failure to remove dysfunctional mitochondria by mitophagy results is an accumulation of damaged mitochondrial, which together magnify the death-signaling pathway and accelerate sarcopenia. Thus, progression of the steps leading to sarcopenia hinge upon whether dysfunctional mitochondria can be removed or if they are be permitted to continue with their apoptotic death signaling cascade. In young cells, signaling for mitochondrial disassembly via fusion and autophagy occurs when ROS damage is high or when the mitochondrial membrane potential (Δψm) is lost. Mitophagy is the selective removal of dysfunctional mitochondria by autophagic processes [139,140]. Mitophagy requires tagging of dysfunctional mitochondria so that they can be recognized for disassembly. The selectivity of mitophagy is controlled by the loss of the mitochondrial membrane potential (Δψm) and by several important proteins including: function mutations of phosphatase and tensin homolog induced putative kinase 1 (Pink1), Parkin, B-cell lymphoma (Bcl)-2 and 19 kDa interacting protein-3 (Bnip3), and Bnip3L/Nix [141,142]. After identification, dysfunctional mitochondria are engulfed in the double membrane autophagosome. The autophagosome fuses with a lysosome and the proteolytic contents of the lysosome are emptied into the autophagosome, which then digests the dysfunctional mitochondria [139,140]. Although it is not meant to be exhaustive, an overview of some of the elements that are part of the mitophagy signaling pathway are shown in Fig. 26.3. Pink1 acts as a primary regulator to target mitophagy in dysfunctional mitochondria. Recent work indicates that under basal physiological conditions in healthy mitochondria, Pink1 is imported to the mitochondria. After it is imported, Pink1 becomes localized to the inner mitochondrial membrane and the intermembrane space and matrix [143]. In healthy mitochondria from young hosts, Pink1 is quickly degraded and therefore does not induce mitophagy. However, Pink1 is activated as a result of a loss of Δψm, which results from mitochondrial damage [144], depolarization [143], or an increase in mitochondrial pH [145] that can occur in aging and disease. Depolarized mitochondria prevent the import of Pink1 [143], which results in an accumulation of Pink1 on the outer mitochondrial membrane. In addition,

L

mitochondrial membrane. Parkin ubiquitinates outer mitochondrial membrane proteins (shown by a green ring around the mitochondria), resulting in recruitment of ubiquitin-binding autophagy receptors such as p62, Bnip3, and Bnip3L/Nix to the mitochondria. Bcl-2 prevents the induction of autophagy by binding Beclin-1. Displacement of Bcl-2 leads to activation of the phagophore nucleation. Elongation of the membrane requires 2 ubiquitin-like conjugation systems: autophagy proteins (ATG) and activation of microtubule-associated protein 1 light chain 3 from its inactive form LC3-I to LC3-II where it is conjugated to PE to form lipidated (activated) LC3II (LC3-II-PE) LC3-II-PE. LC3-II-PE can bind autophagy receptors, which contain transmembrane domains and localize to the outer mitochondrial membrane. The p62 receptor contains an ubiquitin-binding domain, which localizes it to Parkin-ubiquitinated mitochondria. Furthermore, LC3-II-PE participates in autophagosomal membrane elongation (shown in light purple). Bnip3 and its homologue Bnip3L/Nix are also mitophagy receptors that when expressed, localize to the outer mitochondria membrane. It is not clear if it is necessary, but some data suggest that p62 can also bind to ubiquitinated chains (Ub) that are attached to the mitochondria. LC3-II-PE binds to the autophagy receptors (e.g., p62, Bnip3, Bnip3L/Nix, and others) (as depicted by the red double arrows) which then tethers the developing membrane of the autophagosome to the outer mitochondria membrane. LC3-II-PE participates in elongation of the double-layered membrane that surrounds the damaged mitochondria. After maturation of the autophagosome, it fuses with a lysosome, which injects its contents into the mitochondria, to degrade it. ROS, reactive oxygen species; Bcl, B-cell lymphoma; PE, phosphatidylethanolamine.

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Pink 1 that is inside the mitochondria is not degraded but it translocates to the outer mitochondrial membrane of a depolarized mitochondria [143]. At the surface of dysfunctional mitochondria, Pink1 recruits the cytosolic ubiquitin ligase Parkin to the outer mitochondria membrane. Pink1 then activates Parkin [143,146] and phosphorylates ubiquitin [147] to initiate the formation of ubiquitin chains on mitochondria [148]. The phosphorylated form of Parkin accelerates formation of ubiquitin links on the outer mitochondrial membrane, which then further feeds Pink1 as a substrate. Furthermore, Pink1 activates Drp1 [139]. The fission property of Drp1 has a role in the degradation of mitochondria as part of the mitophagy process [139,140]. The autophagy adaptor protein p62 binds to the ubiquitin tags on the mitochondria membrane and links to the activated form of microtubuleassociated protein 1 light chain 3 (LC3). In addition, Bnip3 and Nix translocate to the mitochondria where they act as other autophagy receptors to connect the mitochondria to the autophagosome. Elongation of the double layered membrane is controlled through linkage of autophagy-related (ATG) proteins 12-ATG5-ATG16L and the conjugation of microtubule-associated protein 1 LC3-I with phosphatidylethanolamine (PE) to form the activated lipidated LC3-II-PE, which forms a vital membrane component of the autophagosome and binding partner for autophagy receptors. Elongation of the lipid membrane then engulfs the dysfunctional mitochondria (Fig. 26.3). After forming the mature autophagosome, it fuses with the lysosome to degrade the isolated mitochondria by proteolytic enzymes that are contained in the lysosome [141,142]. Mitophagy is not a simple process in aging, because it has been both reported to increase [100] or decrease [18,149,150] in muscle with aging. Although there is evidence for increasing the migration of Parkin and p62 to the mitochondria with aging, suggestive of increased mitophagy, there is also a paradoxical accumulation of lipofuscin granules, which suggests an impairment in lysosomal function in aging [100]. These data implicate a failure for the lysosome to remove dysfunctional mitochondrial as this would account for the increase in lipofuscin granules. Indeed, lipofuscin deposits have been previously reported in many tissues including muscles of aged rodents or other mammals [151]. Improved removal of dysfunctional mitochondria perhaps via increased activation of lysosomal activity [149] may include exercise and nutritional interventions, although the responses might be regulated at least in part in a fiber type specific fashion [152]. Further work is needed to determine if failure of mitophagy progression in sarcopenia may lie at least in part with lysosomal dysfunction [153] or if improper signaling upstream of the lysosome is more important in regulating lysosome function.

Insufficient Mitophagy Allows Unhealthy Mitochondria to Persist in Aging Muscles Failure to regulate proper mitophagy and proteasome removal of mitochondria, are both upstream of sarcopenic muscle fiber loss. There is strong evidence for an aging-suppression of mitophagy in skeletal muscle and motor neurons [18,149,150]. Not only does Mfn2 have a role in mitochondrial fusion, Mfn2 but also has an important role in acting as a receptor for Pink1 and Parkin targeted mitophagy [154]. Mfn2 abundance declines in muscle with aging and loss of Mfn2 produces an aging-like phenotype,

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including age-associated mitochondrial dysfunction, higher ROS accumulation, and muscle fiber atrophy [155]. Thus, an aging-associated deficiency in Mfn2 might provide the link between impaired mitophagy and sarcopenic muscle loss [155]. However, it should be pointed out that other evidence found no change in Mfn2 and an increase in the Mfn2 to Drp1 ratio that was associated with muscle atrophy in aged mice [64]. Further work is needed to determine the mechanism for loss of Mfn2 and mitophagy and to determine if failure of mitophagy progression in sarcopenia may lie at least in part with lysosomal dysfunction [153] or if improper signaling upstream of the lysosome, and particularly loss of Mfn2 or some other proteins involved in mitochondrial dynamics that in turn leads to loss of mitophagy to induce pathways leading to sarcopenia. Similar to aging muscles, dysfunctional mitochondria and Mfn2 has been implicated in the loss of mitophagy in motor neurons. For example, a loss in inhibition of the E3 ligase Omi/HtrA2 in the mitochondria of neuronal cells has been shown to contribute to a decrease in Mfn2 leading to reduced mitophagy [156]. Furthermore, it is well known that defective mitophagy is an underlying process that contributes to the progression of motor neuron death in ALS [157]. Furthermore, lithium-induced induction of mitophagy [157,158] and mitochondrial biogenesis [158] improve mitochondrial morphology in motor neurons of a G93A SOD-1 ALS mouse model. Other autophagy proteins have also been implicated in loss of mitophagy in aging-associated neuron diseases including Parkinson’s disease. For example, the cleavage product of mitochondrial autophagy protein Pink1, Pink152 can exit the mitochondria in neuron cells and cleave Parkin, which then suppresses its translocation to the mitochondria and autophagic signaling, to prevent elimination of the dysfunctional mitochondria by mitophagy [159]. Thus, reduced mitophagy appears to be a common mechanism in both muscle and neurons that can potentially be permissive for apoptosis, and therefore result in cell death and therefore contribute to loss of muscle mass and function.

LINKING AUTOPHAGY AND APOPTOSIS BIM and Bcl-2 The proapoptotic BH3 only protein Bcl-2-like protein 11, commonly called BIM (BCL2L11) is an important link between autophagy and apoptotic signaling pathways. BIM is a member of the Bcl-2 apoptotic protein family that can interact with the antiapoptotic proteins Bcl-2 and Bcl-2 extralarge (Bcl-xL). However, BIM can also function independently through regulation of the autophagy protein Beclin-1 [160]. This occurs when BIM is removed from its interaction with dynein LC 1 (LC8), which can induce apoptosis by inactivating Bcl-2. In turn, this activates Bax
Bcl-2 antagonist/killer (Bak) or Bax
Bax homodimerization, to induce mPTP pore opening in the outer mitochondria membrane. The resulting permeabilized mitochondria empties its contents to the cytosol to initiate apoptotic-signaling cascades. Thus, BIM appears to have roles in both inhibiting autophagy and promoting apoptosis [160] Bcl-2 is also an important protein in both autophagy and apoptotic pathways, which highlights the crosstalk between these two death-signaling pathways. It is likely that there

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is a required coordination between autophagy and apoptosis to maintain a healthy overall tissue environment. We speculate that disruption of the coordination between autophagy and apoptosis, perhaps by BIM or other proteins, permits dysfunctional mitochondria from being removed by mitophagy, and in turn, the permeabilized mitochondria will perpetuate the apoptotic death signal.

PGC-1α PGC-1α is normally considered to be as a master regulator of mitochondrial biogenesis, but it has recently been shown to increase levels of autophagic proteins in skeletal muscle [103,137]. Indeed overexpression of PGC-1α in denervated mouse muscle leads to increased lysosomal capacity, reduced localization of the autophagy protein p62, and reduced lipidation of LC3-II and subsequent binding to mitochondria [137]. While PGC-1α overexpression did not reduce the Bax/Bcl-2 ratio and caspase-3 activity in immobilized mouse muscles [86], it is not known if other apoptotic proteins in the mitochondrial pathway might have been suppressed in this model. Together, the data support the idea that some mitochondrial proteins such as PGC-1α, Bcl-2, and BIM, and perhaps, others may have multiple roles in regulating both the autophagy and the apoptotic death signaling pathways. These autophagy-apoptosis signaling connections are critically linked through their mitochondrial targets.

UBIQUITIN PROTEASOME SYSTEM Muscle Wasting Although it is clear that mitochondrial centered signaling pathways are important for initiating cellular destruction, another major factor believed to contribute to age-related muscle loss is an acceleration of protein turnover. Enhanced protein loss without a concomitant increase in protein synthesis results in a net loss of protein, which leads to muscle wasting. The Forkhead box class O proteins (FOXO) transcription factor family regulates two main proteolytic systems; the ubiquitin proteasome system (UPS) and the autophagylysosome system. FOXOs regulate the mammalian target of rapamycin complex 1 (MTORC1) signaling, which is associated with muscle hypertrophy [161] and inhibition of mitochondrial autophagy (mitophagy) in skeletal muscle [162]. FOXO proteins are thought to regulate the UPS, which is a tightly controlled system responsible for intracellular protein turnover, including regulation of normal protein turnover and elimination of misfolded and dysfunctional protein [163
165]. Damaged proteins are targeted by the conjugation of a polyubiquitin chain for stepwise proteasomal degradation into individual peptides and then, eventually, into individual amino acids (Fig. 26.3). Ubiquitinated proteins are recognized by the 26S proteasome, which consists of a 20S core and two 19S regulatory subunits [166]. The 19S subunits recognize and bind the ubiquitinated proteins and begin their ATP-dependent disassembly and removal [167]. In skeletal muscle, polyubiquitination-targeting of proteins for degradation and disassembly by the 26S proteasome [168], occurs through Muscle RING Finger 1

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(MuRF1) and muscle atrophy F-box (MAFbx/Atrogin-1) [165], which are muscle-specific E3 ubiquitin ligases [168]. Studies have shown that the inhibition of MAFbx/Atrogin-1 complex reduces muscle wasting [169,170]. Recent reports also indicate that a loss of MuRF1, which reduced skeletal muscle protein degradation in aging [171], may be linked to inhibition of m-TOR signaling in muscles of older individuals or animals [172]. These findings indicate that the UPS system may be an important element in disassembly of skeletal muscle proteins under many atrophic conditions, as one of the systems underlying sarcopenia. However, FOXO regulation is not simple, because MTORC1 is upregulated in denervation-induced muscle atrophy [173], which occurs in aging muscles. Interestingly, inhibition of MTORC1 reduces muscle atrophy via inhibition of MuRF1 and MAFbx/ Atrogin-1 in response to denervation [173]. These data suggest that regulation of protein turnover pathways is tightly controlled because MTORC1, which is generally considered important in pathways of anabolism, is also a critical component of muscle atrophy and protein catabolism. It will be important in future studies to understand if failure to regulate FOXO control of MTORC1 properly by additional proteins might contribute to increased degradation of muscle proteins in aging.

UPS and Sarcopenia There are conflicting data pertaining to the role of the UPS in sarcopenia. For example, increased levels of ubiquitin [174], 26S proteasomes and polyubiquitinated proteins [175] along with increased MuRF1 and MAFbx/Atrogin-1 expression were reported to be elevated in hindlimb muscles of sarcopenic aged rats [175,176]. Furthermore, ubiquitin was found to be greater in quadriceps muscles of 70
79 year-old human subjects as compared to 20
29-year-old young adults [174]. In contrast, MuRF1 and MAFBx expression have been reported to be lower in muscles of old rats than young adult rats [177] and not changed with aging in vastus lateralis of humans [123]. The discrepancy between these studies might be due to variability introduced by a low number of subjects in each study, and perhaps by some differences in the age and sex of the older subjects that were studied. It is also possible that MuRF1 and MAFbx/Atrogin-1 regulation were up or down-regulated in aged muscles as a result of life style factors such as activity, nutritional history, health and smoking habits, etc. in these studies. Muscles from older hosts has been reported to have a lower proteasome activity than muscles from younger animals or humans in some [178
180] but not all studies [175,181,182]. Hwee et al. [171] reported that the 20S and 26S proteasome subunit activities were reduced with aging in muscles of old mice but maintained in muscles from MuRF1 null mice. This suggested that deletion of MuRF1 maintains skeletal muscle protein, and muscle mass while oxidative stress in aged muscles. FOXO regulation of the UPS autophagy pathway in skeletal muscle is also thought to occur by MTORC1-mediated phosphorylation of ULK1 at Ser757 and the subsequent activation of the ULK1-ATG13-RP6KB/ribosomal protein p70S6 kinase (RB1CC1). Inhibition of MTORC1 in muscles of TSC knockout mice reduced autophagy and caused a loss of muscle mass and a reduction in the ability to generate muscle force [162]. Thus, the FOXO mediated UPS appears to be an important regulator of muscle mass in aging.

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UPS and Neural Dysfunction in Aging While proper control of the UPS system is important to maintain optimal cellular function and muscle mass in aging [163] reduced proteasome function and the subsequent accumulation of ubiquitin-tagged proteins have been implicated in aging-associated neurodegenerative diseases [183
185]. An aging-associated reduction of UbcM2, an ubiquitin-conjugating enzyme has been linked to lower UPS activity and neural degeneration [186]. Furthermore, decreased proteasome activity has been documented in the spinal cord of old rats, and this was accompanied by evidence for increased ROS damage in spinal neurons [187]. Loss of proteasome activity may be associated with an age-associated loss of the chaperone heat shock protein 70 (HSP70) because restoration of HSP70 was shown to reduce autophagy-associated lipofuscin and increase proteasome activity in neurons [188]. This is similar to muscle cells, in that proteasome activity via the UPS system is suppressed in aging neuronal cells.

MITOCHONDRIA, MITOPHAGY, AND THE UBIQUITIN PROTEASOME SYSTEM (UPS) Mitophagy Regulation and the UPS It is clear that autophagy and proteasome-mediated degradation are interconnected, so that perturbation of one pathway can affect the function of the other. Thus, it is possible that in studies where UPS increased with aging, autophagy signaling was concurrently elevated, but this was not measured. The autophagy proteins ATG12
ATG5 are required for LC3-II conjugation to PE that drives autophagosomal membrane expansion. Besides its essential role in autophagy, the ATG12 also carries out various autophagy-independent roles. These include promotion of mitochondrial fusion and activation of mitochondrial apoptosis [189]. ATG12 conjugation to ATG3 supports mitochondrial fusion, whereas free ATG12 directly regulates mitochondrial apoptosis in a similar manner to proapoptotic BH3-only proteins [190]. However, ATG12 is also an ubiquitin-like protein, which is directly ubiquitinated. Once ubiquitinated ATG12 promotes its proteasomal degradation via the UPS, whereas, accumulation of free ATG12 contributes to proteasome inhibitor-mediated apoptosis [191]. Another important mitochondrial mitophagy protein, Parkin, mediates proteasomedependent degradation of DRP1 [140] and outer mitochondrial membrane proteins such as Tom20, Tom40, Tom70, and Omp25 when mitochondria become depolarized [141]. This is a unique nonautophagy function for Parkin because proteasome-dependent degradation of outer membrane proteins and outer membrane rupture is not required for mitophagy [141]. The loss of ATG7 or Parkin in denervated muscle induces dysfunctional mitochondrial mitophagy, but also an accumulation of polyubiquitinated proteins. Furthermore, accumulation of damaged and dysfunctional mitochondria attenuates transcriptional activity of nuclear factor erythroid-derived 2-related factors (Nrf2), which have been reported to regulate the transcription of proteasome subunits [192]. Together, these findings suggest that Parkin is essential for progression of the UPS degradation in muscle. Further

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connections between autophagy and UPS pathways have been shown including a report that a deficiency of MuRF1 increased the expression of MAFbx/Atrogin-1 and simultaneous reduced Beclin-1 expression in skeletal muscle after denervation [193]. This observation suggests that inhibition of UPS signaling via MuRF1 may decrease autophagic signaling, at least during denervation. Thus, proper mitophagy signaling to remove dysfunctional mitochondria appear to be important for not only regulating mitochondrial quality, but for fine-tuning other proteolytic pathways.

UPS Disruption Increases Dysfunctional Mitochondria in Muscle and Neural Cells Age-associated conditions such as Parkinson’s and Alzheimer’s disease are associated with high ROS levels in neural cells. Reduced Pink1 and Parkin-regulated ubiquitination of the outer mitochondrial membrane of damaged mitochondria in aged neurons [194,195] can result in a greater accumulation of ROS, which in turn can damage more mitochondria and potentially elevate ROS levels even higher. A dysfunctional proteasome system results in a highly oxidant environment [196] that damages the mitochondria and increases ROS production from mitochondria in muscle and neurons [196]. It is therefore likely that proteasome dysfunction contributes to the higher potential for ROS to be generated in muscle and neuron cells of older humans or animals. Although more work is needed, it is likely that higher ROS accumulation increases the oxidant inducing-potential to further damage mitochondria and initiate apoptotic death-signaling cascades leading toward loss of muscle and motor neurons thereby contributing to sarcopenia. UPS and mitochondria are also linked by a splice variant of mitochondrial regulator PGC-1α. PGC-1α2, PGC-1α3, and PGC-1α4 have been shown to stimulate protein synthesis and suppresses UPS activity in cultured myotubes and mouse skeletal muscle [197,198]. Furthermore, PGC-1α reduces muscle protein degradation by the UPS via blocking NF-κB and FOXO3 activity [87,88]. Future studies are needed to determine if one or more of the splice variants of PGC-1α are critical regulators of mitochondrial-mediated UPS function in aging muscles and motor neurons.

LOCALIZED APOPTOTIC SIGNALING SPREADS TO REMOVE THE ENTIRE FIBER IN SARCOPENIA A model for the process by which single muscle fibers are eliminated in sarcopenia is shown in Fig. 26.4. This model assumes that local mitochondrial damage (potentially via ROS, high calcium loads, mtDNA damage, etc.) causes dysfunctional and leaky mitochondria, which are not removed via mitophagy. The accumulation of dysfunctional mitochondria adversely affects other mitochondria within close proximity (via apoptotic signaling, increased Bax, etc.). These adjacent, healthy mitochondria will then have increased mPTP opening to accelerate the wave of apoptotic signaling. Thus, dysfunctional permeabilized mitochondria will initiate a local apoptosis cascade, which when left unchecked, will result in widespread cellular destruction. This concept of regional cellular disruption that III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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FIGURE 26.4 UPS regulation in sarcopenia. Free ubiquitin conjugates to form a polyubiquitin chain and targets damaged proteins in skeletal muscle. Targeting damaged proteins for proteolysis by the 26S proteasome occurs through Muscle RING Finger 1 (MuRF1) and MAFbx/Atrogin [165], which are muscle-specific E3 ubiquitin ligases. The 19S subunits recognize and bind the ubiquitinated proteins and begin their ATP-dependent disassembly and removal in the 26S proteasome. Proteasome degradation results in small peptides that are further digested into amino acids, while lysosomal degradation directly produces single amino acids. Protein degradation from healthy proteasomes results in a reduced cytosolic environment, but dysfunctional proteasomes induce a highly oxidant cytosolic environment, which in turn damages mitochondrial membranes and causes increased ROS production from the damaged mitochondria. Independent from mitophagy, Parkin ubiquitinates outer mitochondrial membrane components for degradation by the proteasome in depolarized mitochondria. While ATG12 participates in mitophagy, when ubiquitinated, it is degraded in the proteasome to prevent its mitophagic functions, and therefore the proteasome has direct and indirect effects on mitochondria function. ATP, adenosine triphosphate.

increases to encompass the entire muscle fiber is strongly supported by the elegant work done by Cheema et al. [62]. They show that regionalized degeneration of individual muscle fibers is associated with mitochondrial dysfunction (as indicated by dysregulation of succinate dehydrogenase and cytochrome c oxidase enzyme levels) in aging. Furthermore, III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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once the apoptotic signals have been accelerated, and perhaps also by the contribution of more widespread ubiquitin initiated proteolysis and widespread autophagy (rather than selective mitophagy of dysfunctional mitochondria), it is possible that genes of necrosis may be activated to contribute to the overall campaign of cellular destruction [62]. This observation provides an important link to our overall hypothesis, by showing that regionalized atrophy occurs concurrent with mitochondrial dysfunction. We speculate that failure of mitophagy to remove these malfunctioning mitochondria initiates cell degradation and cell death pathways including nuclear apoptosis. The initial signaling occurs in a region around the damaged mitochondria, and when this signaling accumulates, the death signaling cascades in a local area of the fiber result in loss of nuclei and other cellular contents within this region. This model explains how specific nuclei might be targeted for apoptotic dismantling, while other nuclei are not (Fig. 26.5). As mitochondrial and contractile protein synthesis rates are depressed in aged muscles along with some level of disruption in proper mitochondrial protein import and translation, there is a reduced anabolic signal, which would result in localized atrophy even if degradation rates remained constant (but degradation increases with aging). Furthermore, an aging-induced reduction in satellite cell function contributes to the inability to repair this area of local damage and the satellite cells themselves may be targets of cellular removal. If this cellular destruction is not corrected, the death signaling cascades may then spread to adjacent areas, resulting in more wide spread fiber atrophy and cellular removal. With sufficient cellular destruction, the entire fiber may be eliminated. This model assumes that failure to regulate proper mitophagy is upstream of sarcopenic muscle fiber loss. Ubiquitin ligases and general autophagy, or cellular degradation occurs to remove proteins within that local area, which in turn results in localized atrophy. Although loss of a few nuclei will not result in death of the completely multinucleated fiber, widespread nuclear loss could lead to removal of the entire myocyte if clearing of the dying mitochondria and the associated death signaling are not corrected. The final steps of degradation of proteins within the muscle fiber occur directly via activating lysosomal cathepsins and cytosolic protease calpains, which progresses toward ATP-dependent ubiquitin-proteasome activation via FOXO3a associated muscle specific MuRF1 and MAFbx/Atrogin-1 regulation (reviewed in [199,200]). Concomitant with the increased degradation is a reduction in protein synthesis, so the net result is loss of the fiber proteins first locally then more longitudinally to encompass the full fiber.

POTENTIAL FOR NUTRITIONAL STRATEGIES TO REVERSE MITOCHONDRIAL DEATH SIGNALING The Polyphenol Green Tea Extract (GTE) Reduces Apoptosis Signaling in Aged Muscles Green tea extract (GTE) which is from the leaves of the Camellia sinensis plant contains high levels of catechins, a class of polyphenols that appears to a significant level of its biological activity. Green tea catechins that are of particular interest include epicatechin,

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FIGURE 26.5 Hypothetical model for eliminating muscle fibers in sarcopenia via localized mitochondrial

associated dysfunction
mitophagy and apoptosis. (A) In healthy muscle, activation of mitophagy eliminates dysfunctional nuclei so that they cannot continue death signaling. (B) Dysfunctional mitochondria that leak their contents to the cytosol will occur in muscle that has received a significant mitochondrial stress (e.g., ROS, inflammatory mediators, etc.). (C
D) This initiates the apoptotic signaling cascades. (E
F) If the dysfunctional mitochondria are not eliminated, apoptotic death signaling may be activated to eliminate myonuclei and this may concurrently or independently result in wide-spread activation of autophagy and the ubiquitin ligase pathway and also, trigger the necrosis signaling pathway to remove muscle proteins, mitochondria and nuclei within the domain of the initial dysfunctional mitochondria (G
H). (I
J) If the death signaling continues to activate other mitochondria, this will increase the extent of dysfunctional mitochondria, which, in turn, will perpetuate signaling for apoptosis, which will remove nuclei, and signaling to eliminate contractile and non-contractile tissue in the fiber segment. This results in eventual elimination of the portion of the fiber and potentially the entire fiber if the signaling spreads unchecked throughout the fiber. We further hypothesize that the initiation of the disassembly and removal of the fiber could be blocked if the dysfunctional mitochondria which initiate the process, are removed (e.g., via exercise) and replaced by healthy mitochondria.

gallocatechin, epigallocatechin, epicatechin-3-gallate, and epigallocatechin-3-gallate (EGCg). EGCg is by far the most abundant and is often believed to be the among the most biologically active catechins in green tea, although this is largely based on its high antioxidant and antiinflammatory properties [201]. Oxidative stress is reduced after eccentric exercise upon green tea supplementation [202] and green tea catechins were shown to

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26. ANTIOXIDANTS AND POLYPHENOLS MEDIATE MITOCHONDRIAL MEDIATED MUSCLE DEATH SIGNALING

reduce the loss of soleus muscle force during a period of hindlimb suspension (HLS) in mice [203]. Furthermore, in vitro studies have shown that purified EGCg can inhibit serum-starvation and staurosporine-induced apoptosis in human myoblasts [204], attenuate muscle protein breakdown, and increase muscle protein synthesis in C2C12 myotubes in response to serum starvation or incubation in TNF-α [205]. As oxidative stress, inflammation, and muscle wasting are all hallmarks of sarcopenia, it is possible that green tea supplementation could suppress death-signaling pathways in sarcopenic muscle. Furthermore, apoptosis is elevated in response to muscle wasting conditions including muscle disuse and early recovery after disuse [23,206
209]. Green tea was able to reduce muscle wasting and loss of function during hindlimb unloading in sarcopenic rats, but it did not provide any further improvement during the reloading period [95]. As green tea has only B50% EGCg, it is possible that either low levels of EGCg are needed to slow muscle wasting, or perhaps, along with EGCg, one or more of the other catechins that are present in green tea [201] may also have an effect on regulating muscle wasting during disuse in old rodents (Table 26.1). The rationale behind a role for apoptosis is that a reduced number of nuclei and decreased DNA accretion during reloading [213] could prevent or at least contribute to the lack of muscle recovery during reloading with aging. We have found that the proapoptotic proteins AIF and Bax were elevated in response to reloading after HLS in muscles from vehicle-treated rats. This observation is similar to that from other studies [50,98,113,119,209]. GTE suppressed Bax protein abundance in reloaded plantaris and soleus muscles [95]. Bcl-xL, a mitochondrial associated antiapoptotic protein, decreased with reloading in the soleus although it was unchanged in plantaris muscles after HLS or reloading, and GTE did not change this response in either muscle after HLS or reloading. Nevertheless, the lower Bax abundance (or the greater Bcl-xL/Bax ratio) in muscles from old animals given GTE would be expected to promote a lower mitochondrial-induced apoptotic environment than that in vehicle treated muscles [95]. While we have previously found that deletion of Bax attenuated denervation-induced muscle wasting [113], it was interesting to note that while GTE suppressed proapoptotic Bax protein abundance in plantaris and soleus muscles of old rats after reloading following HLS, this was unable to improve muscle recovery in the reloaded muscles as compared to vehicle treated animals (Table 26.1). While increases in individual or multiple signaling proteins in apoptotic pathways likely increase the susceptibility for apoptosis, such changes in signaling cannot be interpreted to mean that removal of nuclei (nuclear apoptosis) will absolutely occur. Moreover, the importance or the full role of apoptosis in contributing to a loss of nuclei in postmitotic tissue is not clear and apoptosis has not been reported in all models where muscle loss occurs in young animals [214,215], although this may differ in aging.

Epigallocatechin-3-Gallate (EGCg), Reduces Signaling for Apoptosis in Aged Muscles EGCg is the predominant catechin in green tea. EGCg has strong antioxidant and antiinflammatory properties, and it is believed to be responsible for most of the health benefits linked to green tea. Both disuse and reloading greatly increase the oxidative stress in the

III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

TABLE 26.1

Polyphenols Compounds Regulating Cellular Signaling in Sarcopenia

Nutritional Compound

Model

Tissue

Muscle Responses

Markers of Oxidative Stress

Regulators of Apoptosis

Regulators Apoptosis of Markers Autophagy

EGCg (1.5 mg/kg)

17 months BALB/ cByJ mice

GAS

Muscle weight m

SOD1k

PCG1-αm

PCG1-αm

HNEk

SIRT1m

SIRT1m

Autophagy Markers

UPS Signaling

Reference

[210]

FOXO3EGCg (200 mg/kg diet)

EGCg (50 mg/kg body weight)

20 months SpragueDawley rats

GAS

32 months Fisher Brown Norway Rats

Plantaris and recovery after HLS

IGF-1 m

MuRF1k

IL-15 m

MAFboxk

[211]

Myostatinm Muscle weight m

Bax k

Force m

Caspase 9k

Fiber CSA m

Caspase 3k

Satellite cells m

AIF k

[116]

FADD k TUNEL k GTE (50 mg/kg body wt)

32 months Fisher Brown Norway Rats

Plantaris and Soleus— recovery after HLS

Fiber CSA m

SOD k

p-AKT m

AIF k

Satellite cells m

8-isoPGF2α k

AKT m

Bcl-xL k

GSK3-ß m

Bax k

protein carbonylsk

[95]

444

26. ANTIOXIDANTS AND POLYPHENOLS MEDIATE MITOCHONDRIAL MEDIATED MUSCLE DEATH SIGNALING

affected muscles of rodents [50,216
219]. Therefore, reducing the high basal levels of oxidative stress in aging could potentially reduce muscle loss during disuse conditions and/ or improve muscle recovery during reloading after HLS in muscles from old animals [50]. Oxidative stress is reduced after eccentric exercise upon supplementation with green tea catechins [220]. Furthermore, green tea catechins reduce the loss of soleus muscle force during a period of HLS in mice [203]. EGCg lowers myonuclear apoptosis in the hindlimb muscles of aged rats in response to disuse and improve muscle recovery following reloading [116] (Table 26.2). In these studies, old Fischer 344 3 Brown Norway inbred rats (age 34 months) that were sarcopenic received either EGCg (50 mg/kg body weight) or water daily by gavage [116]. One group of animals received HLS for 14 days and a second group of rats received 14 days of HLS, then the HLS was removed and they recovered from this forced disuse for 2 weeks. Animals that received EGCg over the HLS followed by 14 days of recovery had a 14% greater plantaris muscle weight (P ,0.05) as compared to the animals treated with the vehicle over this same period. Plantaris fiber area was greater after recovery in EGCg (2715.2 6 113.8 μm2) vs vehicle treated animals (1953.0 6 41.9 μm2). Importantly, compared to vehicle treatment, greater Bcl-2 and lower Bax protein abundance (Table 26.2) explain the lower apoptotic potential in the plantaris of EGCg-treated animals during reloading at least in part. The apoptotic index was in fact lower (0.24% vs 0.52%), and the abundance of proapoptotic proteins Bax (222%) and Fas-Associated protein with Death Domain (FADD) (277%) were lower in EGCg-treated plantaris muscles after recovery. Lower AIF and lower FADD abundance, likely contributed to preserve the number of new nuclei (activated myogenic precursor cells) that survived during the reloading period. Presumably, maintenance of a greater pool of these myogenic precursor cells in our model (e.g., satellite cells) that can be activated during periods of muscle reloading after atrophy should improve the ability for muscle to recover from muscle atrophy. While EGCg did not prevent unloading-induced atrophy, it improved muscle recovery after the atrophic stimulus in fast plantaris muscles from rats with sarcopenia. These data represent potentially important observations with clinical implications for the population of elderly persons who suffer from acute disuse (e.g., hospitalization) and then go through some period of rehabilitation in an attempt to recover function. Subsequent studies should be conducted to test if like our observations in old rats, elderly humans have similar benefits from consuming EGCg during a period of rehabilitation following hospitalization or other disuse. Daily ranges of EGCg between 400 and 800 mg has been reported to be safe and mild enough to be consumed by humans with gastric ulcers [222], and ranges up to 3000 mg of green tea catechins have been used in human studies apparently without negative side effects [223]. Assuming an 80 kg human, 3000 mg/d would be equivalent to 37.5 mg/kg of EGCg, but this is less than the 50 mg/kg used in our study in rats. It is possible that very high doses could have undesired effects, because mice that consumed a diet that was very high in EGCg (1% w/w), showed evidence of increased inflammation [224,225]. Thus, while EGCg shows significant promise, the optimal doses of EGCg should be established in elderly humans to obtain the desired biological effects that improve muscle mass after periods of disuse, without incurring undesired side effects.

III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

TABLE 26.2 Resveratrol Regulation of Cellular Signaling in Sarcopenia Nutritional Compound

Model

Tissue

Muscle Responses

Markers of Regulators of Oxidative Stress Apoptosis

Regulators of Autophagy

Apoptosis Markers

Autophagy Markers

RSV (125 mg/kg/day

32 months Fisher Brown Norway rats

Plantaris, recovery after HLS

Force -

p-AMPK m

Bcl-2m

Fatigue k

AMPK m

Bax m

Fiber CSA -

PCG1-αm

Bcl-xL -

Satellite cell proliferation -

SIRT1-

Cleaved caspase 3 k

UPS Reference Signaling [212]

Cleaved caspase 9 k RSV (50 mg/kg/day)— 6 weeks

27 months Fisher Brown Norway rats

WG

Mitochondrial respiration-

SIRT1m

[41]

Bcl-2m Bax m Bcl-2/Bax Cleaved DNA -

RSV (0.05%; B46
60 g/kg/d), 10 months

18 and 28 months C57BL/6 mice

GAS and plantaris

Mass -

MnSOD m

SIRT1m

Force -

CuZnSOD m

PCG1-α -

H2O2 k

Citrate synthase -

HNE k

Cytochrome c -

Protein carbonyls RSV (500 mg/d)—12 weeks 1 exercise

Men and women 65
80 years

Vastus lateralis

Torquem Powerm

[49]

Nuclear 8-OHdG -

BAK1 k

[221]

Bcl-2m

[41]

Fiber CSAm Total myonuclei m Satellite cells m COX10 m RSV (50 mg/kg/day) 1 20%CR—6 weeks

27 months Fisher Brown Norway Rats

WG

Mitochondrial respiration-

SIRT1-

SIRT1-

Bax m Bcl-2/Bax Cleaved DNA -

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26. ANTIOXIDANTS AND POLYPHENOLS MEDIATE MITOCHONDRIAL MEDIATED MUSCLE DEATH SIGNALING

Potential for Resveratrol (SIRT1 Activation) to Reduce Apoptosis and Oxidative Stress in Aged Muscles Resveratrol (RSV) (3,5,40 -trihydroxystilbene) is a compound found largely in the skins of red grapes. RSV has been shown to inhibit protein degradation and attenuate atrophy of skeletal muscle fibers in several in vitro studies [226
229]. A relatively high dose (400 mg/kg/day) of RSV, in vivo has been shown to attenuate slow muscle fiber atrophy following HLS [230] in rodents (Table 26.2). In agreement with these data, we have found that a low dose (12.5 mg/kg/day) of RSV [50] had a trend (P 5 0.06) to blunt fast muscle losses during HLS-induced muscle wasting. Our lab tested the possibility that RSV has the potential to improve recovery of muscle after wasting conditions by reducing apoptotic signaling in aging [212]. This seems plausible because RSV has been shown to improve noncancer cell viability, reduce apoptosis [231,232] and promote stem cell proliferation and cell repair [233,234], but this is cell type and dose dependent. Nevertheless, the function of RSV on skeletal muscle stem cell function during muscle repair is less clear. Sirtuin 1 (SIRT1), a presumed target of RSV, has been reported to increase proliferation [235] of skeletal muscle stem cells/myoblasts in culture, whereas RSV treatment has been found to increase differentiation of myoblasts rather than induce cell proliferation in vitro [233]. It is possible that RSV will function differently in animal and human cells and in skeletal muscle satellite cells in vivo as compared to in vitro. In sarcopenic rodents, the proapoptotic Bax protein abundance was lower in RSV than vehicle-treated plantaris muscles [212]. In a similar pattern, proapoptotic proteins cleaved caspase 3 and cleaved caspase 9 were increased with muscle disuse but the increases in cleaved caspase 9; a mitochondrial-associated proapoptotic protein was suppressed by RSV. RSV suppressed both cleaved caspase 3 and cleaved caspase 9 in the plantaris muscles of the animals that were allowed to recover from disuse as compared to the vehicle treated group (Table 26.2). Bcl-2 was elevated in a similar fashion in vehicle treated and RSV treated plantaris muscles during HLS [212]. During recovery, the Bcl-2 protein abundance returned to control levels in the vehicle-treated plantaris muscle, but it remained elevated in plantaris muscles that were treated with RSV. The protein abundance of the antiapoptotic Bcl-xL was significantly increased in the RSV group following HLS, while no changes were observed in the vehicle (0.20 6 0.16) treated group as compared to the cage control (0.28 6 0.11) animals [212]. During recovery, the abundance of Bcl-xL was elevated in muscles from both the old vehicle (0.83 6 0.11) and old RSV (0.91 6 0.09) treated animals, although there were no differences observed between these two groups [212]. During recovery after disuse in old sarcopenic rats, RSV tended to increase in the apoptotic index by 29% in the vehicle (1.35 6 0.49) treated animals compared to the RSV (0.96 6 0.50) treated animals. Coupled with the increases in Bcl-2 and the reductions in Bax, cleaved caspase 3, and cleaved caspase 9, it is possible that these changes to a muscle environment that is less favorable for apoptosis could have prevented the loss of some nuclei. More nuclei that are available for transcription may improve in muscle recovery, especially in type II fibers, after disuse in the animals of this study. These data support the idea that nutritional intervention with RSV that might both reduce oxidative stress and apoptotic signaling and therefore be beneficial for reducing sarcopenic effects in aging.

III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

POTENTIAL FOR NUTRITIONAL STRATEGIES TO REVERSE MITOCHONDRIAL DEATH SIGNALING

447

Dietary supplementation with RSV also has the potential to exert beneficial effects not only through its’ ability to directly scavenge free radicals [236,237] and upregulate components of the endogenous antioxidant system [235] but also by its’ capacity to modulate the signal transduction and gene expression of several pathways regulating cellular proliferation [238,239]), mitochondrial biogenesis [239,240], metabolism [241], and apoptosis [238]. Specifically, RSV administration significantly increased MnSOD activity and protein content but not CuZnSOD activity in old HLS animals [50,237]. This is analogous with data showing that RSV protected against oxidative stress through the specific induction of MnSOD [237]. Additionally, we have found that RSV administration increased the content and activity of catalase in muscles from old nonsuspended animals, but it did not increase catalase activity or expression further following HLS [50]. This may be in part because catalase activity is already increased with aging and HLS, so it may have reached a maximal point of induction. Perhaps most importantly, RSV administration reduced indices of oxidative stress in gastrocnemius muscles of old HLS animals, as estimated by H2O2 levels and the lipid peroxidation byproducts malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4HNE) [50]. This is congruent with data from our laboratory and others that have shown that RSV protects against H2O2-mitigated lipid peroxidation in vivo [237] and in vitro [242]. The induction of catalase by RSV has previously been demonstrated [237] and, along with increases in MnSOD, may represent important mechanisms by which RSV acts to reduce H2O2 and H2O2-mediated damage. However, these protective effects of increases in antioxidant enzyme activity and concomitant decreases in markers of oxidant load were not seen in young animals administered RSV, suggesting that there is a differential effect of RSV in young and old animals. The fact that RSV only seems to have an effect in old animals is interesting and is plausibly due to different underlying signaling mechanisms that may occur in old animals during disuse [50]. It is also possible that younger animals are able to handle the stress of HLS and the subsequent detrimental effects that are associated with skeletal muscle disuse, and therefore, the preconditioning effect of RSV administration helps to augment the oxidative stress response in mitochondria from old, but not young animals where it is not needed. This is congruent with our finding that H2O2 concentrations were not increased in muscles from young animals following HLS. Although lipid peroxidation markers were increased in muscles from young suspended animals, despite no increases in H2O2 concentrations, this might indicate a temporal role of oxidative stress in young animals during muscle disuse (Table 26.2). In looking at the effects of RSV in humans, older men (n 5 12) and women (n 5 18) 65
80 years of age who completed exercise and took either a placebo or RSV (500 mg/d) were evaluated to test the hypothesis that RSV treatment combined with exercise would increase mitochondrial density, muscle fatigue resistance, and cardiovascular function more than exercise alone [221]. Twelve weeks of aerobic and resistance exercise coupled with RSV treatment did not further improve cardiovascular risk indices of mitochondrial density, or muscle fatigue resistance more than placebo and exercise only treatments. However, RSV -mediated an increase in knee extensor muscle peak torque (8%), average peak torque (14%), and power (14%) after training, whereas exercise did not increase these parameters in the placebo-treated older subjects. Nevertheless, the impact of RSV on sarcopenia was quite interesting. Exercise combined with RSV significantly improved mean fiber area and total myonuclei by 45.3% and 20%, respectively, in muscle fibers from the

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26. ANTIOXIDANTS AND POLYPHENOLS MEDIATE MITOCHONDRIAL MEDIATED MUSCLE DEATH SIGNALING

vastus lateralis of older subjects. Together, these data indicate a novel anabolic role of RSV in exercise-induced adaptations of older persons. However, it might be that to see the effect of RSV on sarcopenia, it must first have an underlying anabolic stimulus such as exercise. If this is found to be a necessary “priming” stimulus, it might be the case that RSV cannot mimic exercise but rather requires exercise to be effective. Thus, RSV combined with exercise might provide a better approach for reversing sarcopenia than exercise alone (Table 26.2).

β-Hydroxy-β-Methylbutyrate (HMB) Reduces Muscle Mass Loss and Myonuclear Apoptosis It should be noted that other compounds have also been shown to reduce apoptotic signaling in the HLS model of unloading and reloading after HLS. This includes β-hydroxy-β-methylbutyrate (HMB) [98] and, therefore, mitochondrial regulation of apoptotic signaling might be a good target to reduce muscle wasting and/or improve muscle recovery after disuse in aging. HMB is a metabolite of the essential branched-chain amino acid leucine. HMB has been found to reduce muscle wasting associated with trauma and cancer cachexia [243
245]. Furthermore, HMB supplementation has been reported to attenuate fiber atrophy and damage during limb immobilization of adult mice [246] and reduce blood urea and urea nitrogen excretion in bed-confined elderly subjects [247]. Although HMB has been shown to reduce apoptosis in human myoblasts under conditions of serum-starvation or staurosporine-induced apoptosis [204], the potential for HMB to improve muscle recovery or to alter muscle apoptotic signaling in response to unloading and reloading following disuse in aging was not known. To address this, we tested the hypothesis that HMB would reduce myonuclear apoptosis in the hindlimb muscles of aged rats in response to disuse and reloading following HLS-induced muscle disuse. Our data show that HMB coupled with exercise attenuated the decrease in muscle fiber area and improved recovery of muscle mass [98,248]. This was associated with reduced myonuclear apoptosis and abundance of proapoptotic proteins Bax and caspase-3 in skeletal muscle (Table 26.3). HMB supplementation has been previously shown to blunt muscle loss in critically ill patients [247,250] including patients with inflammatory diseases [251
253]. This is due, at least in part, to reductions in proteasome activity [254]. In addition, there is evidence that HMB has the potential to improve strength gains in response to resistance exercise in 70year-old males and females [255] and in functional tests of 76-year-old female subjects [249]. Our data build upon these observations by showing that HMB can reduce unloading-reloading muscle fiber area losses in fast skeletal muscle, and this is associated with reduced apoptotic signaling in aged fast (i.e., plantaris) skeletal muscles. In contrast, the positive effects of HMB were minimal in the soleus. The effect of HMB also appeared to suppress apoptotic signaling largely in the predominantly fast myosin containing plantaris muscle compared to the predominately slow myosin containing soleus muscle. The data from this study do not provide an explanation for this finding. Although speculative, it is possible that the rat plantaris muscle is more sensitive to HMB, perhaps the degree of apoptosis was more severe in the plantaris as compared to the rat soleus muscle (apoptotic

III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

TABLE 26.3 HMB Regulation of Cellular Signaling in Sarcopenia Nutritional Compound

Model

HMB (340 mg/ kg) 35 days

34 months Soleus, Fisher reloading Brown after HLS Norway rats

HMB (340 mg/ kg), 35 days

Tissue

34 months Plantaris Fisher reloading Brown after HLS Norway rats

Muscle Responses

Fiber CSA -

Markers of Oxidative Stress

Regulators of Apoptosis

Regulators Apoptosis of Markers Autophagy

p-AKT -

Autophagy Markers

UPS Reference Signaling

[117]

mTOR Satellite cells -

4EPBP1 -

Force m

p-AKT -

Fiber CSA m

mTOR -

Satellite cells m

4EPBP1 -

p-4EBP1 [117]

MyoD labeling m Id2 m HMB (40 mg/ kg body wt)

34 months Plantaris Fisher reloading Brown after HLS Norway rats

p-4EBP1 -

Force m

TUNEL k

Fiber CSA m

Bcl-2 m

[98]

Bax Bcl-2k Bax k Cleaved caspase 3 k Cleaved caspase 9 k (Continued)

TABLE 26.3 (Continued) Nutritional Compound

Model

Tissue

HMB (40 mg/ kg body wt)

34 months Soleus Fisher reloading Brown after HLS Norway rats

Muscle Responses

Markers of Oxidative Stress

Regulators of Apoptosis

Regulators Apoptosis of Markers Autophagy

Force m

TUNEL k

Fiber CSA m

Bcl-2 m

Autophagy Markers

UPS Reference Signaling

[98]

Bax Bcl-2k Bax k Cleaved caspase 3 k Cleaved caspase 9 k

HMB (2 g /d), Women arginine, (2 g/d) 65
90 years lysine (1.5 g/d)

Functional tests m Muscle Force m Protein Synthesis m

[249]

POTENTIAL FOR NUTRITIONAL STRATEGIES TO REVERSE MITOCHONDRIAL DEATH SIGNALING

451

index, 9.9-fold vs 3.2-fold). HMB can buffer oxidative stress in muscle cells [256], suppress oxidative stress-induced apoptotic signaling pathways. For example, total protein degradation and elevated caspase-3 that are induced by lipopolysaccharide-initiated oxidative stress [256] and TNF-α [257] are reduced in myotubes that have been incubated with HMB. Oxidative stress would be expected to be greater in the plantaris than the soleus, because slow muscles like the soleus have a greater antioxidant capacity [258] (e.g., greater mitochondria localized manganese superoxide dismutase, glutathione, etc.), than fast muscles like the plantaris. In this scenario, the soleus would have a lower need for buffering oxidative stress via an external means such as a nutritional intervention. Therefore, HMB would have a lower impact in the soleus than in the plantaris muscle. Another possibility is that mechanisms that regulate muscle loss have some differences in the fast plantaris and slow soleus muscles. For example, the frequency of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive myonuclei as an indication of nuclear apoptosis appeared to be greater in the plantaris vs the soleus muscle after HLS and reloading after HLS. It is therefore possible that multiple apoptotic pathways including cytokine and mitochondrial pathways were activated in the atrophied plantaris muscle [113,259], whereas primarily mitochondrial associated signaling was more prevalent in the soleus muscle during HLS and reambulation after HLS [119]. Although speculative, HMB may have been more effective at suppressing apoptotic pathways associated with muscle loss in the plantaris as compared to pathways regulating muscle loss in the soleus (Table 26.3). Kornasio et al. found that HMB reduces staurosporine- or starvation-induced apoptosis in cultured human myoblasts and mouse C2C12 cells [204]. They also found that HMB could increase the antiapoptotic proteins Bcl-2 and decrease the proapoptotic protein Bax [204]. Our data [98] show that HMB can suppress apoptotic signaling in vivo in response to disuse/unloading or reloading in aged rat muscles. The mechanism by which HMB regulates apoptosis is not clear, but because mitochondrial apoptotic signaling proteins are altered by HMB, it is likely that HMB can regulate mitochondrial signaling and perhaps mitochondrial structure and/or functions. This is possible since HMB stabilizes sarcolemma HMG-CoA reductase (reviewed in [60]). Mevalonic acid, produced from HMGCoA reductase, is a precursor of coenzyme Q and dolichols, which play a major role in mitochondrial electron transport function and myocyte proliferation. Additional studies are needed to determine if HMB affects apoptosis by stabilizing the function of mitochondria in skeletal muscle under conditions of disuse and reloading. Although apoptosis is an important signaling process that occurs during unloading, clearly apoptosis is not the only contributor to muscle wasting, especially in the soleus muscle where muscle loss is typically more severe than in the plantaris during disuse.

Antioxidant Therapies to Reduce Mitochondrial-Induced Apoptosis Signaling in Sarcopenia Mitochondria are targets for ROS damage and are high producers of ROS in aging muscles [86,260]. Reducing mitochondrial ROS production attenuates mitochondria oxidative damage and improves mitochondrial function [54].

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26. ANTIOXIDANTS AND POLYPHENOLS MEDIATE MITOCHONDRIAL MEDIATED MUSCLE DEATH SIGNALING

Vitamin E (i.e., α-tocopherol) and vitamin C (i.e., ascorbic acid) are antioxidants that appear to have a protective effect by either reducing or preventing oxidative damage in skeletal muscle of old animals. Lipid soluble vitamin E prevents lipid peroxidation chain reactions in cellular membranes by interfering with the propagation of lipid radicals. There is a partial ability for oxidative enzymes to compensate for the increased oxidative insult in tibialis anterior muscles of aged rats in response to chronic repetitive loading as compared to young adult animals [51]. This raises the possibility that a greater nutritionally mediated antioxidant profile may reduce the functional losses in sarcopenia. To test this possibility, old rats were given a dietary supplementation with vitamins E and C (Table 26.4). In this study the tibialis anterior was also exercised for 4.5 weeks using repetitive loading in the aged rats to determine if the antioxidant supplements would be additive to exercise and reduce sarcopenia even further [52]. This study [52] showed that vitamins C and E antioxidant supplementation improved indices of oxidative stress associated with repetitive loading exercise and aging (Table 26.4). Furthermore, antioxidant supplementation improved muscle mass and work output in dorsiflexor muscles of aged animals [52]. Thus, antioxidants may provide a good strategy for reducing ROS damage and improve recovery from sarcopenia especially under conditions of loading or exercise where we might expect an acute elevation in ROS accumulation. Like other membranes, mitochondrial membrane composition is affected by the content and type of dietary lipids. Membranes with a high polyunsaturated fatty acid (PUFA) content are more susceptible to oxidative damage, and therefore, diets with a low PUFA are more likely to have membranes that will resist ROS damage. Dietary consumption of fish oil that accompanied caloric restriction was shown to improve muscle fiber cross sectional area and reduce apoptotic signaling or caspase-3, caspase-8/10, caspase 9, and Bax and an increased Bcl-2/Bax ratio in 10-week-old C57BL/6 [261]. The lower cytosolic cytochrome c levels with dietary addition of fish oil is consistent with a reduced mPTP opening. Together, these data suggest that dietary fats such as fish oil can reduce mitochondrialassociated apoptotic signaling in muscles from young animals [261], and we would anticipate that dietary fats should also improve mitochondrial membranes in muscles from old animals and attenuate apoptotic signaling in sarcopenia. However, studies in humans are more mixed. For example, a study using 53 elderly subjects that provided a diet with a high polyunsaturated omega-3 fatty acid (PUFA) level that included 660 mg EPA (eicosapentaenoic acid), 440 mg DHA (docosahexaenoic acid), 200 mg other omega-3 fatty acids and 10 mg of vitamin E during or immediately after meals [264] found no difference in muscle mass or performance as compared to subjects that did not take the PUFA diet (Table 26.4). In contrast, another study reported that healthy, but inactive, elderly individuals (mean age: 71.0 years) that consumed a PUFA diet of 1.86 g EPA and 1.5 g DHA daily for 8 weeks improved muscle protein synthesis [265]. Furthermore, men and women between the ages of 60 and 85 who were given a fish oil derived PFUA diet (1.86 g EPA and 1.50 g DHA) had greater thigh muscle mass, hand grip strength, knee extension torque, muscle power than placebo fed subjects [266] (Table 26.4). This suggested that antioxidants could reduce or reverse sarcopenic muscle loss. The importance of antioxidants could also be due in part to their actions on satellite cells. This is because PUFA supplementation was shown to reduce TNF-α-induced inflammation, caspase-3 activity in the apoptotic pathway, reduce cell death and improve muscle

III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

TABLE 26.4

Antioxidant Regulation of Cellular Signaling in Sarcopenia

Nutritional Compound XJB, 4 weeks

Model

Muscle Tissue Responses

29 GAS months old F344/BN rats

Markers of Oxidative Stress

Muscle Vo m

Muscle and mitochondria Carbonyl levels k

Power m

MnSOD -

Regulators of Apoptosis

Regulators Apoptosis of Autophagy Markers

Autophagy Markers

UPS Signaling Reference [54]

Mitochondria complex activity m Dietary fish oil (PUFAs: 18% EPA, 12%) 1 CR

10-week- GAS old C57BL/6*

Fiber CSA

Lipid hydroperoxides k

Caspase-3k

[261]

Caspase k8/10 Caspase-9 Bax k BCl-2 BCl-2/Bax m AIF k Cytosolic cytochrome c k

5% sodium butyrate for 10 months

Cystine (3 mg/kg/d)

Catalase m

C57Bl/6J GAS mice. Aged 26 months

Grip strength-

C57BL6 mice aged 17 months (middle aged)

Muscle mass m

MnSOD m

Fiber CSA m

GSH/GSSG m p-AMPK m

Serum IL6 k

HNE k

GAS

DNA fragmentation k

SOD1 m Carbonyls k

[262]

XIAP k AMPK m

TUNEL k

[263]

p-AKT m

FAS k (Continued)

TABLE 26.4

(Continued)

Nutritional Compound

Model

Muscle Tissue Responses

Vitamin E (DL-alpha F344/BN TA tocopherol acetate; rats, 30 30,000 mg/kg) and months vitamin C (L-ascorbic acid; 2% by weight) E and 0% vitamin C

Markers of Oxidative Stress

Muscle mass m

GSH m

Work m

GSH/GSSG m

Regulators of Apoptosis

Regulators Apoptosis of Autophagy Markers

Autophagy Markers

UPS Signaling Reference [52]

HNE k DNA Damagek GPx Activitym GPX-1m Catalase activity m Catalase protein m SOD 1 m

1.3 g of PUFA and 10 mg of vitamin E

Men and Thigh women aged 75 years

Muscle mass -

[264]

Power Strength -

1.86 g EPA and 1.5 g DHA

Men and Thigh women aged 71 years

Muscle mass m Power m Strength m Protein synthesis

[265,266]

POTENTIAL FOR NUTRITIONAL STRATEGIES TO REVERSE MITOCHONDRIAL DEATH SIGNALING

455

regeneration and muscle transcription factors that regulate muscle regeneration under a high fat (palmitate)-induced conditions in muscle cell cultures [267]. However, one needs to interpret this with caution because in vitro data exposures may not be the same as in vivo exposures to the antioxidants in animals or humans. Nevertheless, the data are supportive of the idea that antioxidants provide a potential improve to the muscle environment and might reduce the impact of sarcopenia in aging, especially in conditions, which recruit satellite cells such as loading exercises. The importance of reducing ROS production has been demonstrated in old rats that were given a mitochondria-targeted ROS and electron scavenger, XJB-5-131 (XJB), and ROS damage to mitochondria and skeletal muscle protein were reduced, while complexes I, III, and IV activity of mitochondria were improved in aged muscles. Maximal muscle shortening velocity (Vo) and muscle power are reduced with aging in skeletal muscle from old animals or humans [268
274] but XJB improved Vo without improving muscle mass in aging [54]. These observations suggest that preserving mitochondria function by reducing ROS improves muscle function in sarcopenia. Although not strictly an antioxidant, histone deacetylases play a critical role in skeletal muscle that includes antioxidant roles. The class II HDACs regulate muscle formation and repair through the transcription factor myocyte enhancer factor-2 [275]. As neural disruption and elimination occurs in aging, the HDAC4 is relevant to sarcopenia because it is involved in innervation-regulated gene transcription [276]. Pharmacological inhibition of HDAC by butyrate has been shown to reduce muscle loss in aged mice via inhibition of both oxidative stress markers including catalase and SOD1 and lower carbonylated proteins and lower markers of apoptosis including DNA fragmentation and XIAP [262]. A cysteine-based antioxidant given to middle-aged mice reduced the oxidative redox environment of the gastrocnemius muscle as shown by greater MnSOD, GSH/GSSG ratios, and HNE levels of lipid peroxidation [263]. The cysteine antioxidant also reduced serum inflammatory cytokine levels IL6 improved apoptotic regulators AMPK, p-AMPK, and p-AKT and markedly reduced TUNEL positive apoptotic nuclei in muscles of middleaged mice [263] (Table 26.4). Although these studies were done in middle-aged mice, it is likely that antioxidants provide a powerful tool for reducing ROS-mediated apoptosis and thereby reduce sarcopenia in aging.

Caloric Restriction Regulates Apoptosis and Autophagy in Sarcopenia A 30%
40% reduction in calorie consumption from ad libitum feeding without malnutrition attenuates age-associated muscular atrophy and reduces the loss of fibers in aging rodents and nonhuman primates [79,80,277]. Caloric restriction also attenuates the age-induced decrease in muscle force production per cross-sectional area in rodents [278]. Moreover, caloric restriction improved mitochondrial oxidative capacity [279,280] and reduced apoptosis signaling in aged muscles [261]. Part of the improvement in muscle function has been attributed to decreased ROS production and oxidative damage in aging

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26. ANTIOXIDANTS AND POLYPHENOLS MEDIATE MITOCHONDRIAL MEDIATED MUSCLE DEATH SIGNALING

[281,282], increased antioxidant production (to reduce oxidative damage), and attenuated acceleration of the UPS system (reviewed in [283]). A combination of caloric restriction and aerobic exercise has been shown to reduce body fat without losing lean body mass in obese older women with a starting BMI $ 30 kg/m2 and a fat mass .40% [284]. Several groups have investigated the effects of caloric restriction on mitochondrial health. Recently, long-term 30% caloric restriction has been investigated in a middle-aged men and women (52 years of age) [285]. Data from muscle biopsies showed an increase in autophagy markers, Beclin-1 and LC3 and an elevation in antioxidant enzymes MnSOD in muscles from calorically restricted human subjects [285] (Table 26.5). Alternations in mitochondrial membrane integrity during caloric restriction also may be an important deterrent to apoptosis [261] by decreasing long chain n-3 PUFA content and increasing the degree of membrane saturation [289]. This change is hypothesized to affect aging beneficially by protecting membranes against lipid peroxidation and preventing oxidative damage [290,291]. Mitochondrial dynamics, which are closely associated with autophagy signaling, appears to be altered by caloric restriction. In rodents, caloric restriction increased mitochondrial biogenesis and attenuates the decrease in PGC-1α gene expression in skeletal muscles of old rodents [279,280]. The relatively greater level of PGC-1α may be at least partially responsible for maintaining the oxidative capacity in muscles of old calorically restricted animals [279]. In young humans (36.2 years), caloric restriction has been shown to increase muscle transcripts from several genes involved in mitochondrial biogenesis, including PGC-1α, transcription factor A, mitochondrial, and SIRT1 [288]. However, it is not known if similar changes would be found in sarcopenic muscles of aged persons undergo nutritionally balanced caloric restriction. Caloric restriction appears to work at least in part via the mitochondrial metabolic regulator AMPK because when AMPK is knocked out in mice muscles, the anticipated increase in autophagy proteins Beclin-1, ATG5, and LC3II/LC3I were all blunted [292]. Caloric restriction has also been reported to attenuate apoptosis susceptibility by reducing AIF, APAF-1, DNA fragmentation and several caspases and also reduce mPTP opening, which results in a reduced migration of cytochrome c release from the mitochondria to the cytosol [105,293
295]. These findings are particularly important because they suggest that reducing calorie intake, while maintaining an adequate nutritional homeostasis, can act as a mediator to reduce mitochondrial dysfunction and apoptosis and improve mitophagy in sarcopenia (Table 26.5). It would be particularly interesting to investigate the effects of caloric restriction that is combined with nutritional supplements such as HMB, EGCg, RSV, or GTE to determine if the benefits of caloric restriction and the nutritional interventions work in the same or different pathways to reduce sarcopenia. In conditions of starvation, which is not the same as caloric restriction, BIM is phosphorylated by MAPK8/JNK, which removes the BIM-LC8 interaction, allowing dissociation of BIM and Beclin-1. Bcl-2 sequesters Beclin-1 to reduce its availability for initiating autophagy. Thus, starvation is a negative regulator of autophagy, whereas caloric restriction is a positive moderator of autophagy.

III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

TABLE 26.5 Nutritional Compound 20% CR

8% lifelong CR (10 weeks
24 months)

Regulation of Cellular Signaling by Caloric Restriction in Sarcopenia

Model

Tissue

27 months WG Fisher Brown Norway Rats 24 months F344 Rats,

Muscle Responses Mitochondrial respiration-

Markers of Oxidative Stress

Regulators Regulators Apoptosis of Apoptosis of Autophagy Markers

SIRT1-

SIRT1-

Autophagy Markers

UPS Signaling Reference [18,41]

PCG1-αm

Plantaris

Endo G -nucleosome k

[286]

AIF k HSP 27 m HSP70 -

8% lifelong CR (10 weeks
24 months)

24 months F344, rats

Plantaris Fiber CSA m

[287]

Endo G -nucleosome k AIF k

Connective tissue space in muscle k

HSP 27 m HSP70 -

30% CR, 3
15 years

Women and Men aged 52 years

Vastus lateralis

Serum cortisol m

MnSOD m

ULK1 m

[285]

LC3m Beclin-1m

12.5% CR

Men and women aged 36.8 yearsa

Vastus lateralis

TFAM m

Sirt1 m

[288]

PCG1-αm (Continued)

TABLE 26.5

(Continued)

Nutritional Compound

Model

Tissue

25% CR
40% CR

26 months F344 rats

GAS

Muscle Responses Muscle mass k

Markers of Oxidative Stress

Regulators Regulators Apoptosis of Apoptosis of Autophagy Markers Procaspase-3 k

Autophagy Markers

UPS Signaling Reference [132]

Cleaved caspase 3 k Procaspase-9Cleaved procaspase-9 Procaspase-12 k Cleaved caspase-12 k XIAP k DNA fragmentationk Mitochondria ARC k APAF-1k AIF k

a

Not from aging studies. Protein kinase B, (AKT); apoptosis inducing factor (AIF); AMP-activated protein kinase (AMPK); 4-hydroxynonenal (HNE, lipid marker for oxidative stress); BCL2 antagonist/killer 1 (BAK1), caloric restriction (CR); CuZn-SOD, manganese superoxide dismutase 1, (SOD1); eukaryotic initiation factor 4E binding protein 1 (4E-BP1); total superoxide dismutase (SOD) activity (CuZn-SOD, manganese-SOD, an extracellular-SOD); MnSOD-iso-prostaglandin F(2α) (8-iso-PGF2α); Fas-Associated protein with death domain (FADD); fatty acid synthesis (FAS); Forkhead box protein O3 (FOXO3); Bcl-2-associated X protein (Bcl-2); reduced glutathione (GSH); oxidized glutathione (GSSG); GAS, gastrocnemius; Mammalian target of rapamycin (mTOR); mammalian target of rapamycin complex 1 (MTORC1); muscle RING-finger protein-1 (MuRF-1); muscle atrophy F-box (MAFbx/Atrogin-1) tibialis anterior (TA); hindlimb suspension (HLS); β-Hydroxy-β-methylbutyrate (HMB); IGF1, insulin growth factor-I; Interlukin-15 (IL15: transcription factor A, mitochondrial (TFAM); terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) a marker for DNA fragmentation in nuclear apoptosis; mitochondrial targeted electron scavenger XJB-5-131 (XJB); maximal unloaded shortening velocity (Vo); X-linked inhibitor of apoptosis protein (XIAP).

CONCLUSIONS

459

CONCLUSIONS The data summarized in this chapter build on the hypothesis that optimal mitochondrial function, health, and mitochondrial biogenesis are important for maintaining overall muscle mass. Dysfunctional mitochondria contribute to muscle wasting in aging. If this is true, optimizing nutrition (e.g., antioxidants, SIRT1 activators, HMB, etc.) could initiate selective targeting and removal of the dysfunctional mitochondria via increasing the abundance of mitophagy specific proteins and thereby activating mitophagy to reduce apoptotic signaling. Some nutritional interventions might need an underlying anabolic signal to be in place (“priming” the system) such as that invoked from exercise, before they become effective for reducing sarcopenia. A hypothetical model for the potential intervention sites in the process of regulating mitochondrial-initiated signaling for sarcopenia is shown in Fig. 26.6. Fuchsia-colored lettering and arrows identify the nutritional compounds and their corresponding signaling. The site of most mitochondrial damage is the induction of ROS. Antioxidants such as vitamins C, E, and PUFUs can in turn induce or enhance intrinsic antioxidants including MnSOD, catalase, GPx1, and increase the GSH/GSSG ratio [54,263]. The inclusion of antioxidants also results in less oxidant damage to DNA, protein, and lipids including the lipid membranes of mitochondrial [263]. The lower impact of oxidant stress to mitochondria reduces mitochondrial damage and ROS production. In addition, RSV and caloric restriction increase antioxidants such as MnSOD to reduce the oxidant burden of mitochondria. EGCg increases signaling for autophagy through PGC1-α, SIRT1, and FOXO3a to induce mitophagy removal of mitochondria that are irreparably damaged [95,210]. Removing damaged mitochondria and potentially repairing or reversing the damage in repairable mitochondria will be an important role for any nutritional supplement that is expected to reduce sarcopenia effectively. EGCg, GTE, and HMB have been shown to reduce mPTP opening and reduce the apoptotic signaling cascade, which results in a reduction of DNA fragmentation and TUNEL positive nuclei [98,116,212]. RSV and CR also increase antioxidants induce mitochondrial biogenesis [41,49] to replace unhealthy mitochondrial that were removed via mitophagy and this slows muscle wasting. In addition, EGCg, GTE, HMB, and RSV all act to reduce DNA fragmentation [41,117,221], and EGCg reduces UPS signaling to reduce the extent of muscle atrophy in aging [95,98,211,249]. Thus, in total, sarcopenia may be slowed because muscle atrophy is reduced by nutritional intervention especially if the anabolic signal from loading or exercise first primes the muscle for improved responses to nutritional intervention. Nutritional interventions coupled potentially with exercise could increase selective mitophagic targeting of dysfunctional mitochondria by elevating Bcl-2 and 19 kDa interacting protein-3 (Bnip3) levels. Bnip3 functions as mitophagy receptor to recruit autophagosomes selectively for clearance. Alternatively, nutrition-exercise-induced Pink1 activation of DRP1-associated fission machinery [139] in response to mitochondrial damage [144] or depolarization [143] would also be expected to result in targeted mitophagy. Indeed, exercise has been shown to increase DRP1 and Bnip3 [296
300], which suggests a potential mechanism whereby exercise may target dysfunctional mitochondria for elimination. Once targeted mitophagy has been initiated, exercise and nutritional interventions

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26. ANTIOXIDANTS AND POLYPHENOLS MEDIATE MITOCHONDRIAL MEDIATED MUSCLE DEATH SIGNALING

FIGURE 26.6 Nutritional targeting of mitochondrial initiated sarcopenia. Nutritional interventions target different sites along the pathways leading to sarcopenia. Fuchsia colored lettering and arrows identify the nutritional compounds and their corresponding sites where the influence signaling. The site of most mitochondrial damage is the induction of ROS. Antioxidants such as vitamins C, E, and PUFUs can reduce oxidant damage to DNA, protein and lipids including the lipid membranes of mitochondrial. RSV and CR increase antioxidants to reduce the oxidant burden of mitochondria. EGCg increases signaling for autophagy. EGCg, GTE, and HMB reduce mPTP opening, and the corresponding apoptotic signaling cascade, which results in a reduction of DNA fragmentation. EGCg reduces UPS signaling to reduce the extent of muscle atrophy in aging. ROS, reactive oxygen species; RSV, resveratrol; CR, caloric restriction; GTE, green tea extract; HMB, β-hydroxy-β-methylbutyrate; UPS, ubiquitin proteasome system.

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working together might also elevate the abundance of other more general autophagy proteins to help accelerate removal of dysfunctional mitochondrial and other proteins. Although speculative, another way that nutrition could target mitochondria for elimination is via the chaperone mediated autophagy (CMA) pathway. CMA involves the binding of heat shock protein 8 (HSPA8/HSC70), to selected proteins, which are targeted to the lysosomal membrane, where they interact with membrane receptor lysosomal-associated membrane protein 2A (LAMP2A). Aging causes an increased degradation and subsequently decreased availability of LAMP2A in the lysosomal membranes [301], which reduces the effectiveness of CMA. Future studies should evaluate the role of LAMP2A and determine if exercise or nutritional interventions increase CMA and targeted mitochondrial mitophagy in muscles of old animals or humans. Nutritional interventions could selectively target dysfunctional mitochondria through degradation of PARK7/DJ-1 [138]. PARK7 has been studied extensively in Parkinson disease, but it has been also shown to regulate skeletal muscle contractile protein synthesis and hypertrophy [302]. PARK7 is also an antioxidant protein that limits mitochondrial damage to oxidative stress [303]. Overexpression of PARK7 has been shown to reduce mitochondrial dysfunction under oxidative stress [138] which shows a direct link between autophagy and mitochondrial function. Future studies should identify if exercise or nutritional intervention (e.g., antioxidants, caloric restriction) improves PARK7 expression in muscles of old hosts and if the change in PARK7 directly mediates improvements in skeletal muscle mitochondrial function to attenuate or reverse sarcopenia. It is important to know if there becomes a point when it is too late to repair or replace cells that are involved or targeted for destruction. It will also be important to understand if satellite cells have a role in replacing muscle cells and repairing these fiber sections, as satellite cells will also be affected adversely by death signaling [22,95,117,221,304]. Dysfunctional mitochondria are hypothesized to be the initiators of signaling that leads to sarcopenic muscle loss. If this is proven to be correct, the best approach for reducing the incidence and impact of sarcopenia should include the global goal of understanding the mechanisms by which nutrition targets muscle and potentially neural mitochondria and optimize this information for clinical outcomes that improve mitochondria health and maximize muscle mass in aging populations.

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β-Conglycinin and Skeletal Muscle Satoshi Wanezaki* and Nobuhiko Tachibana* Research Institute for Creating the Future, Fuji Oil Holdings Inc., Osaka, Japan

LIST OF ABBREVIATIONS AMPK AICAR AR1 GLUT4 PI3K LDL SPI SREBP1 VLDL

AMP-activate protein kinase Acadesine/AICA riboside adiponectin receptor 1 glucose transporter-4. phosphatidylinositol 3-kinase. Low-density lipoprotein soy protein isolate. sterol regulatory-element binding protein 1. very low-density lipoprotein

INTRODUCTION The Leguminosae family has approximately 600 lines (including 13,000 species) worldwide; these plants are highly useful. Soybean (Glycin max L. Merrill) is most common representative of the legume family and contains linoleic oils, dietary fibers, polysaccharides, and storage proteins. Based on previous reports concerned with soy, the soy proteins have many physiological functions, particularly plasma cholesterol lowering effects [1
14]. Intake of plant proteins has been found to suppress blood cholesterol levels compared with intake of animal protein, and plant proteins also reduce the thickness of the arterial wall in the cardiac aortic arch [1
3]. A meta analysis of 38 articles demonstrated a significant improvement of blood lipid profiles following intake of soy proteins [5,6]. Indeed, studies have shown that consumption of soy protein significantly decreases blood cholesterol, low-density lipoprotein (LDL)-cholesterol, and triglycerides and significantly increases high-density lipoprotein-cholesterol [5]. The physiological functions of soy protein isolate (SPI) in the liver *

These authors contributed equally to this review.

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00027-0

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© 2019 Elsevier Inc. All rights reserved.

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have been extensively studied [13
16]. For example, a comprehensive analysis using a DNA microarray revealed the influence of SPI intake on homeostasis, such as glycolysis, gluconeogenesis, fatty acid metabolism, cholesterol de novo synthesis, and catabolism [16]. Soy proteins are composed of several protein complexes, not a single protein. Soy protein possesses three major storage proteins: lipophilic protein, 11S globulin (glycinin), and 7S globulin (β-conglycinin) [17
20]. β-Conglycinin is a structural protein consisting of 7S globulin α-, α0 -, and β-subunits.

EFFECTS OF β-CONGLYCININ ON LOWERING OF BLOOD TRIGLYCERIDES The intake of β-conglycinin has several physiological effects, including beneficial health effects, [21], prevention of age-related hearing impairment [22], improving renal functions [23], suppression of blood triglyceride levels [15,24
30], improvement of obesity-induced metabolic abnormalities [31], lowering of body fat [32,33], and improvement of blood glucose control [34,35]. In an experiment in old adult rats, β-conglycinin intake strongly lowered plasma triglyceride levels through suppression of triglyceride synthesis in the liver and of dietary fatty acid absorption in the intestine [25]. Similar phenomena induced by β-conglycinin consumption were shown in diabetic fatty rodents [26]. The mechanism of lowered blood triglycerides is thought to be related to activation of acyl-CoA oxidase, which controls hepatic fatty acid beta-oxidation, and suppression of fatty acid synthase. Interestingly, in normal ICR mice and diabetic kk-Ay mice, significant decreases in blood glucose and plasma insulin levels were observed following β-conglycinin intake compared with casein intake and other soy protein fractions (e.g., glycinin). The decrease in triglyceride levels, particularly triglycerides contained in Very low-density lipoprotein (VLDL), is closely related to glycemic control [27]. In oral administration tests of dietary glucose in normal rats, a significant suppression of the hyperglycemic effect was observed following β-conglycinin intake, suggesting progression of glucose metabolism in several tissues. In addition, β-conglycinin induced significant suppression of blood glucose levels in insulin tolerance tests. These results demonstrated that the skeletal muscle, which makes up 60% of body tissues, is the main organ of glucose metabolism and enhance glucose uptake in the muscle by consumption of β-conglycinin. The decrease in triglyceride release from the liver through β-conglycinin intake is thought to prevent hypertriglyceridemia. The mRNA expression of fatty acid synthase, which contributes to triglyceride content in the liver, is mainly regulated by sterol regulatory-element binding protein 1 (SREBP1) in an insulin-dependent manner [36]. Moreover, intake of β-conglycinin suppresses hepatic triglyceride synthesis, and this process depends on the concentration of insulin. Thus, the reduction of triglyceride release from the liver is assumed to be related to blood triglyceride suppression by β-conglycinin [27].

PROMOTION OF ADIPONECTIN PRODUCTION β-Conglycinin consumption modulates various metabolic factors related to adipose tissues, such as adipocytokines. Adiponectin, a member of the adipocytokine family, activates glucose metabolism in the skeletal muscle and accelerates lipid oxidation in the III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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liver [37
42]. These effects of adiponectin involve signal transduction pathways related to the adiponectin receptors of target cells [43
45]. In 3T3-L1 adipocytes, hydrolysis of β-conglycinin, but not glycinin, promotes adiponectin production and suppresses fat accumulation [46]. Intake of the β subunit of β-conglycinin-specific pentapeptide (YPFVV) may increase adiponectin in diabetic kk-Ay mice [47]. In general, the production of adiponectin is negatively correlated with the fat content of adipose cells [38,39]. The suppression of lipid accumulation in the fat pad induced by β-conglycinin may increase adiponectin levels [28]. Indeed, feeding of β-conglycinin for 4 weeks was found to increase plasma adiponectin levels and improve glucose tolerance (oral glucose tolerance tests and insulin tolerance tests) [27]. In addition, the results of respiratory quotient analysis showed that β-conglycinin-induced upregulation of glucose metabolism was closely related to the increase in adiponectin [35].

β-CONGLYCININ INFLUENCE GLUCOSE METABOLISM IN SKELETAL MUSCLE Interestingly, assessment of energy consumption at 24 hours indicated that glucose, but not lipids, was significantly consumed in the β-conglycinin group in the dark phase [35]. These results suggested that the ingested glucose energy was used without being converted into fatty acids, an optimized form of energy accumulation. Thus, the lower lipid utilization in the respiratory quotient was induced by the decrease in internal fat accumulation following β-conglycinin consumption. Muscles account for the majority of glucose consumption. Glucose uptake in skeletal muscle is precisely controlled by insulin. Consumption of β-conglycinin in diabetic Goto
Kakizaki rats suppresses fasting glucose levels compared with consumption of a casein control diet [34]. In skeletal muscle tissues, glucose transporter-4 (GLUT4) translocation to the skeletal muscle cell surface was induced in the β-conglycinin group (Fig. 27.1). In contrast to the glucose uptake enhancement and hypoglycemic activity of β-conglycinin, plasma insulin levels were significantly lower following consumption of β-conglycinin than those of the control. In a study aiming to clarify the influence of β-conglycinin on the insulin signaling cascade, researchers showed that insulin receptor (IR) substrate-1 mRNA and phosphorylation of phosphatidylinositol 3-kinase (PI3K) were not different between animals consuming a β-conglycinin diet and a casein diet. Because there were no changes in the phosphorylation of Akt between the two groups, the authors concluded that β-conglycinin promoted GLUT4 translocation to the skeletal muscle cell surface in an insulinindependent manner under the fasting conditions. There are two signaling pathways that induce GLUT4 translocation to the skeletal muscle cell surface: the insulin-dependent IR signaling cascade and the insulin-independent AMP-activated protein kinase (AMPK) pathway [48
54]. Many studies have reported that exercise enhances glucose uptake through activation of AMPK [51
54]. The consumption of β-conglycinin promotes the phosphorylation of AMPK [34]. Adiponectin receptor 1 (AR1) is predominantly expressed in muscle cells and is known to contribute to glucose uptake [45]. A significant increase in AR1 mRNA expression in the β-conglycinin group was found to contribute to AMPK activation through the adiponectin signal in skeletal III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE

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FIGURE 27.1 Consumption of β-conglycinin influenced GLUT4 translocation in skeletal muscle, [34]. GLUT4 transferred from GLUT4 vesicle to PM promoted by various signals. GLUT1 represents as a housekeeping protein. Part (A) showed GLUT4 contents at the PM. Part (B) showed GLUT4 contents in the Cy. The column and the error bars represent mean and standard error (SE) (n 5 3), respectively. Open column and striped column represent the Cas group and the β-conglycinin group (βCG), respectively. *The significant difference analyzed by Welch test (P , 0.05). glucose transporter-4; PM, plasma membrane; Cy, cytosol; Cas, casein.

muscle [34]. Therefore, regulating blood glucose by β-conglycinin facilitates improvement of glucose metabolism in skeletal muscle, which does not cause insulin overload.

PEPTIDES DERIVED FROM β-CONGLYCININ MODULATE GLUT4 TRANSLOCATION The hydrolysate of βCG (βCGP) may directly improve glucose metabolism in skeletal muscle cells [55]. The translocation of GLUT4 onto the L6 skeletal muscle cell surface is strongly induced by βCGPs, mimicking digestion using pepsin and trypsin. Glucose uptake into L6 cells using 2-deoxyglucose is strongly increased by βCGP addition (Fig. 27.2). Thus, βCGPs strongly enhance glucose uptake through GLUT4 translocation. Induction of GLUT4 translocation by βCGPs is dependent on the AMPK signaling pathway, not PI3K activation [55]. Although the mechanism through which βCGPs activate AMPK is still unclear, this mechanism is unlikely to involve signal pathway activation through nitric oxide. Thus, there are two potential alternatives: AMPK activation via the AR1 signaling pathway and direct effects of βCGPs on AMPK.

A NOVEL PROTEIN SIMILAR TO β-CONGLYCININ: MUNG BEAN PROTEIN As previously described, β-conglycinin is a structural protein consisting of 7Sα-, 7Sα0 -, and 7S β-subunits. However, another protein has been found to have high homology with

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CONCLUSIONS

1.5

d

2-DG uptake in L6 cells (AU)

b, d a

a, c

b, c

1

3

a

1.0

0.5

0.0 Control insulin

0.3

10

βCG peptides (mg/mL)

FIGURE 27.2 Effects of β-conglycinin peptides on glucose uptake in L6 myotubes [55]. Glucose uptake was measured in L6 myotubes treated with 0.3
10 mg/mL β-conglycinin (βCG) peptides for 4 h. The cells were also treated with Dimethyl sulfoxide (DMSO) and 100 nM insulin as the negative and positive controls, respectively. Values are represented as means 6 SE (n 5 3). Different letters indicate significant differences between the groups (P , .05; Tukey honestly significant difference (HSD) test).

7S globulin in the amino acid sequence [56]. Indeed, mung bean protein, which mainly consists of 8S globulins, has high amino acid sequence homology with β-conglycinin (similarity 68%, Fig. 27.3). Feeding a diet containing mung bean protein to normal rats decreases plasma insulin levels and blood triglyceride levels through suppression of fatty acid synthesis in the liver [57]. Additionally, consumption of mung bean protein has been shown to alleviate symptoms of nonalcoholic fatty liver [58]. Interestingly, despite administration of a mixture containing the same amino acid composition as that of mung bean protein, the mixture had no triglyceride-lowering effects. These findings suggested that mung bean protein may also include functional peptides having physiological effects similar to those of β-conglycinin. Therefore, the mung bean 8S globulin, similar to β-conglycinin, is also likely to improve glucose metabolism in skeletal muscle.

CONCLUSIONS β-Conglycinin is one of the components of soy bean protein. Intake of β-conglycinin affects various metabolic phenomena, such as lowering blood triglyceride levels, decreasing body fat, and normalizing glucose homeostasis. Fig. 27.4 shows an outline of metabolic fluctuations in skeletal muscle by ingestion of β-conglycinin. The action of β-conglycinin (or its peptides) induce GLUT4 translocation by

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27. β-CONGLYCININ AND SKELETAL MUSCLE

FIGURE 27.3 Amino-acid sequence alignment of 8Sα globulin and other seed storage 7S globulins obtained using ClustalW (http://align.genome.jp/) [56]. 8S, 8Sα globulin of mung bean (accession no. PRF3021374A); Conβ, soybean β-conglycinin β (accession no. P25974); Cana, jack bean canavalin (accession no. P50477); Pha, kidney bean phaseolin (accession no. AAC04316). 8Sα globulin has high amino acid sequence homology with β-conglycinin β-subunit (similarity 68%).

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CONCLUSIONS

481

FIGURE 27.4 Outline: β-Conglycinin and/or its peptides modulate glucose uptake in skeletal muscle through AMPK activation [34,55]. Intake of β-conglycinin increases glucose uptake in skeletal muscle. Under the condition of β-conglycinin-fed, GLUT4 translocation is induced by AMPK activation, but is not controlled by IR signaling cascade. Activation of AMPK is promoted various signals such as exercise, medicine Acadesine/AICA riboside (AICAR), and unknown activating components. Adiponectin, which is an activator of AMPK through AdipoR1, is one of them. Intake of β-conglycinin increases adiponectin in vivo. Artificial enzyme-hydrolyzed β-conglycinin peptides promote uptake of 2-deoxyglucose, a glucose analog, accompanied by translocation of glucose transporter 4 in skeletal L6 myotubes. Although the mechanism is unclear, β-conglycinin peptides activate AMPK in vitro. IR, insulin receptor; AdipoR1, adiponectin receptor-1; IRS1, insulin substrate-1; PI3K, phosphatidylinositol 3-kinase; GLUT4, glucose transporter-4.

AMPK activation, in contrast to the insulin signaling cascade, and activates glucose uptake into the skeletal muscle [34,55]. In this case, it is not clear whether AMPK is activated through adiponectin signaling pathways or by β-conglycinin peptide itself as a signal. In either case, there is no doubt that β-conglycinin plays an important role in various organs by affecting metabolism in skeletal muscle. Further studies will clarify what is the key factor of the β-conglycinin and how β-conglycinin modulates the glucose metabolism in the skeletal muscle.

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60.

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C H A P T E R

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Effects of Quercetin on Mitochondriogenesis in Skeletal Muscle: Consequences for Physical Endurance and Glycemic Control Alfredo Ferna´ndez-Quintela1,2, In˜aki Milton-Laskibar2, Leixuri Aguirre1,2, Saioa Go´mez-Zorita1,2, Marcela Gonza´lez3 and Maria P. Portillo1,2 1

CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Spain 2Nutrition and Food Sciences Department, Faculty of Pharmacy, University of the Basque Country, Paseo de la Universidad 7, 01006, Vitoria-Gasteiz, Spain 3Nutrition and Food Science Department, National University of Litoral, National Council for Scientific and Technical Research (CONICET), Santa Fe, Argentina

LIST OF ABBREVIATIONS ACO Akt AUC BMI CD36 COX2 CPT-1b CS EGCG GIR

acyl-CoA oxidase protein kinase B Area under the curve body mass index cluster of differentiation 36 cyclooxygenase 2 carnitine palmitoyltransferase I citrate synthase epigallocatechin 3-gallate glucose infusion rate

Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00028-2

485

© 2019 Elsevier Inc. All rights reserved.

486 GRAS status MRP-2 mtDNA PGC-1α PPARα PI-3K PUFA SGLT-1 SIRT1 TFAM UCP3

28. EFFECTS OF QUERCETIN ON MITOCHONDRIOGENESIS IN SKELETAL MUSCLE

generally recognized as safe multidrug resistance protein 2 mitochondrial DNA content peroxisome proliferator-activated receptor gamma coactivator 1 alpha peroxisome proliferator-activated receptor alpha phosphoinositol 3-kinase polyunsaturated fatty acids sodium-dependent glucose transporter-1 sirtuin 1 Mitochondrial transcription factor A uncoupling protein 3

INTRODUCTION Quercetin (3,30 ,40 ,5,7 pentahydroxyflavone) is a phenolic compound belonging to the family of flavonoids, and within this family, to the group of flavonols (Fig. 28.1). In food stuffs, it can be present as aglycone or as glycoside (rutin and quercitrin). Common food sources of quercetin are onions, apples, various types of berries, coffee, and tea [1] (Table 28.1). This flavonoid is also available in dietary supplements and included in functional foods [2]. The average dietary intake of quercetin not including supplements is between 6 and 31 mg/day [2]. Approximately 60% of quercetin from diet is absorbed. It passes through the small intestine, is hydrolyzed to aglycone by enterobacteria in the cecum and colon, and is absorbed into epithelial cells of the colon via lipophilicity-dependent simple diffusion because, once aglycosylated, the molecule becomes more lipophillic [3]. Quercetin glucosides can also be directly absorbed via the sodium-dependent glucose transporter-1 (SGLUT1) or excreted into the lumen via multidrug resistance protein 2 [4]. In this case, quercetin glycosides can be hydrolyzed by intracellular β-glucosidases [5]. Quercetin absorbed from the intestinal lumen is mostly converted into conjugated metabolites before entering circulation. Both, quercetin and its metabolites, can be accumulated in tissues in different proportions. It has been reported that quercetin exhibits a wide range of biological functions including antiinflammatory, anticancer, immunoregulatory, anticoagulant, antihypertensive, neuroprotective and antioxidant activities. It has also been shown that quercetin can increase mitochondrial biogenesis [6]. Quercetin has generally recognized as safe, and there are no reports of harmful side effects in animals or humans at doses of several grams per day [7]. This chapter focuses on the effect of quercetin on skeletal muscle mitochondria biogenesis and consequences for physical endurance and glycemic control in rodent models and human beings.

FIGURE 28.1 Chemical structure of quercetin (PubChem web page).

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TABLE 28.1 Weighta) [1]

Quercetin Content (mg per 100 mL or 100 g of Fresh

Lovage, fresh

170.00

Dill, fresh

55.15

Fennel, fresh leaves

46.80

Angelica, fresh

41.53

Lettuce (red), raw

40.27

Pak choy, raw

39.00

Dill, dried

36.00

Chilli pepper (yellow), raw

32.59

American cranberry

17.34

Onion (red), raw

17.22

Bog bilberry, raw

17.03

European cranberry

16.39

Onion (yellow), raw

12.65

Lingonberry, raw

12.04

Half-highbush blueberry

10.25

Tarragon, fresh

10.00

Chilli pepper (green), raw

9.93

Black chokeberry

8.90

Sweet rowanberry

8.50

Evergreen huckleberry

8.41

Highbush blueberry, raw

7.82

Kale, raw

7.71

Swiss chard leaves (red), raw

7.50

Blackcurrant, raw

7.10

Lowbush blueberry, raw

6.70

Rowanberry, raw

6.30

Sea-buckthorn berry

6.20

Spinach, raw

5.87

Black huckleberry

5.77

Black crowberry

5.45

Tomato (cherry), whole, raw

4.56

Oval-leaf huckleberry

4.49 (Continued)

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28. EFFECTS OF QUERCETIN ON MITOCHONDRIOGENESIS IN SKELETAL MUSCLE

TABLE 28.1 (Continued) Broccoli, raw

4.25

Rabbiteye blueberry

4.03

Watercress, fresh

4.00

Fennel, dried

3.60

Chives, fresh

3.57

Cascade huckleberry

3.41

Onion (white), raw

3.25

Green currant

3.20

Green bean, raw

3.12

Arctic blackberry

3.10

Coriander, dried

3.00

Sour cherry

2.92

Chilli pepper (red), raw

2.92

Bilberry, raw

2.53

Coriander, fresh

2.50

Apple (dessert), whole, raw

2.47

Swiss chard leaves (white), raw

2.20

Plum, fresh

2.10

a

Data obtained by chromatographic methods after hydrolysis.

QUERCETIN AND PHYSICAL ENDURANCE It is well accepted that one of the main determinants of individual variations in endurance performance are the metabolic properties of skeletal muscle, particularly its mitochondrial oxidative potential. Exercise training is the best strategy to increase muscle mitochondria number and function. One of the numerous adaptations to regular aerobic exercise (endurance) is improved skeletal muscle capacity for oxygen consumption, which in turn is a direct result of higher mitochondrial content. However, the mechanism by which endurance exercise affects mitochondrial remodeling, leading to higher mitochondrial content, remains to be clearly elucidated [8]. In spite of this interesting effect on exercise, the maintenance of a regular program is somewhat difficult, and thus, other strategies have received increasing attention. One of them is the use of phenolic compounds such as quercetin. In 2009, Davis et al. [9] carried out a study devoted to evaluating the effects of shortterm quercetin supplementation on skeletal muscle mitochondrial biogenesis and endurance exercise tolerance in a mouse model (8-week-old Institute of Cancer Research (ICR)

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489

male mice). For this purpose, the animals were housed individually in two types of cages (with or without a running wheel) in order to test voluntary and involuntary running performance. The mice in the regular cages were supplemented with quercetin at doses of 12.5 or 25 mg/kg body weight/day or with placebo. In the case of the mice housed in the cages with the running wheel, quercetin was administered at a dose of 25 mg/kg body weight/day. These doses were considered by the authors as safe and practical for use as a dietary supplement. After 7 days of treatment, the expression of genes related to mitochondrial biogenesis, such as peroxisome proliferator-activated receptor gamma coactivator 1 alpha (pgc-1α), sirtuin 1 (sirt1), mitochondrial DNA content (mtDNA), and cytochrome c enzyme concentration, were assessed in soleus muscles of the animals housed in the regular cages. This muscle was chosen because of its obvious relevance to endurance exercise capacity. PGC-1α is considered the master regulator of mitochondria biogenesis following physiological demands and nutritional inputs [10]. In addition, it drives the formation of slowtwitch muscle fibers [11] and is a critical regulator of skeletal muscle fuel stores, all of which are essential for endurance exercise capacity [12]. SIRT1 functions together with PGC-1α to promote mitochondrial biogenesis [13]; in fact, it interacts with and deacetylates PGC-1α at multiple lysine sites, thus increasing PGC-1α activity (Fig. 28.2). An increase in mtDNA shows an increase in relative copy number of mtDNA per diploid nuclear genome and cytochrome c oxydase, a component of the electron transport chain, which in general increases in conjunction with similar increases in other mitochondrial

FIGURE 28.2 Mitochondriogenesis pathway.

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markers and the oxidation pathway that lead to an overall increase in mitochondrial capacity. In this study, increased mRNA levels of pgc-1α and sirt1 were observed in quercetinsupplemented mice, with no differences between both doses. In the case of mtDNA, it was increased only in the group supplemented with the highest dose (25 mg/kg body weight/day). Moreover, the activity of cytochrome c oxydase was increased by both doses of quercetin. According to these results, the authors proposed that the absence of an increase in mtDNA content with the 12.5 mg/kg dose of quercetin could be due to the short feeding duration. Indeed, increases in mitochondrial enzymes have been shown to occur more quickly and with less stimuli than increases in mitochondrial replication [14]. Then, in order to know the influence of these quercetin-induced effects on maximal endurance capacity, mice were run until fatigue on a treadmill. The run time of the mice on both quercetin supplementation doses (12.5 and 25 mg/kg) was greater than in the controls, with no differences between the former. As far as voluntary wheel-running activity is concerned (cages with running wheel), the mice supplemented with quercetin showed greater activity than those in the control group. Taking together, these results suggest that short-term supplementation with quercetin can enhance mitochondria in skeletal muscle, as demonstrated by the increased expression of mitochondriogenesis markers. This increase led to higher maximal endurance running capacity and voluntary physical activity. Although the authors did not measure fatty acid oxidation, they suggested that the mechanism involved in this improvement was, at least in part, increased oxidative metabolism in muscle. If these results could be translated clinically, the benefits of quercetin may have important implications for enhancing athletic performance. In this context, Nieman et al. [15] carried out an interventional study to test the influence of a quercetin supplement, either alone or in combination with other compounds [epigallocatechin 3-gallate (EGCG), isoquercetin, n-3 polyunsaturated fatty acids (PUFA), vitamin C and folic acid] on mitochondrial biogenesis in skeletal muscle. Supplements were ingested by participants twice daily for 2 weeks before, during, and 1 week after the 3-day period of exercise, with the last daily dose taken 1 hour before exercise to ensure high plasma quercetin levels. For this purpose, trained cyclists (males and females) were divided into three groups: placebo group, quercetin group supplemented with 1000-mg quercetin/day and quercetin-EGCG group supplemented with 1000-mg quercetin/day, 120-mg EGCG/day, 400-mg isoquercetin/day, and 400-mg n-3 PUFA/day. The three days of exercise training consisted of cycling for 3 hours (from 3 to 6 p.m.) at B57% Vmax. At the end of the experimental period gene expression of citrate synthase (CS), cytochrome c oxidase I and pgc-1α, well-known markers of mitochondrial biogenesis, was analyzed in vastus lateralis muscle biopsies obtained from the participants. The authors observed no changes in mRNA levels of the aforementioned genes among the three experimental groups. In addition, no effects were appreciated in physical performance enhancement after quercetin supplementation (alone or combined with other compounds). Thus, the interesting effects induced by quercetin in mice were not reproduced in humans. This difference between mice and humans could be due to a species specific response, but also to differences in the experimental designs in terms of doses of quercetin, experimental period length, type of muscle analyzed, and training status among others. Consequently,

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further studies are needed in order to know whether quercetin may be useful in increasing muscle mitochondriogenesis and physical endurance in humans. In order to know whether training status was an important factor influencing the effects of quercetin on muscle mitochondriogenesis, the same research group carried out a later randomized, double-blinded placebo-controlled, crossover design, in subjects with a body mass index (BMI) lower than 27 kg/m2, without diseases or disease risk factors [16]. One of the objectives of this study was to analyze the effect of 2 weeks of quercetin ingestion on skeletal muscle mitochondrial biogenesis in untrained subjects. The participants were supplemented with 1 g/day of quercetin or placebo for 2 weeks, and they had a 2-week washout period before the crossover. Skeletal muscle expression of genes related to mitochondrial biogenesis sirt1, pgc-1α, cytochrome c oxidase, and CS tended to increase after 2 weeks of quercetin supplementation, compared to placebo group, although without reaching statistical significance. When mtDNA was quantified in this tissue, no differences between quercetin and placebo groups were found. Once again, the interesting results obtained in rodents were not replicated in human beings. The authors proposed the testing of higher doses and longer experimental periods in further studies, in order to find significant effects.

QUERCETIN AND GLUCOSE TOLERANCE For decades, mitochondria were considered to be simply the cell powerhouse. Recent findings have demonstrated that these organelles play a key role in many common diseases in our society, among them insulin resistance and type 2 diabetes. Ectopic fat deposition (triglyceride accumulation) in nonfat-storage tissues, notably skeletal muscle, occurs when white adipose tissue reaches its physical and functional limits, promoting insulin resistance and type 2 diabetes. At the cellular level, lipid-induced skeletal muscle insulin resistance has been associated with ceramide accumulation, and diacylglycerol generation, which lead to the impairment of insulin signaling cascade [17]. In this situation, skeletal muscle exhibits decreased respiration rates and lower oxidative enzyme levels due to the dysfunction of the mitochondrion itself and/or to a decrease in total mitochondrial number within the tissue [18]. Quercetin has been reported to interact with many molecular targets in the small intestine, pancreas, skeletal muscle, adipose tissue, and liver to control whole-body glucose homeostasis. Mechanisms of action of quercetin are pleiotropic and involve the inhibition of intestinal glucose absorption, insulin-secreting, and insulin-sensitizing activities as well as improved glucose utilization in peripheral tissues [19]. As quercetin is able to induce mitochondriogenesis, several studies have been carried out in rats and mice, whose purpose was to analyze the potential involvement of this effect in the hypoglycemic action of quercetin (Fig. 28.3). Henagan et al. [20] investigated the effect of two doses of quercetin on insulin resistance induced by high-fat feeding, as well as on mitochondrial function and pgc-1α expression in quadriceps muscle after 3 and 8 weeks of supplementation. The study was performed in C57BL/6J mice fed a high-fat diet (45% kcal from fat) and distributed in three experimental groups: a group fed the high-fat diet, a group fed the high-fat diet supplemented

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FIGURE 28.3

28. EFFECTS OF QUERCETIN ON MITOCHONDRIOGENESIS IN SKELETAL MUSCLE

Lipotoxicity in skeletal muscle and insulin resistance. Effects of quercetin.

with the high dose of quercetin (24.5 mg/kg body weight/day), and a group fed the highfat diet supplemented with the low dose of quercetin (2 mg/kg body weight/day). After 3 weeks of supplementation with the lowest dose of quercetin, an increase in energy expenditure without changes in body weight or composition was observed. However, after longer supplementation (8 weeks), the increase in energy expenditure was maintained and a decrease in final body weight, accompanied by a reduction in fat mass and slight but significant decrease in lean mass, was observed. Moreover, quercetin attenuated high-fat diet induced insulin resistance. In the case of the highest dose of quercetin, at 3 and 8 weeks, energy expenditure was decreased, beta oxidation was incomplete, and no improvement in insulin resistance was observed. With regard to pgc-1α gene expression, no changes were observed in mice receiving quercetin for 3 weeks, independently of the dose used. When the experimental period length was prolonged for 8 weeks, while the lowest dose of quercetin significantly increased pgc-1α expression, the highest did not modify it. Based on these results, the authors concluded that quercetin improved insulin resistance in a time- and dose-dependent manner. The protective benefits of quercetin in preventing high-fat diet-induced insulin resistance occurred only with a low dose of quercetin after a longer supplementation period and were associated with increased skeletal muscle pgc-1α expression and more complete beta-oxidation. Thus, low doses used during long experimental periods seem to be more useful than high doses. In another study carried out by the same research group and the same animal model and also using a high-fat diet, the authors compared the effects of quercetin and a quercetin-rich red onion extract. In this case, the dose was 17 mg quercetin/kg body weight/day and the experimental period length was 9 weeks. In both experimental

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groups, obesity and insulin resistance were reduced. Moreover, similar increases in energy expenditure and mitochondrial adaptations were observed. Both treatments increased mitochondrial number and improved mitochondrial function, suggesting that the major bioactive component of red onion may indeed be quercetin [21]. Thus, the authors concluded that the benefits of purified quercetin from red onion extract supplementation may be partially due to the increase in skeletal muscle mitochondrial number, which improves fat metabolism in a manner that limits mitochondrial lipid overload. Stewart et al. [22] aimed to determine whether quercetin supplementation was able to reduce the progression of insulin resistance and to modify mitochondrial lipid catabolism in a moderately high-fat diet fed mouse model. For this purpose, 6-week-old male C57BL/ 6J mice were divided in three experimental groups: a group fed a standard-fat diet (10% of energy as fat) (low-fat: LF), a group fed a high-fat diet (45% of energy as fat) (high-fat: HF), and a group fed a high-fat diet supplemented with quercetin (approximately 182 mg/kg body weight/day) (high-fat 1 quercetin: HFQ). Insulin resistance was analyzed at weeks 3 and 8 with an euglycemic-hyperinsulinemic clamp. In order to evaluate the effects of the diets administered, 16 animals of each group were sacrificed after 3 and 8 weeks of treatment at the peak (01:00 hours) and at the lowest point (13:00 hours) of their metabolic cycle. No differences in body weight were observed among the three experimental groups after 3 weeks of treatment. By contrast, at the end of the experimental period (8 weeks), both groups of mice fed the high-fat diet showed greater energy intake, body weight, and adiposity than those from the standard-fat group. In the case of the HFQ group, no differences in energy intake were observed when compared with the other groups after 3 weeks of treatment, while the energy consumption on this group was greater than in the LF group and similar to the HF group after 8 weeks. No differences in glucose and insulin levels were observed after 3 weeks among the three experimental groups. However, after 8 weeks, although blood glucose levels remained unchanged, fasting insulin levels significantly increased in mice fed the high-fat diet when compared with mice fed the low-fat diet. Insulin in the mice fed the high-fat diet supplemented with quercetin was similar to mice from the low-fat diet fed group. Euglycemic-hyperinsulinemic clamp was performed at the same time points in order to evaluate insulin sensitivity. The glucose infusion rate (GIR) recorded on the high-fat fed groups was higher after 3 and 8 weeks of treatment when compared with the low-fat fed group. GIR was lower in mice treated with quercetin for 3 weeks when compared with high-fat fed mice. However, at the end of the experimental period, no differences were observed between both high-fat fed groups. In order to know whether insulin resistance observed in the clamp coincided with impaired fatty acid catabolism, the profile of 45 acylcarnitines of different size and saturation was analyzed in muscle after 8 weeks of treatment. Among them, three (long-chain species) were increased in HF fed groups (with no difference between them) in comparison with the low-fat fed group. Regarding muscle triacylglycerol content, no differences were observed among the three experimental groups after 3 weeks, but at week 8, muscular triacylglycerol content in both groups fed the high-fat diet was higher than that found in mice fed the low-fat diet, with no differences between them. The lack of quercetin effect on muscle triacylglycerol content suggests that the beneficial effect of this molecule on glycemic control was not related to a

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reduction in lipotoxicity induced by high-fat feeding. This result is in good accordance with that obtained by our group in rats [23]. Finally, insulin signaling was studied by western blot. No differences were observed in phosphoinositol 3-kinase among the three groups. Protein kinase B phosphorylation was increased in the high-fat fed group when compared with the other two groups, but its activation induced by insulin was similar in the three groups. No differences were observed regarding different intermediates of insulin signaling. In a study from our laboratory [23], we analyzed the effects of quercetin on glycemic control and the potential involvement of skeletal muscle fatty acid oxidation in this effect. For this purpose, we fed 6-week-old Wistar rats ad libitum with a high-fat, high-sucrose diet for 6 weeks. Quercetin was orally administered daily with the diet in amounts that guaranteed a dose of 30 mg/kg body weight/day. A group fed the same diet, but not receiving quercetin, was used as a control. At the end of the experimental period, no differences in food intake or final body weight were observed between groups but interestingly, quercetin improved glycemic control as observed by changes in several blood parameters. Thus, serum basal glucose and insulin levels were decreased by the treatment and in term the homeostasis model assessment of insulin resistance (HOMA-IR) showed a reduction. Also, fructosamine, an indicator of the average blood glucose concentration over a short-medium period, was reduced. The week prior to sacrifice, a glucose tolerance test was carried out and the area under the curve was significantly lower in quercetintreated group than in the control group. In this study, we set out to elucidate the possible mechanism underlying this effect. It is well known that increased intramyocellular triacylglycerol levels result in insulin resistance by perturbing the insulin signaling pathway. In this context, previous to the present study, Shen et al. [24] had observed that quercetin increased the expression of genes involved in fatty acid oxidation, such as pparα, pgc-1α, carnitine palmitoyltransferase I (cpt-1b), and acyl-CoA oxidase (aco), in C2C12 myotubes, and thus, they proposed the reduction in muscle triacylglycerol accumulation as a mechanism involved in the beneficial action of this flavonoid on glycemic control. Thus, we wanted to check whether this effect was also observed in gastrocnemious muscle from our rats; however, we did not find it. In fact, triacylglycerol levels remained unchanged. In addition, the activity of enzymes involved in fatty acid oxidation, such as CPT-1b, and in mitochondrial density, such as CS, was measured and no changes were detected. Finally, gene expression of cluster of differentiation 36 (cd36) (a fatty acid transporter), genes related to fatty acid oxidation (peroxisome proliferator-activated receptor alpha (pparα), pgc-1α, cpt-1b, aco and uncoupling protein 3 (ucp3)), or associated to mitochondria number (mitochondrial transcription factor (tfam) and cyclooxygenase 2 (cox2)) were not affected by quercetin treatment. In view of these results, we concluded that quercetin was effective as an antidiabetic agent and that this effect was not mediated by a lipid reduction in skeletal muscle. As shown in the above described studies, controversial results have been reported in rodents concerning the involvement of the effects of quercetin on muscle triglyceride content and mitochondriogenesis on the improvement induced by this phenolic compound in insulin resistance. By analyzing the studies carried out in mice, it can be observed that those where the highest doses were used (24.5 and 182 mg/kg body weight/day), no positive effects induced by quercetin were reported, suggesting as proposed by Henagan et al.

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[20] that the effects are dose-dependent, in the sense that apparently there is a threshold above which quercetin becomes ineffective. In the case of the study carried out in rats, no positive effects were observed. Three reasons could explain this fact: (1) the dose used may be too high (higher than the effective doses in mice), (2) the experimental period was too short (shorter than those used in mice studies), or (3) the rat is less sensitive than the mouse. As far as human beings are concerned, very little information has been reported so far concerning the effects of quercetin on glycemic control. Rezvan et al. [25] observed that HOMA-IR levels were significantly lower in quercetin-treated group compared to placebo group in women suffering polycystic ovary syndrome. Oral quercetin (1 g/day) supplementation for 12 weeks also increased serum adiponectin levels without inducing changes in BMI and waist circumference. Unfortunately, in this study, the effects of quercetin on mitochondriogenesis were not addressed.

CONCLUDING REMARKS Quercetin, a flavanol belonging to the group of flavonoids, is able to increase mitochondria biogenesis, in a specific range of doses. This effect results in interesting effects in healthy subjects, such as the increase in physical endurance. Moreover, this effect can also be useful to ameliorate a pathological metabolic situation, insulin resistance, which precedes type 2 diabetes. All these findings have been observed in rodents, and more studies are needed to know whether they can be reproduced in human beings.

Acknowledgments This research has been supported by MINECO (AGL-2015-65719-R), Fondo Europeo de Desarrollo Regional (FEDER) University of the Basque Country (ELDUNANOTEK UFI11/32), Instituto de Salud Carlos III (CIBERobn), and Basque Government (IT-572-13). In˜aki Milton-Laskibar is a recipient of a predoctoral fellowship from the Basque Country Government.

References [1] Neveu V, Perez-Jime´nez J, Vos F, Crespy V, du Chaffaut L, Mennen L, et al. Phenol-Explorer: an online comprehensive database on polyphenol contents in foods. Database (Oxford) 2010;2010:bap024. [2] Vargas AJ, Burd R. Hormesis and synergy: pathways and mechanisms of quercetin in cancer prevention and management. Nutr Rev. 2010;68(7):41828. [3] Bokkenheuser VD, Shackleton CH, Winter J. Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans. Biochem J. 1987;248(3):9536. [4] Murota K, Terao J. Antioxidative flavonoid quercetin: implication of its intestinal absorption and metabolism. Arch Biochem Biophys. 2003;417(1):1217. [5] Ne´meth K, Plumb GW, Berrin JG, Juge N, Jacob R, Naim HY, et al. Deglycosylation by small intestinal epithelial cell beta-glucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans. Eur J Nutr. 2003;42(1):2942. [6] Davis JM, Murphy EA, Carmichael MD. Effects of the dietary flavonoid quercetin upon performance and health. Curr Sports Med Rep. 2009;8(4):20613.

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[7] Harwood M, Danielewska-Nikiel B, Borzelleca JF, Flamm GW, Williams GM, Lines TC. A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties. Food Chem Toxicol. 2007;45(11):2179205. [8] Eynon N, Ruiz JR, Meckel Y, Mora´n M, Lucia A. Mitochondrial biogenesis related endurance genotype score and sports performance in athletes. Mitochondrion. 2011;11(1):649. [9] Davis JM, Murphy EA, Carmichael MD, Davis B. Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance. Am J Physiol Regul Integr Comp Physiol. 2009;296(4):R10717. [10] Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F, et al. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell. 2006;127(6):110922. [11] Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O, et al. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature. 2002;418(6899):797801. [12] Wende AR, Schaeffer PJ, Parker GJ, Zechner C, Han DH, Chen MM, et al. A role for the transcriptional coactivator PGC-1alpha in muscle refueling. J Biol Chem. 2007;282(50):3664251. [13] Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature. 2005;434(7029):11318. [14] Menshikova EV, Ritov VB, Ferrell RE, Azuma K, Goodpaster BH, Kelley DE. Characteristics of skeletal muscle mitochondrial biogenesis induced by moderate-intensity exercise and weight loss in obesity. J Appl Physiol 1985;103(1):217 2007. [15] Nieman DC, Henson DA, Maxwell KR, Williams AS, McAnulty SR, Jin F, et al. Effects of quercetin and EGCG on mitochondrial biogenesis and immunity. Med Sci Sports Exerc. 2009;41(7):146775. [16] Nieman DC, Williams AS, Shanely RA, Jin F, McAnulty SR, Triplett NT, et al. Quercetin’s influence on exercise performance and muscle mitochondrial biogenesis. Med Sci Sports Exerc. 2010;42(2):33845. [17] Boulinguiez A, Staels B, Duez H, Lancel S. Mitochondria and endoplasmic reticulum: targets for a better insulin sensitivity in skeletal muscle? Biochim Biophys Acta. 2017;1862(9):90116. [18] Devarshi PP, McNabney SM, Henagan TM. Skeletal muscle nucleo-mitochondrial crosstalk in obesity and type 2 diabetes. Int J Mol Sci. 2017;18 (4):E831. [19] Eid HM, Haddad PS. The antidiabetic potential of quercetin: underlying mechanisms. Curr Med Chem. 2017;24(4):35564. [20] Henagan TM, Lenard NR, Gettys TW, Stewart LK. Dietary quercetin supplementation in mice increases skeletal muscle PGC1α expression, improves mitochondrial function and attenuates insulin resistance in a time-specific manner. PLoS ONE. 2014;9(2):e89365. [21] Henagan TM, Cefalu WT, Ribnicky DM, Noland RC, Dunville K, Campbell WW, et al. In vivo effects of dietary quercetin and quercetin-rich red onion extract on skeletal muscle mitochondria, metabolism, and insulin sensitivity. Genes Nutr. 2015;10(1):451. [22] Stewart LK, Wang Z, Ribnicky D, Soileau JL, Cefalu WT, Gettys TW. Failure of dietary quercetin to alter the temporal progression of insulin resistance among tissues of C57BL/6J mice during the development of dietinduced obesity. Diabetologia. 2009;52(3):51423. [23] Arias N, Macarulla MT, Aguirre L, Martı´nez-Castan˜o MG, Portillo MP. Quercetin can reduce insulin resistance without decreasing adipose tissue and skeletal muscle fat accumulation. Genes Nutr. 2014;9(1):361. [24] Shen JZ, Ma LN, Han Y, Liu JX, Yang WQ, Chen L, et al. Pentamethylquercetin generates beneficial effects in monosodium glutamate-induced obese mice and C2C12 myotubes by activating AMP-activated protein kinase. Diabetologia. 2012;55(6):183646. [25] Rezvan N, Moini A, Janani L, Mohammad K, Saedisomeolia A, Nourbakhsh M, et al. Effects of quercetin on adiponectin-mediated insulin sensitivity in polycystic ovary syndrome: a randomized placebo-controlled double-blind clinical trial. Horm Metab Res. 2017;49(2):11521.

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Statins and Muscle Damage Matthew J. Sorrentino Section of Cardiology, University of Chicago Medicine, Chicago, IL, United States

The recognition and treatment of coronary artery disease risk factors such as dyslipidemia has made a substantial impact on reducing coronary heart disease events. The National Cholesterol Education Program guidelines recommend low density lipoprotein cholesterol (LDL-C) as the primary target of therapy to reduce cardiovascular events. The HMG CoA reductase inhibitors or statins significantly lower LDL-C and have been shown to reduce coronary events. These medications are recommended as drugs of first choice for treating dyslipidemias and reducing cardiovascular risk. Many individuals, however, are unable to tolerate statins due to side effects. Muscle side effects are the most common adverse effects of statin therapy preventing many patients from achieving benefit from these agents.

SCOPE OF THE PROBLEM The incidence of statin-induced muscle side effects in randomized controlled trials was found to be low, reported in 1% 5% of patients [1]. Observational studies, however, have reported a higher incidence of muscle-related toxicity varying from about 10% 22% [2,3]. Variance in side effect rates may reflect a selection bias in patients enrolled in clinical trials. Clinical trials tend to exclude patients with additional comorbidities that may have increased the likelihood for muscle toxicity. Analysis of the cholesterol treatment guidelines estimate that up to 56 million Americans may be eligible for statin therapy [4]. The high incidence of muscle toxicity from the observational studies would prevent a large number of individuals from obtaining benefit from these medications.

DEFINITION OF MUSCLE TOXICITY Statin-induced muscle toxicity can be categorized into five different groups (Table 29.1). Myalgias are the most common statin-induced muscle toxicity and are described as muscle

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TABLE 29.1 Definition of Statin-Induced Muscle Toxicity Myalgias

Muscle symptoms (aching, stiffness, cramping) without elevation in CK

Myopathy

Muscle weakness without further symptoms and without elevation in CK

Myositis/myonecrosis

Inflammation of muscles with elevation in CK Some clinical trials define myositis and CK $ 10 3 ULN

Rhabdomyositis/rhabdomyolysis

CK $ 10 3 ULN with myoglobinuria and/or renal impairment

Autoimmune-mediated necrolizing myositis

Muscle weakness, elevated CK, persistence of symptoms despite discontinuation of statin

CK, creatine kinase; ULN, upper limit of normal.

soreness, aches, cramping, or stiffness noted either at rest or brought on by exertion. Significant inflammation is not present and elevation in muscle enzymes such as creatine kinase (CK) is not present. Myopathy can be defined as muscle weakness without symptoms. Some individuals note weakness with exercise that was not present before initiation of the statin medication. Myositis or myonecrosis is inflammation of the muscles and is diagnosed by an elevation of the CK enzyme with or without muscle symptoms. Myositis has been defined as a CK elevation greater than 10 times the upper limit of normal for CK. Rhabdomyositis or rhabdomyolysis can be defined as a CK greater than 10 times the upper limit of normal with myoglobinuria and/or renal impairment. Autoimmunemediated necrotizing myositis is a more recently described rare condition exemplified by muscle weakness, elevated CK, and persistence of symptoms for greater than 2 months despite statin discontinuation [5].

MYALGIAS Myalgias are the most commonly reported statin-induced adverse reaction and the most common reason that statin medications are discontinued. The diagnosis of myalgias is subjective since there is no clinical test that can diagnose myalgias. Muscle enzymes such as CK are not elevated in patients complaining of myalgias. Since muscle symptoms are common in patients not taking statins, it can be difficult to determine if symptoms are due to the medication. The National Lipid Association (NLA) has proposed a statin myalgia index score to help clinicians diagnose statin-induced myalgias (Table 29.2) [1]. The scoring system was based on results from observational studies and randomized studies such as the Effects of Statins on Skeletal Muscle Performance (STOMP) study [6] but has not been prospectively validated. Patients with statin associated myalgias complain of muscle tenderness, aches, cramping, and weakness [7]. Symptoms tend to be bilateral and involve large muscle groups such as thigh muscles. Joint pain is usually not a manifestation of statin-induced

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TABLE 29.2

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NLA Statin Myalgia Clinical Index Score

Clinical symptoms (new or increased unexplained muscle symptoms) REGIONAL DISTRIBUTION/PATTERN Symmetric hip flexors/thigh aches

3

Symmetric calf aches

2

Symmetric upper proximal aches

2

Nonspecific asymmetric, intermittent

1

TEMPORAL PATTERN Symptom onset ,4 weeks

3

Symptoms onset 4 12 weeks

2

Symptoms onset .12 weeks

1

DECHALLENGE Improves upon withdrawal of statin (,2 weeks)

2

Improves upon withdrawal of statin (2 4 weeks)

1

Does not improve upon withdrawal of statin ( . 4 weeks)

0

RECHALLENGE Same symptoms recur upon rechallenge ,4 weeks

3

Same symptoms recur upon rechallenge 4 12 weeks

1

STATIN MYALGIA CLINICAL INDEX SCORE Probable

9 11

Possible

7 8

Unlikely

,7

From Rosenson RS, Baker SK, Jacobson TA, Kopecky SL, Parker BA, The National Lipid Association’s Muscle Safety Expert Panel. An assessment by the Statin Muscle Safety Task Force: 2014 update. J Clin Lipidol 2014;8(3 Suppl):S58 71.

symptoms. Patients who are active may notice more symptoms with exercise. Symptoms are usually evident within weeks of starting a statin and typically resolve quickly after stopping the drug. Symptoms may be noticeable at any statin dose although there may be an increased incidence of symptoms at higher doses or with longer acting statins. Patients may have similar symptoms to all statins or may tolerate certain statins and not others. Since muscle symptoms are common, stopping a statin to allow the symptoms to go away and then rechallenging the patient with the statin to see if symptoms recur is the only way to be certain that the symptoms are statin related. If the symptoms persist for greater than 2 months after stopping the statin, then other muscle diagnoses should be considered.

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MYOPATHY The term myopathy is frequently used in a general sense to mean any muscle symptoms. We are using the term myopathy to indicate muscle weakness from statin use without symptoms of muscle pain or signs of myositis. A number of patients have reported fatigue and exertional intolerance when treated with statins. A prospective study of over 1000 individuals treated with modest dose statins showed an unfavorable statin effect on energy and exertional fatigue that was more notable in women [8]. The STOMP trial demonstrated that high-dose statin treatment increased average CK levels suggesting that statins may produce low-level muscle injury even in healthy individuals without muscle symptoms [6]. Muscle strength and exercise capacity, however, was not changed by statin use in this study. In addition, physically active individuals may experience a higher prevalence of musculoskeletal injury when treated with statins [9]. Acute physical activity increases the CK response to exercise in participants on statins [10]. After intense and prolonged exercise such as marathon running, exercise-related increase in CK levels at 24 hours were greater in statin users than in adjusted controls [11]. These observations suggest that statins therapy, even when well tolerated, may exacerbate CK release and damage skeletal muscle with acute exercise.

MYOSITIS AND MYONECROSIS Myositis is a term used to describe muscle inflammation and can only be definitively diagnosed with a skeletal muscle biopsy or with magnetic resonance imaging (MRI). Myositis is commonly associated with muscle pain and tenderness although it can be asymptomatic. The NLA uses the term myonecrosis based on CK elevation (also termed hyperCKemia) [1]. Myonecrosis is considered mild if there is a greater than 3-fold increase in baseline CK, moderate if there is a 10-fold or greater CK increase, and severe if there is a 50-fold or greater increase in CK above baseline.

RHABDOMYOSITIS Rhabdomyositis is diagnosed as a CK greater than 10 times the upper limit of normal with myoglobinuria or renal impairment. Patients may have severe CK elevations without evidence of renal compromise. Extreme exercise can increase the CK level greater than 10 times the upper limit of normal without causing renal impairment. The incidence of a CK greater than 10 times the upper limit of normal is about 0.1% from clinical trials [12]. The incidence of rhabdomyositis is estimated to be about 1/10,000 person-years. The incidence may be slightly higher for high-dose simvastatin (80 mg) which led to the FDA recommending avoiding this dose as a starting dose for patients.

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AUTOIMMUNE-MEDIATED NECROTIZING MYOSITIS The majority of patients that develop muscle damage with statins recover quickly after the statin medication is stopped. Rarely, some patients have persistent muscle symptoms months after cessation of the statin. In rare case, patients may develop an autoimmune myopathy due to statin therapy. The incidence is estimated to be 2 or 3 out of 100,000 patients treated [5]. Symptoms may begin soon after starting a statin but have also been described after years of therapy. Muscle pain and weakness is described. Weakness persists despite stopping the statin. Physical examination can reveal proximal muscle weakness. CK levels are persistently elevated and in nearly 90% of cases are higher than 10 times the upper limit of normal. Muscle edema is noted on MRI. Muscle biopsy can show muscle necrosis and regeneration with cellular infiltrates composed largely of macrophages in the endomysial and perivascular regions. CD4 1 and CD8 1 lymphocytes and CD123 1 plasmacytoid dendritic cells are also noted. Major histocompatibility complex class I molecules are upregulated. These findings indicate an immune-mediated necrotizing myopathy and can be distinguished from polymyositis from the lack of lymphocytes invading nonnecrotic muscles cells or rimmed vacuoles [5]. Patients diagnosed with autoimmune necrotizing myositis have been found to have antibodies against HMG-CoA reductase [13]. These antibodies have not been detected in individuals on statins without muscle symptoms or with myalgias or myopathy that quickly resolves with cessation of the statin medication. Anti-HMG-CoA reductase autoantibodies have been described in patients with an autoimmune myopathy who have never been described a statin as well. These patients tend to be younger than patients with autoimmune myositis associated with statin use [14]. The reason autoimmunity develops against HMG-CoA reductase is unclear. Certain HLA alleles have been found to be associated with autoantibodies even in individuals not exposed to statins suggesting a genetic predisposition. Statins cause an upregulation of HMG-CoA reductase. This increased expression of HMG-CoA reductase in genetically susceptible individuals may lead to autoantibody production. It is also possible that the binding of the statin to the enzyme may change the conformation of the protein complex generating a site for antibody production. These autoantibodies are likely damaging to muscles cells causing the necrotizing myositis and CK release. Antibody levels have correlated with CK levels and the degree of muscle weakness [5]. Autoimmune necrotizing myositis is diagnosed in patients with persistent muscle pain and weakness and CK elevations for greater than 2 months. If the symptoms are mild, improvement in symptoms can occur over time with cessation of the statin therapy. Patients with severe symptoms or symptoms that fail to improve over time may need treatment with immunosuppressive therapy similar to treatments for other autoimmune muscle disorders. Prednisone has been used with clinical improvement. Combination therapy has been used in over half of the patients described including intravenous immune globulin to obtain benefit. Immune therapy can be tapered once strength is recovered. Relapse can occur. Some patients have persistent CK elevations despite improvement in symptoms suggesting that active muscle necrosis is ongoing.

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RISK FACTORS FOR MUSCLE TOXICITY Certain patient groups may be more susceptible to statin-induced muscle toxicity than others. Older individuals, women, and individuals with reduced muscle mass may be at increased risk for statin muscle toxicity [7]. Patients with renal or hepatic dysfunction are at increased risk because of increased serum concentration of the drugs. Hypothyroidism may increase muscle symptoms by delaying statin metabolism. Statin therapy may bring out symptoms from an underlying muscle disorder. Patients with vitamin D deficiency may be more prone to muscle symptoms. Most statins are metabolized in the liver by the cytochrome P450 (CYP) system of isoenzymes (the exception is pravastatin). Lovastatin, simvastatin, and atorvastatin are metabolized by the 3A4 isoenzyme which is a commonly used metabolic pathway responsible for the metabolism of approximately one-third of medications prescribed to patients [15]. Common drugs metabolized by 3A4 include macrolide antibiotics such as clarithromycin, azole antifungals, tricyclic antidepressants, protease inhibitors, calcium channel blockers, gemfibrozil, amiodarone, cyclosporine, tacrolimus, sildenafil, and warfarin. CYP 3A4 is also present in the intestine. Inhibition of intestinal 3A4 reduces intestinal metabolism of 3A4 metabolized statins and increases statin absorption raising serum concentrations. A significant increase in serum concentration of a statin can increase the potential for drug toxicity. Grapefruit juice contains intestinal CYP 3A4 inhibitors and a significant intake of the juice can increase the serum concentration of lovastatin, simvastatin, and atorvastatin potentiating toxicity [16]. Drug drug interactions can increase the incidence of muscle symptoms and the possibility of rhabdomyositis. This possibility was most dramatically noted with the interaction between cerivastatin, a statin no longer on the market, and gemfibrozil which led to a significant increased risk of rhabdomyositis compared with other statins. There is evidence that simvastatin, especially at higher doses, may have more musclerelated side effects. A genomewide association study in 85 subjects with statin-induced myopathy on simvastatin 80 mg daily identified a strong association with a singlenucleotide polymorphism located within SLCO1B1 on chromosome 12 [17]. SLCO1B1 encodes an organic anion-transporting peptide known to regulate the hepatic uptake of statins. Individuals that inherit the C allele were found to have higher statin serum concentrations. The prevalence of the C allele in the population is approximately 15%. Genotyping of SLCO1B1 may identify individuals at increased risk for statin toxicity. Although this study evaluated individuals with myopathy on simvastatin 80 mg, the results will likely apply to other statins as well.

MECHANISM OF MUSCLE TOXICITY The mechanism of statin-induced muscle toxicity in unknown and may involve multiple pathways. Initial studies found that statins induced the expression of atrogin-1, a key gene involved in skeletal muscle atrophy. Atrogin-1 is a muscle-specific F-box protein that was found to be strongly induced in catabolic states and plays a key role in muscle atrophy [18]. Expression of atrogin-1 appears at an early stage before muscle loss is detectable.

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Atrogin-1 promotes muscle protein degradation by the ubiquitin proteasome pathway. Statin administration has been shown to increase atrogin-1 expression. Furthermore, muscle damage was shown to be markedly reduced in atrogin-1 knockout cells suggesting a direct mechanistic role of atrogin-1 in statin-induced myopathy [19]. Not all studies, however, have shown increased expression of atrogin-1 in statin-treated cohorts and statins have not clearly been shown to cause muscle atrophy [20]. Statins may impair mitochondrial function. Statins may reduce the content of coenzyme Q10 (CoQ10) in muscles although not all tissue studies have found evidence for a fall in CoQ10 levels [21]. In addition, a decrease in mitochondrial oxidative phosphorylation capacity in skeletal muscle has been reported as a consequence of statin treatment [22]. A decrease in CoQ10 content in skeletal muscle has been shown to limit maximal oxidative phosphorylation capacity in patients treated with simvastatin [23]. This results in a compromised energetic state within skeletal muscle and may explain weakness associated with exercise in some statin users. Additional muscle toxic mechanisms have been suggested. A reduction in sarcolemmal or T-tubule cholesterol may disrupt calcium release needed for muscle contraction. Increased myocyte concentration of plant sterols may be myopathic [7]. Further work is needed to elucidate these possible mechanisms.

MANAGEMENT Myalgias are the most common adverse effect causing discontinuation of statins. Before abandoning statin therapy, it is important to determine if the muscle complaints are due to the statin. A muscle and joint pain history prior to prescribing a statin should be reviewed so that subsequent muscle complaints can be compared to the initial report to determine if new symptoms have developed with introduction of the statin. A drug free period for a few weeks followed by reintroduction of the statin can determine if the muscle complaints are due to the statin medication. Changing statin medications may reduce adverse effects of statins since a statin metabolized by a different enzymatic pathway may be better tolerated. Lovastatin, simvastatin, and atorvastatin are metabolized by the CYP 3A4 cytochrome system in the liver, whereas pravastatin, rosuvastatin, and pitavastatin are metabolized by alternate pathways. Less drug drug interactions may occur if medications are chosen that are metabolized by different enzymatic systems. The hydrophilicity or lipophilicity of statins may correlate with muscle symptoms. Highly lipophilic statins such as simvastatin and lovastatin may increase the risk for CK release compared with low lipophilic statins such as pravastatin and rosuvastatin [24]. Statins with a longer half-life may be used with alternative dosing schedules such as a few times a week or every other day dosing. Rosuvastatin with a 19-hour-half life and atorvastatin with an 11 24-hour half-life (parent drug and active metabolite) are likely to have the best efficacy using intermittent dosing. A pilot study using every other day atorvastatin dosing showed comparable LDL-C lowering as daily dosing [25]. A prospective trial of once a week rosuvastatin in individuals with a history of myalgias on statin therapy achieved a greater than 12% LDL-C lowering and 80% of the patients were able to

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continue rosuvastatin without developing myalgias [26]. A review of patients with statin side effects found that over 72% of individuals were able to tolerate an every other day regimen of low dose of rosuvastatin with a mean LDL-C reduction of about 34% [27]. Combination therapy with a statin plus an intestinal agent can be used in individuals that can only tolerate a low dose statin. The combination ezetimibe or a bile acid sequestrant with a statin can achieve an additional 20% LDL-C reduction [28]. Intestinal agents do not have systemic exposure and would not be expected to worsen myalgias. Finally, newer agents such as the PCSK9 inhibitors can achieve a greater than 50% reduction in LDL-C and are recommended therapies for high-risk individuals that cannot tolerate any dose statin [29].

CONCLUSIONS Statin-induced muscle toxicity may impact up to 20% of treated patients. Many patients will experience mild muscle aches and weakness that can be tolerated or alleviated by changing statin dose or dosing schedules. More severe muscle toxicity includes myositis, rhabdomyositis, and autoimmune-mediated necrolizing myositis. These conditions may lead to significant muscle damage and be life threatening. The exact mechanism underlying myonecrosis is not fully understood but may involve pathways that have roles in muscle atrophy. Statins remain the medications of first choice in reducing cardiovascular risk since they are generally well tolerated and have shown efficacy in reducing LDL-C and in preventing cardiovascular events in multiple studies. Newer agents, such as the PCSK9 inhibitors, may have similar LDL-lowering efficacy without muscle toxicity but evidence for cardiovascular risk reduction is only recently becoming available and the inhibitors are significantly more expensive than the statin medications.

References [1] Rosenson RS, Baker SK, Jacobson TA, Kopecky SL, Parker BA, The National Lipid Association’s Muscle Safety Expert Panel. An assessment by the Statin Muscle Safety Task Force: 2014 update. J Clin Lipidol 2014;8(3 Suppl):S58 71. [2] Bruckert E, Hayem G, Dejager S, Yau C, Begaud B. Mild to moderate muscular symptoms with high-dosage statin therapy in hyperlipidemic patients—the PRIMO study. Cardiovasc Drugs Ther 2005;19(6):403 14. [3] Buettner C, Davis RB, Leveille SG, Mittleman MA, Mukamal KJ. Prevalence of musculoskeletal pain and statin use. J Gen Intern Med 2008;23(8):1182 6. [4] Pencina MJ, Navar-Boggan AM, D’Agostino Sr. RB, et al. Application of new cholesterol guidelines to a population-based sample. N Engl J Med 2014;370(15):1422 31. [5] Mammen AL. Statin-associated autoimmune myopathy. N Engl J Med 2016;374(7):664 9. [6] Parker BA, Capizzi JA, Grimaldi AS, et al. Effect of statins on skeletal muscle function. Circulation 2013;127 (1):96 103. [7] Thompson PD, Panza G, Zaleski A, Taylor B. Statin-associated side effects. J Am Coll Cardiol 2016;67 (20):2395 410. [8] Golomb BA, Evans MA, Dimsdale JE, White HL. Effects of statins on energy and fatigue with exertion: results from a randomized controlled trial. Arch Intern Med 2012;172(15):1180 2. [9] Mansi I, Frei CR, Pugh MJ, Makris U, Mortensen EM. Statins and musculoskeletal conditions, arthropathies, and injuries. JAMA Intern Med 2013;173(14):1 10.

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[10] Thompson PD, Zmuda JM, Domalik LJ, Zimet RJ, Staggers J, Guyton JR. Lovastatin increases exerciseinduced skeletal muscle injury. Metabolism 1997;46(10):1206 10. [11] Parker BA, Augeri AL, Capizzi JA, et al. Effect of statins on creatine kinase levels before and after a marathon run. Am J Cardiol 2012;109(2):282 7. [12] Ganga HV, Slim HB, Thompson PD. A systematic review of statin-induced muscle problems in clinical trials. Am Heart J 2014;168(1):6 15. [13] Mammen AL, Chung T, Christopher-Stine L, et al. Autoantibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase in patients with statin-associated autoimmune myopathy. Arthritis Rheum 2011;63 (3):713 21. [14] Werner JL, Christopher-Stine L, Ghazarian SR, et al. Antibody levels correlate with creatine kinase levels and strength in anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase-associated autoimmune myopathy. Arthritis Rheum 2012;64(12):4087 93. [15] Guengerich FP. Cytochrome p450 and chemical toxicology. Chem Res Toxicol 2008;21(1):70 83. [16] Fuhr U. Drug interactions with grapefruit juice. Extent, probable mechanism and clinical relevance. Drug Saf 1998;18(4):251 72. [17] Link E, Parish S, Armitage J, et al. SLCO1B1 variants and statin-induced myopathy—a genomewide study. N Engl J Med 2008;359(8):789 99. [18] Gomes MD, Lecker SH, Jagoe RT, Navon A, Goldberg AL. Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci USA 2001;98(25):14440 5. [19] Hanai J, Cao P, Tanksale P, et al. The muscle-specific ubiquitin ligase atrogin-1/MAFbx mediates statininduced muscle toxicity. J Clin Invest 2007;117(12):3940 51. [20] Mallinson JE, Marimuthu K, Murton A, et al. Statin myalgia is not associated with reduced muscle strength, mass or protein turnover in older male volunteers, but is allied with a slowing of time to peak power output, insulin resistance and differential muscle mRNA expression. J Physiol 2015;593(5):1239 57. [21] Hargreaves IP, Duncan AJ, Heales SJ, Land JM. The effect of HMG-CoA reductase inhibitors on coenzyme Q10: possible biochemical/clinical implications. Drug Saf 2005;28(8):659 76. [22] Sirvent P, Bordenave S, Vermaelen M, et al. Simvastatin induces impairment in skeletal muscle while heart is protected. Biochem Biophys Res Commun 2005;338(3):1426 34. [23] Larsen S, Stride N, Hey-Mogensen M, et al. Simvastatin effects on skeletal muscle: relation to decreased mitochondrial function and glucose intolerance. J Am Coll Cardiol 2013;61(1):44 53. [24] Dale KM, White CM, Henyan NN, Kluger J, Coleman CI. Impact of statin dosing intensity on transaminase and creatine kinase. Am J Med 2007;120(8):706 12. [25] Matalka MS, Ravnan MC, Deedwania PC. Is alternate daily dose of atorvastatin effective in treating patients with hyperlipidemia? The Alternate Day Versus Daily Dosing of Atorvastatin Study (ADDAS). Am Heart J 2002;144(4):674 7. [26] Kennedy SP, Barnas GP, Schmidt MJ, Glisczinski MS, Paniagua AC. Efficacy and tolerability of once-weekly rosuvastatin in patients with previous statin intolerance. J Clin Lipidol 2011;5(4):308 15. [27] Backes JM, Venero CV, Gibson CA, et al. Effectiveness and tolerability of every-other-day rosuvastatin dosing in patients with prior statin intolerance. Ann Pharmacother 2008;42(3):341 6. [28] Gagne C, Gaudet D, Bruckert E. Efficacy and safety of ezetimibe coadministered with atorvastatin or simvastatin in patients with homozygous familial hypercholesterolemia. Circulation 2002;105(21):2469 75. [29] Bergeron N, Phan BA, Ding Y, Fong A, Krauss RM. Proprotein convertase subtilisin/kexin type 9 inhibition: a new therapeutic mechanism for reducing cardiovascular disease risk. Circulation 2015;132(17):1648 66.

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Alcoholic Myopathy Emilio Gonza´lez-Reimers1, Geraldine Quintero-Platt1, Emilio Gonza´lez-Arnay2, Candelaria Martı´n-Gonza´lez1, Lucı´a Romero-Acevedo1 and Francisco Santolaria-Ferna´ndez1 1

Internal Medicine Service, University Hospital of the Canary Islands, University of La Laguna, Tenerife, Canary Islands, Spain 2Department of Anatomy, Pathology and Histology, University of La Laguna, Tenerife, Canary Islands, Spain

Ethanol is a toxic compound that affects muscle in two ways: as acute disease leading to rhabdomyolysis, a relatively uncommon and potentially life-threatening entity in which muscle fibers suffer a necrotic process, leaking myoglobin and muscle enzymes into the bloodstream; or as chronic myopathy, a very common entity with usually mild clinical expression that is characterized by a gradual onset of muscle atrophy and weakness, especially affecting the proximal muscles [1]. Along time, muscle loss may reach 30% of the entire musculature [2]. While acute alcoholic myopathy is an entity that typically affects malnourished heavy alcoholics after severe binge drinking episodes, chronic myopathy affects chronic alcoholics, and is associated with lifetime ethanol consumption [3]. Although in experimental models alcoholic myopathy may appear without any evidence of associated malnutrition, impaired nutritional status as well as other alterations observed in the alcoholic may play a role in its pathogenesis [1]. Chronic alcoholic myopathy constitutes the core of this review.

CONCEPT AND PREVALENCE Chronic alcoholic myopathy affects heavy alcoholics who generally seek attention for other more impressive and probably more severe entities, such as liver cirrhosis or infection. Because of this, it frequently remains underdiagnosed. Indeed, chronic alcoholic myopathy is a frequent condition. In classic studies performed six decades ago, histologically defined myopathic changes were observed in 17 out of 24 heavy alcoholics, whereas electromyographic changes indicative of myopathy were detected in 11 out of 24 patients [4].

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However, in prospective studies, it has been found in as much as 60% of alcoholics (92 out of 151 patients), when assessed by muscle (quadriceps) biopsy [3]. In another study, Estruch et al. [5] found 117 patients affected by chronic myopathy out of 250 alcoholics; the diagnosis of myopathy was sustained by decreased strength in the deltoid muscle of the nondominant arm. In that study, alcoholic myopathy was the most frequent of the ethanol-related diseases recorded (liver disease, cardiomyopathy, chronic pancreatitis, peripheral neuropathy). Duane and Peters [2] performed quadriceps biopsy (vastus lateralis portion) and found myopathy in 39/81 (48%) cases. By far, chronic alcoholic myopathy constitutes the most frequent form of myopathy in Western countries [1].

HISTOLOGIC FEATURES Histologically, an almost universal finding is type II fiber atrophy, especially type IIb fiber atrophy [6]. Necrosis is uncommon. In a study on 58 alcoholic patients subjected to muscle biopsy, necrosis was observed in only one case [7], in contrast with 33% of cases with fiber atrophy. The scarcity of necrosis phenomena fits well with the usual finding of a normal biochemical pattern in patients with alcoholic myopathy. In contrast, interstitial fibrosis was quite common with 36% of patients showing certain degree of fibrosis. Wang et al. also described increased fibrous tissue deposition in muscles of rats treated with ethanol, a finding that was accompanied by increased expression of matrix metalloproteinase 9 in gastrocnemius and plantaris muscles [8]. Increased muscle fibrosis in ethanol-fed mice was also observed by Dekeyser et al. during the muscle injury repair process, together with increased expression of transforming growth factor (TGF)-beta [9]. Ultrastructural alterations include myofibrillar and mitochondrial abnormalities, and lipid and glycogen accumulation [10]. The accumulation of glycogen may be due in part to decreased activity of muscle glycogen phosphorylase and/or phosphofructokinase, as shown by Martin et al. in 1984 [11], and also by Trounce et al. in 1987, who also reported decreased activity of several other enzymes of the glycolytic pathway [12]. Interestingly, fatty infiltration of muscle fibers was also observed in microscopic sections, affecting 28% of patients. In a more recent study, and using a completely different method, myosteatosis was assessed by computed tomography in 353 out of 678 cirrhotics, 153 of them alcoholics. Among the latter, 86 (56.2%) had myosteatosis [13]. Myonecrosis and signs of apoptosis are also observed. In a study on 30 heavy, wellnourished alcoholics (those who were malnourished were previously excluded), who drank a mean of 159 g ethanol per day during a mean period of 24 years, Ferna´ndez-Sola et al. found decreased deltoid muscle strength compared with controls, as well as histological criteria of myopathy in 50% of them. Patients with histological criteria of myopathy showed even more decreased muscle strength than those without myopathy, especially if the myopathy was moderate or severe [14]. Patients with myopathy showed a higher apoptotic index [assessed by TUNEL (desoxiribonucleotidyl-transferase-mediated-dUTP-biotin nick-end labeling) assay] and alterations of the mitochondrial derived apoptosis-regulating mechanisms B cell lymphoma 2 (Bcl-2) associated X protein (BAX, a proapoptotic protein, and BCL-2, a protein whose overexpression inhibits apoptosis). However, no relationship was found between the alterations observed in the apoptosis-related variables and muscle

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strength or with the degree of ethanol consumption [14]. In alcoholics, other nonmitochondrial pathways that also activate apoptosis may also be involved such as those dependent on caspases, cytokines, or insulin-like growth factors (IGFs) [15]. In studies that used electron microscope analyses, a striking finding is the observation that ultrastructural changes precede muscle fiber atrophy: ultrastructural alterations were present not only in all the patients with fiber atrophy but also in some who did not show fiber atrophy [16].

CLINICAL FEATURES Muscle wasting and weakness may be prominent features, although in 20% of cases it may be asymptomatic [16]. It may exceptionally cause pain. Functionally, weakness may reach such an intensity that impedes walking. Women may be more susceptible than men, as shown by Urbano-Ma´rquez et al. in a study on 50 alcoholic women and 100 alcoholic men, despite a lower lifetime ethanol consumption by women [17]. This result was also reproduced experimentally by Hunter et al. in 2003 [18]. Weakness preferentially affects proximal muscles, especially at the lower limbs and also deltoids, but handgrip strength may become also impaired [19]. In a study on 86 alcoholics, decreased handgrip strength was more marked among those who reported a longer period of addiction (Fig. 30.1). Among alcoholics, there are other frequent conditions in which similar symptoms can be also found and we will now briefly review them.

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P 5 0.001).

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Inverse relationship between handgrip strength (in kg) and years on addiction (rho 5 2 0.35;

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Polyneuropathy Theoretically, alcoholics may develop neuropathy either by a direct effect of ethanol itself or by coexisting vitamin deficiency, notably vitamin B12 and/or vitamin E deficiency. Although sensory alterations may dominate the clinical picture, with distal hypoesthesia, and especially paresthesia or ataxia, sometimes motor symptoms with distal paresis and a steppage gait may be more prominent. This clinical picture may be difficult to distinguish from myopathy, although weakness is more proximal in myopathy and more distal in polyneuropathy. Polyneuropathy is a well-documented complication of alcoholism, although there are not many studies dealing with this problem. Estruch et al., in 1993 [5], described that polyneuropathy was present in 41 out of 250 alcoholics. This proportion (16%) was higher than that regarding cirrhosis (9%), but lower than the proportion of patients with myopathy (47%). Achillean reflexes were absent in 13 out of 33 heavy alcoholics, and distal paresthesia was present in 15 patients; both alterations were more frequently observed among cirrhotics, as well as several findings suggestive of autonomic neuropathy. Indeed, autonomic and peripheral neuropathies were more frequently observed among cirrhotics [20]. Also, a prevalence of around 50% of autonomic or peripheral polyneuropathy among alcoholic cirrhotics was also reported by Thuluvath and Triger in 1989 [21]. A relationship between autonomic dysfunction and liver function impairment was also observed by Bajaj et al. in 2003 [22]. In these studies, the relationship between polyneuropathy and myopathy was not assessed, but others have reported a lack of association between neurophysiologically documented polyneuropathy and histologically proven myopathy [23]. Alcoholics are also affected by several conditions in which walking is impaired, such as cerebellar atrophy, Wernicke’s encephalopathy, or Marchiafava Bignami disease, but these alterations have characteristic clinical features that facilitate the differential diagnosis. It is more complicated to discern whether or not there is vitamin deficiency, especially vitamin E and vitamin B12 deficiency. Although this last alteration is uncommon, vitamin E deficiency is frequent among alcoholics [24] and may affect peripheral nerve and/or Goll and Burdach bundles. Although weakness may ensue, and may be eventually severe, especially in vitamin E deficiency [25], pure muscle alterations, without ataxia or paresthesia, is unusual or does not occur at all.

Protein-Calorie Malnutrition A long-debated question is the role of protein-calorie malnutrition on the development of alcoholic myopathy [26]. Protein-calorie malnutrition is very frequent among alcoholics [27], and muscle wasting is an outstanding feature of this situation, although this finding is not shared by all authors [28]. Moreover, experimental studies have also reported that when rats are fed a 1.5% protein-containing diet, muscle atrophy ensued, especially affecting type IIb fibers [29]. In the classic study by Urbano-Ma´rquez et al., the authors concluded that malnutrition did not play a significant role on alcoholic myopathy and cardiomyopathy [3], and this position is held by other researchers [2,30]. In contrast, in several studies performed by our group, we have reported that myopathy in alcoholics was associated with protein-calorie malnutrition [7,27], and this has also been assessed experimentally [31]. Confirming previous results, we have recently shown a significant,

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FIGURE 30.2 Direct relationship between handgrip strength (in kg) and triceps skinfold (r 5 0.38; P , 0.001), a variable related with subcutaneous fat. This finding supports the existence of a relationship between alcoholic myopathy and nutritional status.

direct relationship between handgrip strength and triceps skinfold (Fig. 30.2), a variable that does not depend on muscle mass, but on subcutaneous fat, and is therefore more closely related to nutritional status. Perhaps the important conclusions are that in some alcoholics, myopathy develops without any evidence of associated malnutrition [5], and that coexisting malnutrition aggravates alcoholic myopathy, as we have shown in rats treated following the classic Lieber DeCarli liquid diet model [31].

Vitamin D Deficiency In the last three decades, the discovery of a vitamin D receptor in muscle has provided a mechanistic explanation for the severe muscle weakness observed in patients with osteomalacia [32]. Today, the role of vitamin D deficiency in proximal muscle wasting and performance is well established [33], as are the benefits derived from vitamin D supplementation [34]. Alcoholics frequently show vitamin D deficiency [35], and therefore, a relationship between muscle weakness and low vitamin D levels has been a matter of research. In 1989, Hickish et al. failed to find an association between muscle strength in alcoholics and vitamin D levels [36], but in an experimental model, serum 1,25(OH)2D3 levels were lower in rats fed ethanol according to the Lieber DeCarli model and were directly related to type II muscle fiber area [37]. Additionally, among 90 alcoholics, we recently reported a significant relationship between decreased lean mass and low vitamin D levels [38].

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Alcohol-Related Pseudo-Cushing Syndrome Pseudo-Cushing syndrome is a well-described complication of alcoholism and probably results from a transient alteration of ACTH secretion [39]. It is considered an uncommon condition—by far less common than chronic myopathy—and it is usually reversible after alcohol abstinence [40]. To our knowledge, there are no studies linking alcoholic myopathy to the so called pseudo-Cushing syndrome, although in a recent review it was recorded that 81% of patients with this entity showed muscle weakness [41]. In any case, alcoholic myopathy and alcohol-induced pseudo-Cushing syndrome seem to be completely different, unrelated, clinical pictures.

PATHOGENESIS Fiber atrophy is the hallmark of chronic alcoholic myopathy. Fiber atrophy can be viewed as the result of an imbalance between protein synthesis and protein breakdown (Fig. 30.3). As commented later in more detail, Lang et al., nearly two decades ago, have shown in several studies that the main mechanism for the development of fiber atrophy in alcoholics is decreased protein synthesis, mainly by a direct effect of ethanol on dephosphorylation of mammalian target of Rapamycin (mTOR), reducing mRNA translation [42]. However, the deleterious effect of ethanol on muscle mass already affects muscle cell development.

FIGURE 30.3

Main mechanisms involved in alcoholic myopathy.

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Cell Development: Ethanol Inhibition of Muscle Repair Development of muscle cells is a complex phenomenon which starts with the condensation of mesenchymal stem cells (MSC) around the paraxial mesoderm during embrionary stages and continues into postnatal life. While axial and body wall musculature is generated by elongation of dermomyotome epithelium, limb musculature formation implies migration of progenitors from ventral dermomyotome [43,44]. This process requires coordination between Wnt-driven mesoderm formation, later Wnt/β-catenin induction of epithelial mesenchymal transition and production of fate controller genes proteins, grouped into the myogenic regulatory factor (MRF) family [45,46]. MRF family genes are upregulated by expression of paired box 3 (PAX3) and mesenchymal-epithelial transition (MET), that are also involved in epithelial mesenchymal transition required for migration. Proper function of PAX3 needs previous phosphorylation by BRAF [43]. Finally, PAX3 and MET promote the expression of genes of the MRF family, like MyoD and MRF-4, thus starting the final transformation of the former MSC into muscle cells through fusion of myoblastic cells into myotubes under the influence of myogenin [43]; (Fig. 30.4). Rearrangement of myofibrils, distribution of nuclei in the periphery, and reorganization of the cytoskeleton all take place during the differentiation process. Most of muscle development takes place during fetal stages, but postnatal muscle growth needs growing and fusion of resident MSC with existing myotubes. Resident MSC

FIGURE 30.4 Simplified schematic representation of muscle development, indicating the central role of the Wnt/beta catenin pathway in myogenesis, activating PAX3/MET signaling pathway and MRFs, and how ethanol inhibits this process. MRFs, myogenic regulatory factors; PAX3, paired box 3PRK, protein kinase K; AMPK, AMP activated protein kinase; NFKB, nuclear factor kappa B.

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seem to be subdivided in different subsets of cells with varying degrees of fate commitment and, then, able to give rise to different subpopulations of mesenchymal cells, including adipocytes and fibroblasts [46]. This differential development appears to happen under competitive rules heavily influenced by environmental factors, like inflammatory or nutritional status [46]. Ethanol inhibits muscle cell differentiation [47] and skeletal muscle repair, by not only interfering with myotube formation and MyoD and myogenin expression but also activating Notch-target genes, leading to inhibition of the myogenic fusion process [48]. In addition, ethanol misuse leads to chronic inflammation, which promotes expression of genes involved in the nuclear factor kappa B pathway, downregulating myogenesis and promoting adipogenesis. Inflammation-related oxidative stress also reduces levels of β-catenin, enhancing adipogenesis. TNF-alpha reduces Protein Kinase A (PKA) /AMPK, favoring MSC commitment to fatty cells [46,49]. Moreover, malnutrition associated with ethanol consumption may lead to a reduction in myogenic commitment for MSC. Indeed, the offspring of mammalians subjected to nutritional deprivation during pregnancy develop a collagen-rich skeletal muscle [46]. Other authors have shown that chronic ethanol consumption alters skeletal muscle regeneration, exaggerating early inflammatory cytokine response and oxidative stress, and leading to a delay in muscle fiber repair, an increase in fibrous tissue deposition—perhaps in association with increased TGF-beta response, and also impairing recovery of muscle activity. Induction of the muscle reparative transcription factor, ciliary neurotrophic factor, implicated in myotube differentiation, was delayed [9]. In a binge drinking model in macaques Simon et al. showed that the expression of several myogenic genes in myoblasts was impaired, leading to decreased myotube formation [50], a finding that undoubtedly explains, at least in part, the muscle atrophy observed in alcoholic myopathy. Possibly in association with these effects, it was described some decades ago that postnatal administration of ethanol produced a selective atrophy and a decrease of the number of type IIb (fast glycolytic) fibers when the animals were sacrificed after 40 days [51].

Reduced Protein Synthesis Decreased protein synthesis affects both the myofibrillar and sarcoplasmic compartments, especially involving muscles rich in type II fibers [52]. Decreased protein synthesis is a direct effect of ethanol, that adds to the contributory roles played by polyneuropathy and likely vitamin D deficiency, and to ethanol-mediated alterations in mediators of protein synthesis such as insulin or IGF-1 signaling [53]. IGF-1 plays an important role on muscle growth. It acts mainly via Akt-mTOR (vide infra). Reduced IGF-1 levels have been described long ago among alcoholics [54], but its role in alcoholic myopathy was stressed some years later [53]. Specifically, IGF-1 stimulates mRNA translation initiation, but ethanol blunts this effect [55]. mTOR is a protein kinase that regulates growth by controlling mRNA translation. This ability resides in the mTOR complex (mTOR C)-1, that phosphorylates the ribosomal protein S6 kinase-1 (S6K-1) and the eukaryotic initiation factor 4E binding protein-1. These two last proteins stimulate cell growth and protein synthesis. Alcohol inhibits mTOR C-1 activity including S6K-1 phosphorylation and thus inhibits protein synthesis [56]. mTOR C-2 regulates the activity of protein kinases, including, among others, serine-threonine IV. ADVERSE EFFECTS DUE TO DRUGS AND ALCOHOL

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specific protein kinase (Akt) phosphorylation in response to growth factors. Akt phosphorylation promotes S6K-1 phosphorylation, which in turn exerts a negative feedback effect on Akt phosphorylation. Inhibition of S6K-1 phosphorylation mediated by ethanol increases Akt phosphorylation, so that ethanol increases the activity of mTOR C-2 [57], repressing the promoting growth effect of mTOR C-1. These effects are independent of IGF-1 [58]. Myostatin is a negative regulator of muscle growth, which acts via the SMAD 3 pathway inhibiting protein synthesis and inducing proteolysis [59]. Through SMAD 2/3 dependent signaling pathways, it inhibits expression of myogenin and MyoD, leading to muscle atrophy. Myostatin upregulation has been documented in alcohol-mediated cardiomyopathy [60], and experimental data also support a role of myostatin on muscle atrophy in alcoholic models. Lang et al., in 2004, showed that gastrocnemius muscle content in myostatin mRNA was markedly increased in alcohol-fed rats, and that this increase was blunted after IGF-1/IGFBP-3 (Insulin-like growth factor binding protein) injection [61]. Elevated levels of myostatin may be associated with obesity, suggesting a relationship between obesity and muscle atrophy [62]. This finding is therefore linked with the role played by adiposity on muscle atrophy, a field largely unknown in alcoholics. Insulin resistance is a common finding in obese patients and altered response to insulin signaling has also been observed in alcoholics [63]. One of the hormones produced in fat tissue is leptin which increases resting energy expenditure. Leptin exerts a positive effect on muscle, increasing fiber size and muscle mass and expression of myogenin. On the contrary, it decreases expression of myostatin and ubiquitin ligases [64]. But leptin also promotes a Th-1 immune response, with production of interferon gamma (IFNG) and inducing macrophages to produce IL-6 and TNF-alpha. Indeed, high IL-6 is usually described in obese patients, in relation to leptin [65], and IL-6 may be associated with increased muscle protein breakdown [64]. Therefore, the altered cytokine profile described in obese patients may contribute to muscle atrophy, although data are not conclusive. In a study on 79 alcoholics, we found low leptin levels in relation with fat mass—that was also reduced [66]. More recently, in 53 alcoholics, a negative correlation was observed between IL-6 and muscle strength (unpublished data, Fig. 30.5).

Increased Protein Breakdown Degradation of muscle proteins might theoretically play a role in ethanol-mediated muscle atrophy. In addition to the ubiquitin proteasome machinery, theoretically inhibited by ethanol [67], there are several cellular mechanisms by which protein breakdown may ensue, including several forms of regulated cell death and/or breakdown of organelles. Regulated cell death involves genetically encoded molecular pathways, distinguishing it from accidental cell death, by which a cell is destroyed after being exposed to overwhelming insults [68]. Several forms of regulated cell death have been described, including apoptosis, autophagy, necroptosis, pyroptosis, mitotic catastrophe, or mitoptosis [69], but the main pathways which take place in alcoholic myopathy are apoptosis and autophagy. This last mechanism should be viewed as an adaptative, prosurvival mechanism in situations of limited nutrition, in which the cell breaks down unnecessary organelles, whereas apoptosis is a programmed cell death. IV. ADVERSE EFFECTS DUE TO DRUGS AND ALCOHOL

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FIGURE 30.5 Relationship between IL-6 and handgrip strength (rho 5 2 0.29; P 5 0.036).

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30

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Apoptosis Apoptosis is an energy-dependent, controlled cell deletion process that plays a major role in ontogenesis, cancer, and several diseases. Two main pathways may be involved in apoptosis activation, including the mitochondrial pathway and death receptor dependent pathways. In the mitochondrial pathway, the initiator caspases activate the proapoptotic proteins Bcl-2 homologous antagonist/killer (BAK) and BAX, which in turn alter mitochondrial permeability causing cytochrome C release. Ferna´ndez-Sola` et al., in 2003, showed by immunohistochemical staining that apoptotic indices, including BAX, were significantly higher in muscle biopsies of well-nourished alcoholics with myopathy [14]. The second pathway activates the death domain containing tumor necrosis factor (TNF) receptor superfamily first apoptosis signal (Fas), TNF related apoptosis induced ligand (TRAIL), TNF receptor (TNFR), ultimately leading to activation of caspase 8 and 10. This so called extrinsic pathway, through caspase 8, also engages the mitochondrial pathway, leading to amplification of the apoptotic response [15]. Oxidative stress and increased TNF-alpha production explains that the extrinsic pathway can be also activated in patients with alcoholic myopathy [70].

Autophagy Another possibility that explains muscle protein loss in alcoholics is autophagy. Thapaliya et al. have recently shown, in muscle samples of five alcoholic cirrhotics and five controls, that ubiquitin proteasome markers were downregulated, but lipidated LC3B—an autophagosoma marker—was increased [71]. The same authors studied the gastrocnemius muscle of alcoholized mice and found increased expression of autophagy-related genes and increased lipidated LC3B. Murine myotubes exposed to ethanol also confirmed

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increased ethanol-mediated autophagy. The authors also showed that this effect was dependent on acetaldehyde rather than on ethanol itself. Autophagy may be considered as a prosurvival mechanism, able to remove damaged organelles and mitochondria potentially capable of triggering apoptosis [72]. von Haefen et al. have shown that ethanol downregulates autophagy markers in various types of cell cultures, and that this downregulation promoted apoptotic cell death that could be blocked after stimulation of autophagy by rapamycin [73]. Therefore, the net effect of ethanol on autophagy and alcoholic myopathy needs further research. Altered Carbohydrate Metabolism Data regarding alterations of carbohydrate metabolism in alcoholic myopathy are less conclusive, and, in some cases, contradictory. Xu et al. (1996) showed that acute ethanol administration caused insulin resistance, leading to impaired uptake of glucose, especially by muscles rich in type I fibers [74]. A similar effect was observed in situations of chronic ethanol exposure, but this time affecting both type I and type IIb fibers. These data are in contrast with the findings described by Boada et al., who found increased glucose utilization by isolated rat diaphragm in the presence of ethanol [75]. In any case, the role of altered glucose utilization in the pathogenesis of alcoholic myopathy is unclear [76]. Inflammation/Oxidative Stress Alcoholism may be viewed as a proinflammatory condition, characterized by increased portal endotoxemia, increased proinflammatory cytokine secretion, and increased production of reactive oxygen species (ROS). Free radicals may lead to increased TNF-alpha secretion, which in turn promotes excessive production of free radicals, closing a positive feedback loop. We already mentioned that increased TNF-alpha activates the extrinsic pathway of apoptosis and exerts a wasting effect on muscle (a synonym for TNF-alpha is cachectin), at least in experimental models [77]. TNF-alpha is high in alcoholics because there is an increase in intestinal permeability that allows gram negative bacteria to reach portal blood, stimulate Kupffer cells, and eventually generate an increase in TNF-alpha serum levels [78]. As mentioned previously, TNF-alpha triggers extrinsic apoptotic pathway and is also a potent inductor of the ubiquitin proteasome catabolic pathway [79], thus contributing to muscle atrophy, although it also inhibits RNA translation [80]. Intimately related to TNF, the inflammatory cytokine TNF-like weak inducer of apoptosis is strongly related to muscle atrophy associated with denervation, immobilization, or unloading [81], but, to our knowledge, there are no data regarding its role in alcoholic myopathy. Activated macrophages not only produce TNF-alpha but also other proinflammatory cytokines, such as IL-1b, IL-6, and IFNG, that may be also involved in muscle atrophy [82]. In a recent study, muscle IL-6 expression and serum IFNG levels increased in elderly healthy adults after 7 days of bed rest and were accompanied by a loss of 4% of leg lean mass [83]. The role of these cytokines in alcoholic myopathy is uncertain, but high IL-6 [84] and/or IFNG have been reported in several studies in alcoholics [85]. One of the triggering factors of cytokine secretion is the excessive presence of ROS. Oxidative damage may be due to an imbalance between decreased activity of the antioxidant systems and increased free radical production. Ethanol and/or its metabolites affect both antioxidant systems and ROS generation. Both ethanol and acetaldehyde metabolism

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by alcohol dehydrogenase and acetaldehyde dehydrogenase, respectively, generate the superoxide anion, whereas induction of microsomal ethanol oxidizing system (MEOS) system by chronic ethanol consumption generates more ROS, notably hydroxyl radicals and superoxide [86]. The effect of proinflammatory cytokines activating macrophages and neutrophils may contribute to the generation of more ROS. Iron overload, frequent in alcoholics, may also contribute to enhanced ROS production [87]. The possibility that other metabolic pathways were also involved in mitochondrial oxidative damage was explored by Nguyen et al. [88] who analyzed the Insulin/IGF signaling pathways in gastrocnemius muscles of rats fed a 35.5% ethanol-containing diet. They found that ethanol strongly impaired insulin/IGF-1 signaling pathway, both by reducing the genetic expression of several proteins involved in the signaling pathway and interfering with the pathway itself, leading to altered synthetic, growth, and reparative functions. Moreover, these alterations caused an increase in mitochondrial oxidative damage [88]. The authors also found that acetylcholinesterase expression in skeletal muscle was impaired, possibly in relation to oxidative stress. Inhibition of acetylcholine-esterase may be related to muscle fiber atrophy [89]. Decreased activity of antioxidant systems are also described among alcoholics. The most important intracellular antioxidant systems include glutathione peroxidase and superoxide dismutase, and their enzymatic activity critically depends on the availability of certain trace elements, such as selenium, copper, zinc, and manganese. Ethanol-mediated deficiency of these elements in muscle tissue has been described in animal models [90], and serum selenium is lower in alcoholics [91,92]. However, zinc supplementation does not improve muscle atrophy, although it did reduce malondialdehyde (MDA) levels [93]. Extracellular antioxidants, including, among many others, vitamin E, ascorbic acid, and retinol, are also low in alcoholics, but data relative to a pathogenetic role on alcoholic myopathy are lacking. A significant decrease in serum tocopherol and selenium in patients with myopathy was reported by Ward and Peters [94], but Ferna´ndez-Sola` et al. report normal muscular and serum levels of alpha tocopherol, retinol, and ascorbic acid [95]. Overall, the role of oxidative stress in the pathogenesis of alcoholic myopathy is still a matter of debate. In an experimental study with rats fed according to the Lieber DeCarli liquid diets, both protein deficiency and ethanol provoked type II muscle fiber atrophy, which was associated with increased muscle MDA levels and muscle iron overload [93]. The finding of increased lipid peroxidation is shared by other authors in experimental models of cancer-associated muscle wasting [96], in contrast with Ferna´ndez-Sola` et al. [97], who failed to find a clear relationship between alcoholic myopathy and oxidative damage. However, reduced mitochondrial Glutathione (GSH) is related to altered mitochondrial permeability and muscle fiber apoptosis [98]. Adachi et al. did find that alcohol was associated with the formation of hydroperoxides in skeletal muscle that alter cell permeability and may contribute to apoptosis [99].

FUTURE PROSPECTS. EFFECTS ON DISTANT ORGANS Although acute alcohol-mediated rhabdomyolysis may kill some patients, especially cirrhotics [100], chronic myopathy is not directly related to mortality. In fact, it may improve, at least partially, with alcohol cessation [101] or by decreasing the amount of ethanol

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consumed [102]. We have described the most important pathways involved in its pathogenesis. Together with well-established, solid knowledge about some processes, there are still many obscure lacunae that need further research. A fascinating new field of research is the analysis of the potential systemic effects caused by alcoholic myopathy. In contrast with the classic concept that muscle was only a relatively inert structural, protein-storing organ, recent research has revealed that it can also be considered an endocrine organ that secretes several molecules and growth factors that may affect structure or function of distant organs. The name myokines was coined by Pedersen in 2003 [103] to refer to these molecules. Therefore, muscle disease, as is the case of alcoholic myopathy, may play causative damaging roles in distant organs. To our knowledge, this is an open field of research that will probably yield important information in the immediate future. Some examples of the importance of the relationships between myokines and the functional alteration of distant organs are outlined below. The most anatomically related organ is bone. Muscle activity undoubtedly affects bone remodeling, both by a direct effect of the increased mechanical force on the Wnt-beta catenin canonical pathway and by indirect effects. In a very recent study, we have shown that serum sclerostin levels are inversely related to handgrip strength and left arm lean mass in 70 alcoholics [104]. In this sense, myostatin not only increases bone resorption but also inhibits bone formation [105]. Indeed, the myostatin receptor has been identified in osteoblasts [106]. The effect of myostatin on bone may be counteracted by follistatin or decorin. These molecules are also secreted by muscle and they are related to muscle hypertrophy [107]. Osteoglycin is a muscle-secreted anabolic factor that also exerts anabolic effects on bone [108]. Irisin is a substance secreted by muscles that exerts, in an autocrine fashion, anabolic effects not only on muscle but also on bone, activating osteoblasts, and on adipocytes. In these last cells, it stimulates uncoupling protein 1 and increases fat browning and thermogenesis [109]. Muscle also produces several interleukins, such as Il-6, IL-7, and IL-15, and hormones such as IGF-1, with wide effects not only on bone and muscle growth, as commented previously, but also on neuronogenesis and on intermediate metabolism. It would be of interest to know to which extent altered secretion of these substances by the damaged muscle are responsible for the cerebellar or cortical atrophy, hippocampal alterations, or osteoporosis observed in these patients. The relationship between muscle and the brain is especially important in alcoholic patients. It is well known that exercise induces neurogenesis, especially in the hippocampus, increasing metabolic activity of neuronal cells with increased expression of enzymes involved in glucose metabolism, and also increasing angiogenesis. Importantly, the exercise-induced new neurons show a lower threshold of excitability and, therefore, have enhanced plasticity to exercise stimulation. These effects on brain are mediated by some growth factors such as brain derived neurotrophic factor (BDNF), IGF-1, vascular endothelial growth factor, or platelet-derived growth factor. Exercise not only induces production of IGF-1 and BDNF in the hippocampus [110], but also exercise training increases mRNA expression and BDNF protein in the soleus muscle of rats [111]. Running also increases secretion of muscle-derived cathepsin B, and this protein enhances the expression of BDNF and doublecortin in hippocampal progenitor cells [112]. IGF-1 levels are related to hippocampal volume changes after a period of controlled exercise among individuals aged 60 77 years [113].

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CONCLUDING REMARKS Chronic alcoholic myopathy is a very frequent complication of alcoholism, affecting more than 50% of heavy alcoholics. It is characterized by muscle fiber atrophy, especially affecting type II-b fibers. Muscle wasting takes place independently of nutritional status, polyneuropathy, pseudo-Cushing, or vitamin D deficiency. Pathogenetically, it may be viewed as the result of an imbalance between decreased muscle protein synthesis and increased protein breakdown. Decreased protein synthesis seems to be more important in muscle wasting than increased breakdown, but both apoptosis and autophagy are enhanced in alcoholic muscle. Possibly, the proinflammatory milieu of the chronic alcoholic and the altered redox state play a contributory role. In addition to impaired walking and reduced muscle strength, it is possible that alcoholic myopathy could play a role in some alterations observed in alcoholics in distant organs, such as brain or bone, given the presence of several myokines that are active in these organs. However, this is still a little explored field.

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IV. ADVERSE EFFECTS DUE TO DRUGS AND ALCOHOL

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A AAs. See Amino acids (AAs) Abdominal obesity, 202, 204 Abscisic acid, 413
414 Absorption, 268
270 Accelerometers, 18
19 ACD. See Acute coronary disease (ACD) Acetoacetate, 266
267 Acetyl CoA, 266
267 Acetyl-1,8-dihydroxy-3-methylnaphthalene, 409 Acetylcholine-esterase expression, 520 aco. See Acyl-CoA oxidase (aco) ACSM. See American College of Sports Medicine (ACSM) Activating transcription factor (ATF), 50
51 Active efflux transporters, 320 Active proliferative retinopathy, 207 Acute adverse effects, 168
169 Acute alcoholic myopathy, 509 Acute arginine supplementation and exercise, 302 Acute coronary disease (ACD), 217 Acute effect of physical activity, 214
215 Acute exercise, 152
157 Acute physiologic responses, 187 Acute resistance exercise, 145 Acute responses to resistance exercise, 187 Acute supplementation with BCAAs, 187 of protein and amino acids, 184
186 Acyl-CoA oxidase (aco), 476, 494 Adaptations, 160
161 to environmental stress, 154
155 stress and, 152
157 Adaptive effects nutrition on adaptive effects of exercise, 151
152 as side effect, 169
172 Adaptive theory, training to based on, 153 Adenosine 50 -monophosphate-activated protein kinase alpha (AMPKα), 129 50 -Adenosine monophosphate-activated protein kinase (AMPK), 40, 51, 141, 402 direct molecular target of phytochemicals activating, 414 phosphorylates TSC2, 40 Adenosine triphosphate (ATP), 298, 322
323, 427 production, 118
120

Adequate carbohydrate, 187 Adequate dietary protein intake, 19
20 Adequate intakes (AIs), 152 average daily intake, 165
166 Adhesion molecules involved in immune cell infiltration, 126
128 Adipokines, 125
126 Adiponectin production, 476
477 Adiponectin receptor 1 (AR1), 477
478 Adipose tissue, 127
128, 476
477 Adiposity, 94, 109 ADMA. See Asymmetric dimethylarginine (ADMA) Advanced-stage COPD, 223 Adverse effects, 166 of supplementation, 168
172 Aerobic exercise(s), 18, 205, 211
212 moderate, 22
23 training studies, 214 Aerobic organisms, 153 Aerobic physical exercise, 228
229 Aerobic training, 207 AGAT. See Arg:glycine amidinotransferase (AGAT) Age-associated changes, 420 Age-associated conditions, 438 Age-related declines in muscle mass, 17 Age-related muscle loss, 435 Age-related muscle wasting, 17 Aged muscles PGC-1α of mitochondria in, 426 polyphenol GTE reduces apoptosis signaling in, 440
442 potential for resveratrol to reduce apoptosis and oxidative stress, 446
448 AGES-Reykjavik study, 18 Aging, 19, 109 aging-associated muscle denervation, 429
430 aging-induced muscle loss, 361
362 altered mitochondrial dynamics with, 424
425 attenuates mitochondrial function, 421
422 mitochondrial regulation of neural dysfunction in, 422
423 muscles mitochondria initiators of cell death signaling, 427
430 mitochondrial function, 421
423

529

530 Aging (Continued) mitochondrial permeability transition accelerates apoptosis, 429
430 potential sources of mitochondrial dysfunction, 423
425 UPS and neural dysfunction in, 437 AIF. See Apoptosis inducing factor (AIF) AIs. See Adequate intakes (AIs) Akt. See Protein kinase B (Akt) ALA. See Alpha-linolenic acid (ALA) Ala-Gln. See L-alanyl-L-glutamine (Ala-Gln) Alanine
serine
cysteine transporters (ASC transporters), 327 Alarm reaction, 153 Alcohol, 25
26 alcohol-related pseudo-cushing syndrome, 514 Alcoholic myopathy, 512
513 clinical features, 511
514 alcohol-related pseudo-cushing syndrome, 514 polyneuropathy, 512 protein-calorie malnutrition, 512
513 vitamin D deficiency, 513 concept and prevalence, 509
510 future prospects, 520
521 histologic features, 510
511 mechanisms, 514f pathogenesis, 514
520 apoptosis, 518 autophagy, 518
520 cell development, 515
516 increased protein breakdown, 517 reduced protein synthesis, 516
517 Alcoholics, 512 Alcoholism, 519 Alfacalcidol, 394 All-cause mortality in older age, 96
100 α-keto-β-methylvaleric acid (KMV), 266 α-ketoisocaproic acid (KIC), 266 α-ketoisovaleric acid (KIV), 266 Alpha-linolenic acid (ALA), 159
160, 359
360 α-methylbutyryl CoA, 266 α-tocopherol, 452 ALS. See Amyotrophic Lateral Sclerosis (ALS) Altered carbohydrate metabolism, 519 American College of Cardiology guidelines, 216 American College of Sports Medicine (ACSM), 214 American Heart Association, 216 Amino (
NH2) group, 263
265 Amino acids (AAs), 38, 71, 75, 139
142, 159, 183, 187, 263
265, 272, 279. See also Branched-chain amino acids (BCAAs); Sulfur amino acids absorption, 35 acute supplementation of protein and, 184
186

INDEX

adaptations to endurance training with protein and AA supplementation, 186 kinetic studies in cancer patients, 71
75, 72t, 73t pool, 280 regulate mTORC1, 38
39 splanchnic sequestration, 35 transporter systems, 326
327 Amiodarone, 504 Ammonia (NH3), 281 AMPK. See 50 -Adenosine monophosphate-activated protein kinase (AMPK) Amyotrophic Lateral Sclerosis (ALS), 425 Anabolic potential determinant, 271
272 Anabolic resistance, 71, 74, 270
271, 361
362 Anabolic stimuli, 361
363 Angiogenic factors, 52 Angiopoietins, 52 Angiotensin II receptor blocker, 127
128 Angiotensin-converting enzyme inhibitor, 127
128 Animal fat, 348 ANSES, 158, 168
169 Anti-HMG-CoA reductase autoantibodies, 503 Antihypersensitive effect, 215 Antihypertensive effect, 207 Antiinflammatory M2 macrophages, 129 properties, 289 Antioxidant, 169
170 defense, 169
170 effect, 173 of omega-3 supplements, 24 systems, 155 therapies to reducing mitochondrial-induced apoptosis signaling, 451
455 antioxidant regulation of cellular signaling, 453t Antiporters, 326
327 Apaf-1. See Apoptosis protease activating factor-1 (Apaf-1) Apoplexy, 210 Apoptosis, 427
428, 440
442, 518 caloric restriction regulates, 455
458 linking autophagy and, 434
435 signaling, 428
429 Apoptosis inducing factor (AIF), 425, 427
428 Apoptosis potential for resveratrol to reducing, 446
448 Apoptosis protease activating factor-1 (Apaf-1), 427
429 Appendicular skeletal muscle mass (ASM), 3
4 mass, 94 Appendicular skeletal muscle mass indices (ASMI), 3
4 AR. See Average requirement (AR)

INDEX

AR1. See Adiponectin receptor 1 (AR1) Arg:glycine amidinotransferase (AGAT), 298 Arginine (Arg), 75, 283, 296 arginine and muscle protein homeostasis in acute injury, 304
305 endothelial function and, 299 homeostasis, 297 metabolism, 296
298, 296f muscle creatine and, 298 muscle function and, 302
303 muscle protein homeostasis and, 299
302 tissue healing and, 303
304 Argininosuccinate lyase (ASL), 297 Argininosuccinate synthase (ASS), 297 ASC transporters. See Alanine
serine
cysteine transporters (ASC transporters) Ascorbic acid, 452 Asian Working Group for Sarcopenia (AWGS), 5
6 ASL. See Argininosuccinate lyase (ASL) ASM. See Appendicular skeletal muscle mass (ASM) ASMI. See Appendicular skeletal muscle mass indices (ASMI) Aspalathin, 406
409, 408f ASS. See Argininosuccinate synthase (ASS) Asthma, 197, 225
226 Asymmetric dimethylarginine (ADMA), 298 ATF. See Activating transcription factor (ATF) ATG. See Autophagy proteins (ATG) ATG13. See Mammalian autophagy-related gene 13 (ATG13) Atherosclerosis, 200, 209, 211 Athletes, 155
156, 174, 184 Atorvastatin, 504
506 ATP. See Adenosine triphosphate (ATP) Atrogin-1, 435
436 Autoimmune disease, 208, 390 Autoimmune-mediated necrotizing myositis, 499
500, 503 Autophagosomes, 430 Autophagy, 38, 142, 430
434, 518
520 adaptor protein p62, 432
433 altered carbohydrate metabolism, 519 caloric restriction regulates apoptosis and autophagy, 455
458 dysfunctional mitochondria removal by mitophagy, 432
433 induction of mitophagy, 431f inflammation/oxidative stress, 519
520 insufficient mitophagy allows unhealthy mitochondria, 433
434 linking autophagy and apoptosis BIM and Bcl-2, 434
435 PGC-1α, 435

531

Autophagy proteins (ATG), 432
433 ATG12, 437 Average requirement (AR), 165
166 AWGS. See Asian Working Group for Sarcopenia (AWGS) Azole antifungals, 504

B Bak. See Bax
Bcl-2 antagonist/killer (Bak) Basic fibroblast growth factor (bFGF), 52 Bax protein, 427
428, 442, 444 Bax
Bax homodimerization, 434 Bax
Bcl-2 antagonist/killer (Bak), 434 BCAAs. See Branched-chain amino acids (BCAAs) B-cell lymphoma interacting protein-3 (Bnip3), 311, 432 B-cell lymphoma-2 (Bcl-2), 432, 434
435, 456 Bcl-2 extralarge (Bcl-xL), 434 protein, 442 Bcl-2. See B-cell lymphoma-2 (Bcl-2) Bcl-xL. See Bcl-2 extralarge (Bcl-xL) BCL2L11 protein, 434 BDNF. See Brain derived neurotrophic factor (BDNF) Beclin-1, 456 β-conglycinin (βCGP), 478 and skeletal muscle effects on lowering of blood triglycerides, 476 influence glucose metabolism in skeletal muscle, 477
478 novel protein similar to, 478
479 peptides derived from βCGP modulate GLUT4 translocation, 478 promotion of adiponectin production, 476
477 β-hydroxy-β-methylbutyrate (HMB), 19
20, 448, 456 reducing muscle mass loss and myonuclear apoptosis, 448
451, 449t bFGF. See Basic fibroblast growth factor (bFGF) BIA. See Bioelectrical impedance analysis (BIA) BIM, 434
435 Bioactive factors, bioassay systems to search for, 402
403 Bioassay systems, 402
403 Bioelectrical impedance analysis (BIA), 3
4, 94
95 BIA-derived SM indices, 10 Blood Arg, 297 Blood brain barrier, 185
186 Blood pressure (BP), 201 Blood triglycerides, βCGP effects on lowering of, 476 Blunted muscle protein synthesis, 19
20 BMI. See Body mass index (BMI) Bnip3. See B-cell lymphoma interacting protein-3 (Bnip3) Body adiposity, 108

532 Body composition indices, 5 phenotypes, 93
94 Body mass index (BMI), 19, 67
68, 83, 93, 95, 109, 491 Body tissues, 279
280 BP. See Blood pressure (BP) Brain derived neurotrophic factor (BDNF), 521 Branched-chain amino acids (BCAAs), 71, 75, 183, 185
186, 189, 263
265, 264f, 279
280 acute effects of BCAA intake on muscle protein synthesis, 270
271 chronic effects of BCAA supplementation, 272
274 and skeletal muscle acute supplementation with, 187 amount of protein and amount of food product, 266t catabolism, 266
267, 267f composition of various food sources and skeletal muscle, 264t and insulin resistance, 274
275 leucine content of dietary protein, 271
272 mTOR signaling and muscle protein synthesis, 267
270 requirement and tolerable upper intake level, 265
266 Bromacology, 414 Bronchial asthma, 225
227 evidence-based physical training, 226 possible mechanisms, 227 type of training, 227 BsmI polymorphism, 383

C C2C12 myoblasts, 363
364 C2C12 myotubes, 363
364 Cachexia, 67 Calcitriol, 394 Calcium channel blockers, 504 Calf skeletal muscle, 100 Caloric restriction, 456 regulates apoptosis and autophagy, 455
458, 457t Calumenin, 311
312 Camellia sinensis (C. sinensis), 440
442 Cancer, 69
70, 197, 227
229, 390 cachexia, 362, 365
366 evidence-based training, 228 possible mechanisms, 228 total and specific AAs in promoting muscle anabolism, 71
75 amino acids, 75 branched chain amino acids, 75 short-term interventions, 71
75 type of training, 228
229

INDEX

Cancer patients muscle anabolism protein anabolism, 70
71 retrospective CT studies, 70 ω-3 fatty acid supplementation role in promoting muscle anabolism in, 77
78 Capillary shear stress, 51 Capillary-to-fiber ratio, 49, 54 Carbamoyl phosphate synthetase I (CPS I), 283 Carbamoyl phosphate synthetase II (CPS II), 283 Carbohydrates, 159, 183 ingestion, 161 threshold, 159 Carbohydrates (CHO), 159
165 5-Carbon amino acid glutamine (C5H10N2O3), 279 Carbon monoxide (CO), 323 Carboxyl (
COOH) group, 263
265 Cardiac rehabilitation (CR), 216 Cardiometabolic risk factors, 11 Cardiomyocytes, 302 Cardiomyopathy, 512
513 Cardiopulmonary fitness, 226 Cardiorespiratory fitness (CRF), 198 Cardiovascular disease (CVD), 93
94, 159
160, 199, 227
228 bronchial asthma, 225
227 cancer, 227
229 cerebral apoplexy, 210
212 COPD, 223
225 coronary heart disease, 215
217 heart failure, 218
221 hypertension, 212
215 intermittent claudication, 221
223 in older age, 96 Cardiovascular Health Study, 96 Cardiovascular risk factors in older age, 95 Carnitine, 329
331, 335
336 Carnitine palmitoyltransferase I (cpt-1b), 494 Carnitine palmitoyltransferase-1 (CPT-1), 118
120 Casein, 184, 271
272 Caspase-3, 427
428 Caspase-independent signaling, 428
429 CASTOR1. See Cellular arginine sensor for mTORC1 (CASTOR1) Catabolic pathway, 274
275 of cysteine, 322
323 Catabolic stimuli, 139
140 response to, 363
366 Catabolic stimulus, 281
283 Catabolism of BCAAs, 266
267, 267f metabolism of intermediate products derived from, 268f Cation transport regulator-like protein 1 (Chac1), 321

INDEX

CBP80. See 80 kDa nuclear cap binding protein (CBP80) CBS. See Cystathionine β-synthase (CBS) CCAAT/enhancer binding protein (C/EBPβ), 129 CCR2. See Chemokine (C-C motif) receptor type 2 (CCR2) cd36. See Cluster of differentiation 36 (cd36) C/EBPβ. See CCAAT/enhancer binding protein (C/EBPβ) Cell development, 515
516, 515f mitochondria initiators of cell death signaling, 427
430 targets, 169
170 types, 297
298 in vitro studies using cell-based model systems, 349 Cellular arginine sensor for mTORC1 (CASTOR1), 39 Cellular degradation, 440 Cellular processes under mTORC1, 36
38 Ceramides by SFA, De novo production of, 351
352 Cerebellar atrophy, 512 Cerebral apoplexy, 197, 210
212 evidence-based physical training, 211
212 possible mechanisms, 212 type of training, 212 Cerebrovascular accident, 210 βCGP. See β-conglycinin (βCGP) Chac1. See Cation transport regulator-like protein 1 (Chac1) CHD. See Coronary heart disease (CHD) Chemokine (C-C motif) ligand 2 (CCL2). See Mice quadriceps gene expression of MCP-1 Chemokine (C-C motif) receptor type 2 (CCR2), 126
127 Chemokine (C-X-C motif) ligand 1 (CXCL1), 126
127 Chemokine (C-X3-C motif) ligand 1 (CX3CL1), 126
127 Chemokines, 126
128 Chemotherapy, 227
228. See also Cancer toxicities, 70 Cholesterol, 200 Chronic adaptations to resistance exercise with protein supplementation, 188 Chronic alcoholic myopathy, 509
510 Chronic arginine supplementation and exercise, 302
303 Chronic conditions, range of, 393 Chronic diseases muscle anabolism in cancer patients, 70
71 omega-3 fatty acid supplementation, 77
78 other than cancer, 68
69 role of total and specific amino acids in promoting muscle anabolism

533

in cancer, 71
75 in chronic disease other than cancer, 76
77 sarcopenia in chronic diseases, 67
68 impact of sarcopenia on disease outcomes in chronic setting, 68
70 Chronic exercise, 152
157 Chronic high-fat diet, 109 Chronic inflammation, 515
516 Chronic kidney disease, 68
69 Chronic obstructive pulmonary disease (COPD), 197, 223
225 evidence-based physical training, 224
225 possible mechanisms, 225 type of training, 225 Chronic overfeeding, skeletal muscle in muscle mass, 109 strength, 108 typology, 111 skeletal muscle protein metabolism and obesity, 109
110 Chronic resistance exercise, 145 Chronic Respiratory Questionnaire (CRQ), 224 Chronic setting, sarcopenia impact on disease outcomes in, 68
70 Circulating growth hormones, 139
140 Cirrhosis, 76
77 Cit. See Citrulline (Cit) Citrate synthase (CS), 490
491 Citrulline (Cit), 75, 297, 309 citrulline-modulated functions, 311
312 modulatse muscle protein synthesis, 310 in muscle functions, 312 muscle protein metabolism, 311
312 Citrullus vulgaris. See Watermelon (Citrullus vulgaris) Citrus aurantium (C. aurantium), 169 c-Jun N-terminal kinase (JNK), 349
350, 381
382 CK. See Creatine kinase (CK) Clarithromycin, 504 Clinical cancer studies, 75 Cluster of differentiation 36 (cd36), 494 CMJ, 190
191 c-Myc expression, 145 Coenzyme A (CoA), 266, 324, 329
331, 335
336 Coenzyme Q10 (CoQ10), 505 Cohort studies, 11 Combination therapy, 503 with statin plus, 505 “Compete-high” approach, 160
165 Compounds metabolically linked to cysteine, 318
324 Computed tomography (CT), 6, 70 Concurrent training (CT), 47 specific responses to, 57
59

534

INDEX

Concurrent training (CT) (Continued) molecular responses to, 58
59 physiological responses to, 57 Control protein degradation processes, 35
36 Controlled cell deletion process, 518 Controlled studies, 209 COPD. See Chronic obstructive pulmonary disease (COPD) CoQ10. See Coenzyme Q10 (CoQ10) Coronary heart disease (CHD), 197, 215
217 evidence-based physical training, 216 possible mechanisms, 216 type of training, 216
217 cox2. See Cyclooxygenase 2 (cox2) CPS I. See Carbamoyl phosphate synthetase I (CPS I) CPS II. See Carbamoyl phosphate synthetase II (CPS II) CPT-1. See Carnitine palmitoyltransferase-1 (CPT-1) cpt-1b. See Carnitine palmitoyltransferase I (cpt-1b) CR. See Cardiac rehabilitation (CR) Creatine (Cr), 296, 298 Creatine, 329, 331
332, 335
336 creatine/phosphocreatine, 331 Creatine kinase (CK), 189, 298, 499
500 elevation, 502 C-reactive protein (CRP), 67
68, 95, 215 CRF. See Cardiorespiratory fitness (CRF) CRP. See C-reactive protein (CRP) CRQ. See Chronic Respiratory Questionnaire (CRQ) CS. See Citrate synthase (CS) CSE. See Cystathionine γ-lyase (CSE) CT. See Computed tomography (CT); Concurrent training (CT) Cultured L6 myotubes, glucose uptake assay in, 403
404, 403f CuZn-superoxide dismutase (CuZnSOD), 423 CVD. See Cardiovascular disease (CVD) CX3CL1. See Chemokine (C-X3-C motif) ligand 1 (CX3CL1) CXCL1. See Chemokine (C-X-C motif) ligand 1 (CXCL1) Cyclooxygenase 2 (cox2), 494 Cyclosporin-sensitive phosphatase, 52
53 Cyclosporine, 504 CYP. See Cytochrome P450 (CYP) Cystathionase, 318 Cystathionine, 318, 335 Cystathionine β-synthase (CBS), 318 Cystathionine γ-lyase (CSE), 318 Cystatin C, 311
312 Cysteine, 315, 317f, 320
321, 332
336 compounds metabolically linked to, 318
324 catabolic pathways of cysteine, 322
323, 322f coenzyme A, 324

general roles of taurine, hydrogen sulfide, and sulfate, 323
324 glutathione, 319
321 cysteine-based antioxidant, 455 deficiency syndrome, 334 endogenous synthesis, 318 residues, 315
316 Cysteinylglycine, 328 Cystine, 327
328 Cytochrome c, 425, 428
429, 489
490 Cytochrome oxidase, 186 Cytochrome P450 (CYP), 504 CYP 3A4, 504 Cytokines, 218, 519 secretion, 519
520 Cytokinins, 402, 413
414

D DAG. See Diacylglycerols (DAG) DAG O-acyltransferase 1 (DGAT1), 353 Daidzein, 410, 412f, 413f Daidzin, 410 Damaged proteins, 435
436 DAO. See Dynapenic abdominal obesity (DAO) DAP1. See Death-associated protein 1 (DAP1) db/db mice, 131, 409
410 T2D model, 412 DBP. See Diastolic BP (DBP) De novo production of ceramides by SFA, 351
352 of diacylglycerols by SFA, 351 Death-associated protein 1 (DAP1), 38 Defective mitophagy, 434 Deglutathionylation of proteins, 320 Dehydration, 154 Delayed onset muscle soreness (DOMS), 189 Denervation, 68, 422
423, 427
428 aging-associated muscle, 429
430 DEP domain containing mTOR-interacting protein (DEPTOR), 140
141, 268
270 Depolarized mitochondria, 432
433 DEPTOR. See DEP domain containing mTORinteracting protein (DEPTOR) Desulfuration pathways, 322 Detoxification process, 323
324 DGAT1. See DAG O-acyltransferase 1 (DGAT1) DHA. See Docosahexaenoic acid (DHA) Diabetes, 390 type 1 diabetes, 208
210 type 2 diabetes, 205
208 Diacylglycerols (DAG), 117
120, 351 de novo production of, 351 Diastolic BP (DBP), 202
203

INDEX

Diet-induced obesity (DIO), 125 Dietary BCAAs, 274
275 Dietary energy, 19 Dietary fat, 452 and health, 347
348 quality, 347
348 sources, 347
348 Dietary magnesium, 21 Dietary protein, 251
253, 263
265, 268
270 leucine content of, 271
272 Dietary reference values (DRVs), 165
168 for general population, 165
166 Dietary supplements, 173
174 Dietary vitamin D, 379 Differential proteomics approach, 311 Digestible protein, 271
272 Digestion, 268
270 1,25-Dihydroxyvitamin D (1,25D), 21 DIO. See Diet-induced obesity (DIO) Dipeptide form of glutamine, 288 DLT. See Dose-limiting toxicity (DLT) DNA, 475
476 mutations, 423
424 Docosahexaenoic acid (DHA), 359
362, 452 DOMS. See Delayed onset muscle soreness (DOMS) Dose-limiting toxicity (DLT), 69 Drp1. See Dynamin-related protein 1 (Drp1) Drug
drug interactions, 504 DRVs. See Dietary reference values (DRVs) Dual-energy X-ray absorptiometry (DXA), 3
4, 6, 94 Dynamin-related protein 1 (Drp1), 424 Dynapenia, 87 Dynapenic abdominal obesity (DAO), 85 clinical implications, 87
88 Dynapenic obesity, 84t Dysfunctional mitochondria, 420, 432 removal by mitophagy, 432
433 UPS disruption increases, 438 Dysfunctional permeabilized mitochondria, 438
440 Dysfunctional proteasome system, 438

E 4E-binding proteins (4E-BPs), 36 4EBP1, 142 E3 ubiquitin ligases, 139
140 EAAs. See Essential amino acids (EAAs) ECs. See Endothelial cells (ECs) Ectopic fat deposition, 491 eEF2K. See Eukaryotic elongation factor 2 kinase (eEF2K) EFSA. See European Food and Safety Authority (EFSA) EGCg. See Epigallocatechin-3-gallate (EGCg) Eicosapentaenoic acid (EPA), 359
362, 452

535

eIF2. See Eukaryotic initiation factors 2 (eIF2) eIF4E. See Eukaryotic translation initiation factor 4E (eIF4E) Electromyography (EMG), 390
391 Electron transport chain, 49
50 Elite synchronized swimmers, 163
164 EMG. See Electromyography (EMG) EndoG. See Endonuclease G (EndoG) Endogenous antioxidant systems, 169
170 Endogenous nitrogen compounds, 279 Endogenous synthesis, 297 of cysteine, 318 Endonuclease G (EndoG), 428
429 Endothelial cells (ECs), 51 Endothelial dysfunction, 209 Endothelial function, 222
223 Arg and, 299 Endothelial nitrogen oxide, 207 Endothelial NOS (eNOS), 297
299 Endurance exercise MHC composition responses by, 52
53 performance acute supplementation of protein and amino acids, 184
186 adaptations to endurance training, 186 Endurance training (ET), 47 adaptations to ET with protein and amino acid supplementation, 186 muscular adaptation to, 48
53 Energetic macronutrients nutrition strategies, 160
165 requirements, 157
160 intervals, 158
160 principle of setting intervals, 157
158 Energy demanding process, 158, 185 Energy production and mitochondrial biogenesis, 37 ENL. See Enterolactone (ENL) eNOS. See Endothelial NOS (eNOS) Enterolactone (ENL), 410 Environmental stress, adaptation to, 154
155 Enzymatic complex, 51 EPA. See Eicosapentaenoic acid (EPA) Epidemiological studies, 211 Epigallocatechin-3-gallate (EGCg), 440
445, 456, 490 resveratrol regulation of cellular signaling, 445t Epigenetic mechanism, 132 Equol, 410
412 Ergogenic aids and risks, 173
174 ERK. See Extracellular signal-regulated kinase (ERK) Essential amino acids (EAAs), 183, 253
254, 263
265, 270 Estrogen, 319
320 Estrogen-related receptor-α, 49
50

536

INDEX

ET. See Endurance training (ET) Ethanol, 509 ethanol inhibition of muscle repair, 515
516 ethanol-related diseases, 509
510 ubiquitin
proteasome machinery, 517 2-Ethylmalonic encephalopathy protein 1. See Sulfur dioxygenase Etiology-based types of malnutrition, 67 Euglycemic-hyperinsulinemic clamp, 493 Eukaryotic elongation factor 2 kinase (eEF2K), 36 Eukaryotic initiation factor 4E-binding protein 1 (4EBP1), 55, 268
270, 310
311 Eukaryotic initiation factors 2 (eIF2), 55, 286 Eukaryotic translation initiation factor 4E (eIF4E), 142, 268
270 European Food and Safety Authority (EFSA), 157
158, 172
173 European Working Group on Sarcopenia in Older People (EWGSOP), 5
6, 18, 25
26, 94 Eustress, 153 Evidence-based physical exercise, 208
209. See also Physical exercise and abdominal obesity, 204 impact on metabolic control, 206 and insulin resistance/prevention of type 2 diabetes, 202
204 and metabolic syndrome, 202 Evidence-based physical training, 198
201, 211
215, 218
219, 221
222, 224
226, 228 acute effect of physical activity, 214
215 effect on resting blood pressure, 213
214 EWGSOP. See European Working Group on Sarcopenia in Older People (EWGSOP) Exercise, 18, 139
140, 197, 202, 361
362. See also Physical exercise acute Arg supplementation and, 302 Arg in, 302 capacity, 216 chronic Arg supplementation and, 302
303 effect, 228 exercise-induced angiogenesis, 52 asthma, 226 change in micronutrients requirements, 167
168 oxidative stress, 154
155 stimuli, 47 exercise-related requirement justifying micronutrient supplement, 165
172 setting reference values for micronutrients, 165
168 side and adverse effects of supplementation, 168
172

frequency, 57 from general to exercising population, 158
160 nutrition on adaptive effects, 151
152 recovery from, 189
191 response to exercise training, 365
366 Extracellular degradation of glutathione, 320
321 Extracellular signal-regulated kinase (ERK), 40, 381
382 ERK5, 381
382 Extracellular signals, 38

F 4F2hc-xCT, 327
328 Factorial RCT, 23
24 FADD. See Fas-Associated protein with Death Domain (FADD) Falls and vitamin D deficiency, 393
394 FAs. See Fatty acids (FAs) Fas-Associated protein with Death Domain (FADD), 444 “Fast and slow” protein concept, 252
253 Fast digestive protein, 255
256 Fast soluble proteins, 254
255 “Fast” proteins, 252
255 for muscle anabolic response, 253
256 Fasting blood glucose level (FBG level), 405 Fat-free mass (FFM), 75, 107, 188 Fat(s), 160
165, 183
185. See also Obesity animal, 348 dietary, 452 milk, 348 oxidation, 161, 163 vegetable, 348 visceral, 93, 200 Fatty acids (FAs), 159
160, 347
348 composition, 349 MUFA, 347
348 oxidation, 494 PUFA, 452 FBG level. See Fasting blood glucose level (FBG level) FDA, 168
169 Fetuin-A, 311
312 FFA. See Free fatty acid (FFA) FFM. See Fat-free mass (FFM) Fiber atrophy, 514 nonspecific muscle fiber atrophy, 390
391 type 2 muscle fiber atrophy, 390
391, 510 Fibromyalgia, 392 Fis1. See Fission protein 1 (Fis1) Fish oil, 359 dietary consumption of, 452 supplements, 24

INDEX

Fission protein 1 (Fis1), 424 Fission proteins, 424 FokI polymorphism, 383 Food and components, pharmacology of, 414 FOR. See Functional overreaching (FOR) Forkhead box class O proteins (FOXO), 435
436 Free fatty acid (FFA), 161, 185
186 Free sulfur amino acids plasma concentrations of sulfur amino acids, 326 skeletal muscle concentrations of, 325
326 transmembrane transporters, 326
328 Free tryptophan (f-TRP), 185
186 Fructose, 163 f-TRP. See Free tryptophan (f-TRP) Functional food science, 414 Functional foods, 172, 486 Functional overreaching (FOR), 156
157 Fusion proteins, 424

G G protein-coupled receptor 56 (GPR56), 142 γ-glutamyl cycle, GSH metabolism through, 320
321 “Gaseous” signaling molecule, 323 Gastrocnemius muscle, 409 GATOR. See GTPase activating protein activity toward rags (GATOR) GCN2. See General control nonderepressible 2 (GCN2) Gemfibrozil, 504 General autophagy, 440 General control nonderepressible 2 (GCN2), 300
301 Genetic contribution to vitamin D effects, 383 Genome-wide polysome profiling, 37 Genomic effect of vitamin D, 381 GH. See Growth hormone (GH) Gingerol (GIN), 409
410 GIR. See Glucose infusion rate (GIR) GLP-1. See Glucagon-like peptide 1 (GLP-1) GLS. See Glutaminase (GLS) GLU. See Glutamate (GLU) Glucagon-like peptide 1 (GLP-1), 348 Glucose, 162
163 quercetin and glucose tolerance, 491
495 uptake assay in cultured L6 myotubes, 403
404, 403f Glucose infusion rate (GIR), 493 Glucose metabolism, 405
412 βCGP influence glucose metabolism in skeletal muscle, 477
478 in skeletal muscle cells, 405
412 Glucose transporter (GLUT4), 207, 215, 352
353, 402, 404f translocation, 477, 478f peptides derived from βCGP modulate, 478 Glutamate (GLU), 281, 283
284

537

Glutaminase (GLS), 281
283 Glutamine, 190 considerations of glutamine supplementation, 288
289 dipeptides, 288 glutamine-dependent anaplerosis, 281 intertissue metabolic flux, 286
288 and skeletal muscle glutamine metabolic biochemistry, 279
284 glutamine nutritional properties in, 284
288 important considerations of glutamine supplementation, 288
289 transporters, 283
284 Glutamine synthetase (GS), 281 Glutathione (GSH), 169
170, 283, 319
320 antioxidant system, 284 homeostasis, 334 in skeletal muscle, 328 intracellular degradation of, 321 metabolic pathways, 319f metabolism through γ-glutamyl cycle, 320
321 synthesis, 328 Glutathione conjugates (GSX), 319
320 Glutathione homodisulfide (GSSG), 284, 319
320 Glutathione-S-transferase, 319
320 Glutathionylation, 320 Gly-Gln. See L-glycyl-L-glutamine (Gly-Gln) Glycemic index, 159 Glycin max L. See Soybean (Glycin max L.) Glycine, 320
321 Glycogen phosphorylase, 311 Glycogen synthase (GS), 281
283, 406 Glycogenolysis, 311 Glycolysis, 37, 40, 311 Glycolytic enzyme activity, 47 Glycolytic-type fibers, 111 Goto
Kakizaki rats, 477 GPR56. See G protein-coupled receptor 56 (GPR56) Green rooibos extract (GRE), 408
409 Green tea catechins, 440
442 Green tea extract (GTE), 440
442, 456 Growth factors, 39
40 Growth hormone (GH), 139
140, 300 GS. See Glutamine synthetase (GS); Glycogen synthase (GS) GSH. See Glutathione (GSH) GSSG. See Glutathione homodisulfide (GSSG) GSX. See Glutathione conjugates (GSX) GTE. See Green tea extract (GTE) GTP. See Guanosine triphosphate (GTP) GTPase activating protein domain, 141 Rheb protein, 141

538 GTPase activating protein activity toward rags (GATOR), 301 GATOR1, 39, 268
270 GATOR2, 268
270 Guanosine triphosphate (GTP), 329
331, 330f hydrolysis, 141 Gut microbiota, 410

H Hamartin. See Tuberous sclerosis complex (TSC)—TSC1 Hans Selye’s theory, 153 Harmonization, 165
166 HbA1c. See Hemoglobin A1c (HbA1c) HBP. See Hexosamine biosynthetic pathway (HBP) Health claims and applications to sport nutrition, 172
174 Health maintenance, 151
152 Heart failure (HF), 197, 218
221, 302, 493 evidence-based physical training, 218
219 possible mechanisms, 219
220 type of training, 220
221 Heat shock (HS), 284
286 glutamine, 285f response, 284
286 Heat shock protein 70 (HSP70), 437 Heath shock factor 1 (HSF-1), 286 Height squared (ht2), 5 Helsinki Birth Cohort Study, 19 Hemiparesis, 212 Hemoglobin A1c (HbA1c), 201 Hepatotoxicity, 335 Herbal products, 174 Heterotrimer, 141 Hexokinase-II, 40 Hexosamine biosynthetic pathway (HBP), 286 HF. See Heart failure (HF) HFD. See High-fat diet (HFD) HFQ, 493 HGI. See High glycemic index (HGI) HIF-1. See Hypoxia-inducible factor 1 (HIF-1) High glycemic index (HGI), 163 High plasma AA, 253
254 High-fat diet (HFD), 126 High-fat feeding, 118
120, 125
132 High-intensity physical activity, 198 Highly lipophilic statins, 505 Hindlimb suspension (HLS), 440
442 Hindrance of adaptive effects as side effect, 169
172 Histone deacetylases, 455 HLA alleles, 503 HLS. See Hindlimb suspension (HLS) HMB. See β-hydroxy-β-methylbutyrate (HMB)

INDEX

HMG CoA reductase inhibitors, 499, 503 HOMA-IR. See Homeostasis model assessment of insulin resistance (HOMA-IR) Homeostasis, 475
476 Arg, 297 glutathione, 328 Homeostasis model assessment of insulin resistance (HOMA-IR), 411, 494 Homeostatic perturbations, 47 Homeostatic systems, 154
155 Homocysteine, 317
318 Hormesis theory, 152
155 Hospital Patient Health and Territory law, 168
169 HRE. See Hypoxic-response element (HRE) HS. See Heat shock (HS) HS proteins (HSPs), 284
286 HSF-1. See Heath shock factor 1 (HSF-1) HSP70. See Heat shock protein 70 (HSP70) HSPs. See HS proteins (HSPs) Human interventional studies, 75 Human plasma free methionine, 326 Human skeletal muscle, 48, 326 hVPS34. See Mammalian vacuolar protein sorting 34 homologue (hVPS34) Hydrogen peroxide (H2O2), 284 Hydrogen sulfide (H2S), 322
324, 335
336 Hydroxyl radical (OH•), 284 25-Hydroxyvitamin D (25(OH)D), 21
22 Hyperaminoacidemia, 361
362 Hypercholesterolemia, 199 HyperCKemia. See Creatine kinase (CK)—elevation Hyperglycemic effect, 476 Hyperlipidemia, 200
202 evidence-based physical training, 200
201 possible mechanisms, 201 type of training, 201
202 Hyperphosphorylation, 36 Hypertension, 199, 207, 211
215 evidence-based physical training, 213
215 possible mechanisms, 215 symptomatic ischemic heart disease, 201
202 type of training, 215 Hypertensive, 213
214 Hypertensive group, 213
214 Hypertriglyceridemia, 199 Hypertrophy, 3 hypertrophy, 54 muscle mass, 55 of myoblasts, 380 skeletal muscle, 145
146 Hypoglycemia risk, 209 Hypothyroidism, 504 Hypovitaminosis D, 375

INDEX

Hypoxia, 154, 171
172 Hypoxia-inducible factor 1 (HIF-1), 52 HIF1α, 37 Hypoxic-response element (HRE), 52

I IEDs. See Immune-enhancing diets (IEDs) IFN. See Interferon (IFN) IFNG. See Interferon gamma (IFNG) IGFs. See Insulin-like growth factors (IGFs) IHT. See Intermittent hypoxic training (IHT) IL. See Interleukins (IL) IMF mitochondria. See Intermyofibrillar mitochondria (IMF mitochondria) Immobilization, 68 Immune cells, 125, 279, 380 immune and muscle cell cross talk, 129
130 infiltration into skeletal muscle, 126 chemokines, adhesion molecules involved in, 126
128 Immune response, 304
305 Immune system, 286
288 Immune therapy, 503 Immune-enhancing diets (IEDs), 304
305 Immunofluorescence, 130 Immunosuppressive drug rapamycin, 140
141 IMTG. See Intramuscular triacylglycerol (IMTG) In vitro studies using cell-based model systems, 349 InCHIANTI study, 100 Indicator amino acid oxidation method, 265 Indices biological bases underlying, 4
6 clinical implications of indiscriminate use, 10
11 Induced putative kinase 1 (Pink1), 432 Inducible NOS (iNOS), 297
298 Inflammation, 111, 125
126, 190, 286
288, 519
520 Influx of fatty acids, 127 Ingested protein digestion, 35 Inorganic sulfurs, 332 iNOS. See Inducible NOS (iNOS) Inositol polyphosphate monokinase (IPMK), 39 Insulin, 35, 39
40, 71, 109
110, 253
254, 300, 477, 516 secretagogues, 274
275 secretion, 205
206 sensitivity, 349
350 SFA-mediated lipotoxicity and inhibition, 349
350 Insulin receptor (IR), 477 Insulin receptor substrate-1 (IRS-1), 349
350 Insulin resistance, 108, 111, 117
120, 125
126, 199, 301
302, 493, 517 BCAAS and, 274
275 syndrome. See Metabolic syndrome of type 2 diabetes, 202
204

539

Insulin-like growth factors (IGFs), 510
511 IGF-1, 129, 141, 219
220, 521 signaling, 516 INT. See Intense interval training (INT) Intense interval training (INT), 202
203 Interferon (IFN), 128 Interferon gamma (IFNG), 517, 519 Interleukins (IL), 126
127, 521 IL-1b, 519 IL-6, 95, 519 Intermittent claudication, 197, 221
223 evidence-based physical training, 221
222 possible mechanisms, 222
223 type of training, 223 Intermittent hypoxic training (IHT), 154 Intermyocellular nonesterified-free fatty acid, 125
126 Intermyofibrillar mitochondria (IMF mitochondria), 117
118 International Diabetes Federation, 202 International Society of Sports Nutrition, 159 International Working Group on Sarcopenia (IWGS), 5
6 Interorgan exchange, 335
336 Interorgan fluxes, 333
334 Intestinal agents, 505 Intracellular antioxidant systems, 520 degradation of GSH, 321 fatty acyl-CoA, 117 signaling pathways, 57 targets, 381
382 Intramuscular energy stores, 47 Intramuscular lipids, 118
120 Intramuscular triacylglycerol (IMTG), 161, 349
350 Intramyocellular triacylglycerols. See Intramuscular triacylglycerol (IMTG) Intraperitoneal glucose tolerance test (IPGTT), 412 Inverse dose
response association, 208 IPGTT. See Intraperitoneal glucose tolerance test (IPGTT) IPMK. See Inositol polyphosphate monokinase (IPMK) IR. See Insulin receptor (IR) Irisin, 521 IRS-1. See Insulin receptor substrate-1 (IRS-1) Isobutyryl CoA, 266 Isoleucine, 263
265 catabolism, 266
267 Isometric exercise, 213 Isopentenyladenine, 413
414 Isotopic tracer methodology, 71
72 Isovaleryl CoA, 266 IWGS. See International Working Group on Sarcopenia (IWGS)

540

INDEX

J Janus kinase/signal transducers and activators of transcription pathway (JAK/STAT pathway), 128 JNK. See c-Jun N-terminal kinase (JNK) Joint pain, 500
501

K 80 kDa nuclear cap binding protein (CBP80), 36 70-kDa ribosomal S6 kinase (p70S6k), 55 KHH buffer. See Krebs
Henseleit
Hepes buffer (KHH buffer) KIC. See α-ketoisocaproic acid (KIC) KIV. See α-ketoisovaleric acid (KIV) KK-Ay mice, 411 KMV. See α-keto-β-methylvaleric acid (KMV) Korean National Health and Nutrition Examination Survey, 20
23, 26 Krebs cycle. See Tricarboxylic acid cycle Krebs
Henseleit
Hepes buffer (KHH buffer), 403
404

L L6 myoblasts, 402 L6 myotubes, 402
403 L-alanyl-L-glutamine (Ala-Gln), 288 L-arginyl-L-glutamine, 288 LBM index. See Lean body mass index (LBM index) LC3 marker, 456 LC3. See Light chain 3 (LC3) LCHF diet. See Low carb high fat diet (LCHF diet) LCn-3PUFAs, 362 content, 361 supplementation, 363
367 treatment effect, 365
366 treatment under basal/fasting conditions, 362
363 LDL-C. See Low-density lipoprotein-cholesterol (LDLC) LDs. See Lipid droplets (LDs) Lean body mass, 364
365, 364f Lean body mass index (LBM index), 3
4 Leguminosae family, 475 Leptin, 517 signaling, 131 Leucine, 19
20, 71, 253
254, 263
267, 270
273 binding, 268
270 catabolism, 266
267 content of dietary protein, 271
272 leucine-enriched protein supplement, 273
274 leucine-rich amino acids, 190 Leukotriens, 319
320 LF, 493 LGP. See Liver glycogen phosphorylase (LGP)

L-glycyl-L-glutamine (Gly-Gln), 288 Lid-latch mechanism, 268
270 Lieber
DeCarli liquid diet model, 512
513, 520 Ligand
receptor interaction, 381 Light chain 3 (LC3), 432
433 Lipid droplets (LDs), 347
348 storage into, 353 Lipids, 347
348 infusion studies, 351 lipid-protein particles, 200
201 metabolism, 142 soluble vitamin E, 452 Lipopolysaccharide injection, 365
366 lipopolysaccharide-initiated oxidative stress, 448
451 Lipoproteins, 209 Lipotoxicity, 353 Liver cirrhosis, 68
69 Liver glycogen phosphorylase (LGP), 406 LOAEL. See Lowest-observed-adverse-effect level (LOAEL) Local tissue inflammation, 86 Localized apoptotic signaling, 438
440 hypothetical model for eliminating muscle fibers, 441f UPS regulation in sarcopenia, 439f Long-term glucocorticoid therapy, 390 Lovastatin, 504
505 Low aerobic-oxidative capacities, 118
120 Low carb high fat diet (LCHF diet), 162 Low muscle function, 94 Low muscle mass, 94, 365
366 Low-density lipoprotein-cholesterol (LDL-C), 499 Low-load resistance exercise, 140 Lowest-observed-adverse-effect level (LOAEL), 335

M Macrophage-secreted cytokines, 129 Macrophages, 287 MAFbx. See Muscle Atrophy F-box (MAFbx) Magnetic resonance imaging (MRI), 3, 6, 502 Malnutrition, 67, 310 Malondialdehyde (MDA), 520 Mammalian autophagy-related gene 13 (ATG13), 38 Mammalian lethal with Sec13 protein 8 (mLST8), 35
36, 140
141, 268
270 Mammalian skeletal muscles, 58
59 Mammalian target of rapamycin complex 1 (mTORC1), 35
36, 140
142, 268
270, 300
301, 327, 516
517 cellular processes under, 36
38 leucine-induced activation, 269f

INDEX

mTORC1
S6K1 axis, 143
145 pathway, 310
311 signaling, 435 signaling pathway, 142 Mammalian target of rapamycin complex 2 (mTORC2), 35
36 Mammalian vacuolar protein sorting 34 homologue (hVPS34), 39 Mammalian/mechanistic target of rapamycin (mTOR), 35, 140
141, 300
302, 516
517 signaling, 38, 267
270 MAP4K3. See Mitogen-activated protein kinase kinase kinase kinase (MAP4K3) MAPK. See Mitogen-activated protein kinase (MAPK) Marchiafava
Bignami disease, 512 Mb. See Myoglobin (Mb) MCP-1. See Monocyte chemoattractant protein 1 (MCP-1) MD. See Mean difference (MD) MDA. See Malondialdehyde (MDA) Mean difference (MD), 221
222 Mechanotransduction, potential mechanisms involving in, 56 Mediterranean diet pattern, 19 MEF. See Myocyte enhancer factor (MEF) Melanins, 319
320 Membrane-related processes, 117
118 Mesenchymal stem cells (MSC), 515 Messenger RNA (mRNA), 49, 281
283, 476, 490 Metabolic abnormalities, 67 Metabolic adaptations, 155
156 Metabolic control, impact on, 206 Metabolic diseases/disorders, 93 hyperlipidemia, 200
202 metabolic syndrome, 202
205 obesity, 198
199 type 1 diabetes, 208
210 type 2 diabetes, 205
208 Metabolic hypothesis, 111 Metabolic inflexibility, 118
120 Metabolic syndrome, 202
205, 299 evidence-based physical exercise, 202
204 possible mechanisms, 205 type of training, 205 Metabolically related compounds, skeletal muscle requirement in qualitative requirements and interorgan fluxes, 333
334 safety and practical considerations, 335
336 specific situations and potential supplementations, 334 critically ill patients, 334 elderly ill patients, 334 paracetamol treatment, 335

541

Metformin, 203, 402 Methionine, 315, 316f, 333
336 metabolism and specific roles transamination, 318 transsulfuration, 318 transmethylation and compounds metabolically linked to, 317
318 METs, 158, 515 MF. See Myofibrosis (MF) Mfn1. See Mitofusins 1 (Mfn1) MGC4504 gene, 321 MHC. See Myosin heavy chain (MHC) MI. See Myocardial infarct (MI) Mice quadriceps gene expression of MCP-1, 126
127 Microbial metabolites, 410
412 Micronutrients setting reference values, 165
168 adverse effects, 166 DRvs. for general population, 165
166 exercise-induced change, 167
168 supplement, 165
172 Microvascular network responses, 51
52 Milk fat, 348 ingestion, 184 milk-based beverages, 191 proteins, 184 Minerals, 165 MINOS study, 21
22 Minute ventilation at maximal exercise (VEmax), 226 Mithormesis theory, 153
154 Mitochondria permeability transition pore (mPTP), 428
429 Mitochondria(l), 35
36, 424 biogenesis, 37, 421
422, 456, 489
490 biogenesis, 489
490 CK, 298 content, 117
118 dynamics, 425, 456 enzyme activity, 186 factors involved in mitochondria dysfunction, 120
121 function, 48 aging attenuates mitochondrial function, 421
422 in aging muscles and motor neurons, 421
423 housed proteins, 428
429 initiators of cell death signaling, 427
430 membrane potential, 120
121 membrane proteins, 437
438 mitochondrial-induced nuclear apoptosis in aging muscle, 427
429, 428f mitophagy protein, 437
438 morphology, 424 network responses, 49
51, 50f

542

INDEX

Mitochondria(l) (Continued) permeability, 428
429 transition accelerates apoptosis, 429
430 PGC-1α regulation, 425
426 proteins, 37
38, 435 regulation of neural dysfunction, 422
423 respiratory chain, 311 role in type II fibers removal, 430 source, 169 Mitochondrial DNA (mtDNA), 422, 489
491 damage and aging, 423
424 deletions, 423
424 Mitochondrial dysfunction, 118
120 dynamic dysfunction, 425 potential sources altered mitochondrial dynamics with aging, 424
425 mitochondrial DNA damage and aging, 423
424 ROS-induced damage, 423 Mitochondrial transcription factor A (tfam). See Transcription factor A, mitochondrial (TFAM) Mitochondrial-induced apoptosis signaling, antioxidant therapies to reducing, 451
455 Mitochondrial-induced nuclear apoptosis in aging muscle, 427
429 Mitochondriogenesis in skeletal muscle quercetin and glucose tolerance, 491
495 quercetin and physical endurance, 488
491 Mitofusins 1 (Mfn1), 424 Mitofusins 2 (Mfn2), 120
121, 424 Mitogen-activated protein kinase (MAPK), 381
382 Mitogen-activated protein kinase kinase kinase kinase (MAP4K3), 39 Mitogen-activated protein kinase phosphatase-1 (MKP1), 129 Mitophagy, 427, 430 dysfunctional mitochondria removal, 432
433 insufficient mitophagy allows unhealthy mitochondria to persisting, 433
434 regulation and UPS, 437
438 MKP-1. See Mitogen-activated protein kinase phosphatase-1 (MKP-1) mLST8. See Mammalian lethal with Sec13 protein 8 (mLST8) Moderate-vigorous physical activity (MVPA), 18 Molecular mechanisms, 140, 142 involving in muscle responses to ET, 49
53 responses of MHC composition by endurance exercises, 52
53 responses of microvascular network, 51
52 responses of mitochondrial network, 49
51 involving in muscle responses to RT, 55
57

activation of satellite cells and the control of muscle mass, 56
57 control of ribosomal protein synthesis, 55
56 potential mechanisms involving in mechanotransduction, 56 of postmeal regulation of muscle anabolism cellular processes under mTORC1, 36
38 upstream mTOR signaling, 38
40 Molecular responses to CT, 58
59 putative adaptive pathways in response, 58f Monocyte chemoattractant protein 1 (MCP-1), 126
127 Monosaturated fatty acids, 359
360 Monounsaturated fatty acids (MUFAs), 159
160, 347
348 mechanisms of action in SM, 349
353 salvage effect on SFA-mediated lipotoxicity, 352
353 Mortality, 93
94 Motor neurons degeneration models, 422
423 mitochondrial function in, 421
423 PGC-1α regulation of mitochondria in, 426 MPB. See Muscle protein breakdown (MPB) MPO. See Myeloperoxidase (MPO) MPS. See Muscle protein synthesis (MPS) mPTP. See Mitochondria permeability transition pore (mPTP) MRF. See Myogenic regulatory factor (MRF) MRI. See Magnetic resonance imaging (MRI) mRNA. See Messenger RNA (mRNA) MRP. See Multidrug resistance-associated proteins (MRP) MS. See Myosteatosis (MS) MSC. See Mesenchymal stem cells (MSC); Muscle stem cells (MSC) mtDNA. See Mitochondrial DNA (mtDNA) mTOR. See Mammalian/mechanistic target of rapamycin (mTOR) mTORC1. See Mammalian target of rapamycin complex 1 (mTORC1) MUFAs. See Monounsaturated fatty acids (MUFAs) Multidrug resistance-associated proteins (MRP), 320 Multiple logistic regression analysis, 87 Mung bean 8S globulin, 479 Mung bean protein, 478
479 MuRF1. See Muscle RING Finger 1 (MuRF1) Murine C2C12 myocytes, 270 Muscle anabolism in cancer patients protein anabolism, 70
71 retrospective CT studies, 70 “interest of fast” proteins for muscle anabolic response, 253
256 muscle anabolic resistance, 253
254 omega-3 fatty acid supplementation, 77
78

INDEX

Muscle atrophy F-box (MAFbx), 139
140, 435
436, 504
505 Muscle biopsies, 378
379, 503 Muscle capillarity, 47 Muscle cell cross talk during skeletal muscle regeneration process, 129
130 glucose uptake, 405
412 migration dynamics, 380 vitamin D effect on muscle cell metabolism, 382
383 on muscle cell proliferation and differentiation, 381
382 Muscle contraction, 151 Muscle creatine, Arg and, 298 Muscle cytochrome C oxidase activity, 219
220 Muscle damage autoimmune-mediated necrotizing myositis, 503 definition of muscle toxicity, 499
500 management, 505
506 mechanism of muscle toxicity, 504
505 myalgias, 500
501 myopathy, 502 myositis and myonecrosis, 502 rhabdomyositis, 502 risk factors for muscle toxicity, 504 scope of problem, 499 Muscle dysfunction, 349 Muscle edema, 503 Muscle enzymes, 500 Muscle fiber atrophy, 511 Muscle function, 392
393 Arg and acute arginine supplementation and exercise, 302 Arg in exercise, 302 chronic arginine supplementation and exercise, 302
303 plant hormones and, 413
414 Muscle growth, 140, 272
274 Muscle health, 151
152 Muscle hypertrophy, 54, 362 Muscle immune cell infiltration, and activation, 125
128 chemokines, adhesion molecules involved in immune cell infiltration, 126
128 immune cell infiltration into skeletal muscle, 126 skeletal muscle immune cell activation, 128 Muscle loss/atrophy, 187 Muscle mass, 35, 53
54, 54f, 109, 379 age-related declines in, 17 control, 56
57 HMB reducing muscle mass loss, 448
451

543

hypertrophy, 55 regulation, 362
364 response to exercise training and catabolic stimuli, 365
366 responses in healthy, weight stable adults, 364
365 strength and physical function, 366
367 Muscle mitochondrial function, 118
120 Muscle mitochondriogenesis, 491 Muscle oxidative capacities, 118
120 Muscle pain, 391
392 Muscle protection, 256 “interest of fast” proteins for muscle anabolic response, 253
256 skeletal muscle loss and protein nutrition, 251
253 Muscle protein accrual/hypertrophy, 187 content, 310 homeostasis in acute injury, 304
305 Arg, mTOR, and protein synthesis, 300
302 increased secretion of growth factors, 300 increased substrate delivery, 299
300 mass, 324
325 metabolism, 311
312 omega-3 fatty acids effect on muscle protein turnover, 361
362 Muscle protein breakdown (MPB), 35, 53, 70, 187, 361
362 Muscle protein synthesis (MPS), 53, 68, 187, 361
362 acute effects of BCAA intake, 270
271 leucine-induced activation of mTORC1, 269f mTOR signaling and, 267
270 rates, 272 Muscle proteolysis, 311 Muscle quality, 366 Muscle regeneration, 380 capacity, 128
132 immune and muscle cell cross talk, 129
130 effect of obesity and/or high-fat feeding, 130
132 Muscle repair, ethanol inhibition of, 515
516 Muscle responses to ET, 49
53 to RT, 55
57 Muscle RING Finger 1 (MuRF1), 139
140, 435
436 Muscle side effects, 499 Muscle soreness, 190 Muscle stem cells (MSC), 125 Muscle strength, 85, 96, 108, 140, 310, 377 Muscle symptoms, 500
501 Muscle toxicity, 499
500, 504
505 risk factors for, 504 Muscle triacylglycerol content, 493
494 Muscle typology, 111 changes in, 111

544 Muscle volume response to exercise training and catabolic stimuli, 365
366 responses in healthy, weight stable adults, 364
365 strength and physical function, 366
367 Muscle wasting, 435
436 Muscle weakness, 376, 378
379, 393 and vitamin D deficiency, 390
391 Muscles, 477 Muscular adaptation to ET, 48
53 molecular mechanisms involved in muscle responses to ET, 49
53 muscular adaptation to resistance training, 53
57 oxidative phenotype, 48
49 to RT, 53
57 molecular mechanisms involving in muscle responses to RT, 55
57 skeletal muscle microvascular network, 54 training-induced increase in muscle mass, 53
54 Mutant polyglutamines protein aggregation, 280
281 Mutations, 227
228 MVPA. See Moderate-vigorous physical activity (MVPA) Myalgias, 391, 499
501, 505 Myeloperoxidase (MPO), 286
287 MYF. See Myogenic factor (MYF) Myoblast determination protein (MYOD), 130
131 Myocardial infarct (MI), 216 Myocardial ischemia, 215
216 Myocyte enhancer factor (MEF), 50
51 Myocytes, 128 MYOD. See Myoblast determination protein (MYOD) MyoD. See Myogenic differentiation (MyoD) Myofiber phenotype, 48 Myofiber size, 48 Myofibrillar proteolysis, 309
310 Myofibrosis (MF), 83
85 MYOG. See Myogenin (MYOG) Myogenic differentiation (MyoD), 56
57 Myogenic factor (MYF), 132 Myogenic regulatory factor (MRF), 56
57, 515 Myogenin (MYOG), 130
131 transcription factors, 56
57 Myoglobin (Mb), 189 Myokines, 197, 521 Myonecrosis, 499
500, 502 Myonuclear apoptosis, HMB reducing, 448
451 Myopathy, 390, 499
500, 502 falls and vitamin D deficiency, 393
394 muscle pain and vitamin D deficiency, 391
392

INDEX

muscle weakness and vitamin D deficiency, 390
391 vitamin D, 389
390 vitamin D and physical performance measures, 392
393 Myosin heavy chain (MHC), 48
49 composition responses by endurance exercises, 52
53 Myosin isoforms, 49 Myositis, 499
500, 502 Myostatin, 517 expression, 89 Myosteatosis (MS), 83
85, 510 Myotubes, 351
352 fusion, 380 Myristate acid. See Myristic acid Myristic acid, 348

N N-Acetlycysteine, 335 NADPH, 281
283 NADPH oxidase system (NOX system), 283, 423 Nakanojo Study, 18
19 National Cholesterol Education Program, 499 National Health and Nutrition Examination Survey, 100 National Lipid Association (NLA), 500, 502 statin myalgia clinical index score, 501t Natural killer (NK), 126 NCD. See Normal calorie diet (NCD) NEAA. See Non-EAA (NEAA) Nephropathy, 205
206 Nepodin, 409
410 NETs. See Neutrophil extracellular traps (NETs) Neural diseases, 423
424 Neural dysfunction, mitochondrial regulation of, 422
423 Neural dysfunction, UPS and, 437 Neuronal NOS (nNOS), 297
298, 302 Neuropathy, 210 Neutrophil extracellular traps (NETs), 286
287 New Mexico Elder Health Survey, 18 New York Heart Association (NYHA), 218 NF-κB. See Nuclear factor kappa B (NF-κB) NFAT. See Nuclear factor of activated T cells (NFAT) NFOR. See Nonfunctional overreaching (NFOR) Nitric oxide (NO), 279, 283, 296, 309, 323 NK. See Natural killer (NK) NLA. See National Lipid Association (NLA) NMMA. See N-Monomethylarginine (NMMA) N-Monomethylarginine (NMMA), 298 nNOS. See Neuronal NOS (nNOS) NO synthases (NOS), 297

INDEX

No-observed-adverse-effect level (NOAEL), 335 Non-EAA (NEAA), 183, 270 Nonenvironmental-stressing situations, 171
172 Nonessential amino acid, 279 Nonfactorial RCTs, 23 Nonfunctional overreaching (NFOR), 156
157 Nongenomic effect of vitamin D, 381 Nonisocaloric supplements, 185 Nonsmall cell lung cancer (NSCLC), 72
73, 77t Nordic diet, 19 Normal calorie diet (NCD), 126
127 Normotensive group, 213
214 NOS. See NO synthases (NOS) NOX system. See NADPH oxidase system (NOX system) NRF. See Nuclear respiratory factor (NRF) Nrf2. See Nuclear factor erythroid-derived 2-related factors (Nrf2) NSCLC. See Nonsmall cell lung cancer (NSCLC) Nuclear factor erythroid-derived 2-related factors (Nrf2), 437
438 Nuclear factor kappa B (NF-κB), 127
128, 349
350, 423 Nuclear factor of activated T cells (NFAT), 52
53 Nuclear respiratory factor (NRF), 49
50 Nuclear/nucleus, 381 apoptosis, 427
428 genome encodes, 49
50 respiratory factor 1 genes, 118
120 Nutrition(al), 19
22, 154 on adaptive effects of exercise, 151
152 approaches, 362 combining physical activity and, 22
25 consequences on nutrition periodization, 164
165 disorders, 67 requirements, 157
158, 165, 167 science, 414 strategies, 160
165 supplements, 456 Nutritional strategies, 163 to reverse mitochondrial death signaling antioxidant therapies to reducing mitochondrialinduced apoptosis signaling, 451
455 caloric restriction regulates apoptosis and autophagy, 455
458, 457t EGCg, 442
445 HMB reducing muscle mass loss and myonuclear apoptosis, 448
451 polyphenol GTE reduces apoptosis signaling in aged muscles, 440
442 potential for RSV to reducing apoptosis and oxidative stress, 446
448 NYHA. See New York Heart Association (NYHA)

545

O OAT. See Orn aminotransferase (OAT) ob/ob mice, 125
127, 131, 406, 411
412 Obese Zucker rats, 109
110 Obesity, 86
87, 93
94, 117
118, 125
132, 198
199, 201. See also Sarcopenic obesity (SO) and/or high-fat feeding effect on skeletal muscle repair, 130
132 evidence-based physical training, 198
199 obesity-linked inflammation, 125
126 obesity-related disorders, 111 skeletal muscle in muscle mass, 109 muscle strength, 108 muscle typology, 111 skeletal muscle protein metabolism and obesity, 109
110 skeletal muscle insulin resistance, 117 type of training, 199 OCT. See Orn carbamyl transferase (OCT) OCTN2. See Sodium-dependent organic cation transporter-2 (OCTN2) OEA. See Oleoylethanolamide (OEA) 25(OH)D. See 25-Hydroxyvitamin D (25(OH)D) 1α,25(OH)2vitamin D activates, 381
383 1α,25(OH)2vitamin D
VDR complex, 381 Older people, 5
10 Oleic acid, 348, 352 Oleoylethanolamide (OEA), 352
353 O-linked-N-acetylglucosamine (O-GlcNAc), 286 Olive oil, 348 Omega tail, 359
360 Omega-3 fatty acids (ω-3 fatty acids), 359
361, 452
455 effect on muscle protein turnover, 361
362 LCn-3PUFA treatment under basal/fasting conditions, 362
363 regulation of muscle mass, 362
364 response to anabolic stimuli, 363 response to catabolic stimuli, 363
364 supplementation in promoting muscle anabolism, 77
78 Omega-3, 255
256 Omega-6 fatty acids (ω-6 fatty acids), 359
360 Optic atrophy 1 (Opa1), 424 Oral quercetin, 495 Organic precursor, 375 Orn. See Ornithine (Orn) Orn aminotransferase (OAT), 297 Orn carbamyl transferase (OCT), 297 Ornithine (Orn), 297 Osteoglycin, 521 Osteomalacia, adults with, 390

546

INDEX

OTS. See Overtraining syndrome (OTS) Overreaching syndrome, 156
157 Overtraining syndrome (OTS), 156
157 Overweight, 100, 107, 204. See also Obesity Oxidants, 169 Oxidation of indicator amino acid, 265 Oxidative metabolism, 48 Oxidative phenotype, 48
49 Oxidative phosphorylation, 422 Oxidative phosphorylation activity (OXPHOS activity), 118
120 Oxidative stress, 20
21, 111, 120
121, 169, 440
442, 448
451, 519
520 potential for resveratrol to reducing, 446
448 Oxidative-type fibers, 111 Oxidized glutathione derivatives, 320 5-Oxoproline, 321 OXPHOS activity. See Oxidative phosphorylation activity (OXPHOS activity) Oxygen treatment, 224
225

P p38 MAPK, 381
382 p70 ribosomal protein S6 kinase 1 (S6K1), 36, 310
311 p70S6k. See 70-kDa ribosomal S6 kinase (p70S6k) PA. See Phosphatidic acid (PA); Physical activity (PA) Paired-box protein (PAX), 132 p-Akt. See Phosphorylated Akt (p-Akt) Palmitate acid. See Palmitic acid Palmitic acid, 348, 353 Palmitoleate. See Palmitoleic acid Palmitoleic acid, 348 Palmitoyl-CoA, 351
352 p-AMPK. See Phosphorylated AMPK (p-AMPK) PAPS. See 30 -Phosphoadenosine 50 -phosphosulfate (PAPS) Paracetamol, 315
316, 319
320, 323
324 paracetamol-protein adducts, 315
316 treatment, 335 Parkinson’s disease, 423
424 Pathogenesis, 514
520 apoptosis, 518 autophagy, 518
520 cell development, 515
516, 515f increased protein breakdown, 517 reduced protein synthesis, 516
517 PAX. See Paired-box protein (PAX) PCr. See Phosphocreatine (PCr) PCSK9 inhibitors, 505 PDEs. See Phosphodiesterases (PDEs) PE. See Phosphatidylethanolamine (PE)

PEPCK. See Phosphoenolpyruvate carboxykinase (PEPCK) pepT. See Peptide transporters (pepT) Peptide transporters (pepT), 328 pepT1, 289, 328 pepT2, 328 Peptides, 478 Percutaneous transluminal angioplasty (PTA), 222 Perilipin-5 (PLIN5), 353 Perimuscular adipose tissues (PMAT), 126 Peripheral arteriosclerosis, 221 PERKA
TF4. See Protein kinase RNA-like endoplasmic reticulum kinase—activating transcription factor 4 (PERKA
TF4) Peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α), 37, 49
50, 118
120, 352
353, 425
426, 435, 456, 489
490 regulation of mitochondria, 425
426 in aged muscles, 426 in motor neurons, 426 Peroxisome proliferator activated receptor γ coactivator 1β (PGC1-β), 352
353 Peroxisome proliferator-activated receptor gamma coactivator (PGC1), 352
353 Peroxisome proliferator-activated receptors alpha (PPAR-α), 352
353, 494 Persulfide dioxygenase. Sulfur dioxygenase PGC1. See Peroxisome proliferator-activated receptor gamma coactivator (PGC1) Phenotypic plasticity of mitochondria, 48 Phenotypical presentation, 197 Phosphatidic acid (PA), 56 Phosphatidylethanolamine (PE), 432
433 Phosphatidylinositol 3-kinase (PI3K), 310
311, 349
350, 477 Phosphatidylinositol 3,4,5-trisphosphate (PIP3), 131 30 -Phosphoadenosine 50 -phosphosulfate (PAPS), 323
324 Phosphocreatine (PCr), 298 Phosphodiesterases (PDEs), 414 Phosphoenolpyruvate carboxykinase (PEPCK), 406 Phosphorylated Akt (p-Akt), 405 Phosphorylated AMPK (p-AMPK), 405 Phosphorylation, 36 Phosphorylation of 4E-BP1, 268
270 Physical activity (PA), 18
19, 151, 158, 197, 223, 228 acute effect of, 214
215 combining PA and nutrition, 22
25 Physical endurance, quercetin and, 488
491 Physical examination, 503 Physical exercise, 226, 228. See also Resistance exercise and abdominal obesity, 204

INDEX

in chronic diseases cardiovascular and pulmonary diseases, 210
229 metabolic diseases, 198
210 methods, 197
198 perspective, 229 and insulin resistance/prevention of type 2 diabetes, 202
204 and metabolic syndrome, 202 training, changes in nutritional requirements and dietary support acute and chronic exercise, 152
157 energetic macronutrients, 157
165 exercise-related requirement justifying micronutrient supplement, 165
172 health claims and “beneficial” expected effects, 172
174 role of nutrition on adaptive effects of exercise, 151
152 Physical function, strength and, 366
367 Physical inactivity, 68, 211 Physical performance, 151
152, 392
393 Physical therapy methods, 212 Physical training, 209, 217, 221 Physiological adaptations, 152 Physiological regulation of skeletal muscle mass protein synthesis, resistance exercise and, 140
142 ribosomal biogenesis, resistance exercise and, 143
145 satellite cells, resistance exercise and, 145
146 Physiological responses to CT, 57 Physiological stress, 151 Phytochemicals, 410
412 bromacology, 414 direct molecular target activating AMPK, 414 glucose metabolism in skeletal muscle cells, 405
412 aspalathin, muscle cell glucose uptake and glucose metabolism, 406
409 nepodin, gingerol, muscle cell glucose uptake, and glucose metabolism, 409
410 stilbenoids, muscle cell glucose uptake, and glucose metabolism, 405
406 intestinal metabolites, and skeletal muscle function cultured skeletal muscle cells and incubated muscle tissues, 402
403 glucose uptake assay in cultured L6 myotubes, 403
404, 403f microbial metabolites, muscle cell glucose uptake, and glucose metabolism, 410
412 plant hormones and muscle function, 413
414 PI3K. See Phosphatidylinositol 3-kinase (PI3K) Piceatannol, 405
406, 405f, 407f Pink1. See Induced putative kinase 1 (Pink1)

547

PIP3. See Phosphatidylinositol 3,4,5-trisphosphate (PIP3) Pitavastatin, 505 PKC. See Protein kinase C (PKC) Plant extracts, 169 Plant hormones, 413
414 Plasma BCAA concentrations, 274
275 concentrations of sulfur amino acids, 326 glutamine concentration, 280 glutathione, 328 Plasticity of skeletal muscle, 17 Platelet-derived growth factor, 521 PLIN5. See Perilipin-5 (PLIN5) PMAT. See Perimuscular adipose tissues (PMAT) Pol III, 143
145 Polar amino acid, 280
281 Polyamine synthesis, 304 Polyglutamines, 280
281 Polyneuropathy, 512 Polynuclear neutrophils, 129 Polyphenol compounds regulating cellular signaling, 443t GTE reduces apoptosis signaling in aged muscles, 440
442 Polysulfides (H2Sn), 323 Polyunsaturated fatty acids (PUFAs), 159
160, 255
256, 347
348, 359
360, 452, 490 Polyunsaturated omega-3 fatty acids, 24, 359
361, 360f. See also Omega-3 fatty acids (ω-3 fatty acids) muscle mass and volume, 364
366 effect of omega-3 fatty acids on muscle protein turnover, 361
362 series of enzymatic reactions, 361f Population reference intakes (PRI), 165
166 Postprandial lipid response, 348 Postprandial muscle protein synthesis, 35, 270
271 Postural instability, 393 Potassium (K1), 21, 283
284 PPAR-α. See Peroxisome proliferator-activated receptors alpha (PPAR-α) PRAS40. See Proline-rich Akt substrate of 40 kDa (PRAS40) Pravastatin, 505 Precachexia, 67
68 Prednisone, 503 PRI. See Population reference intakes (PRI) Proapoptotic proteins, 427
428 Proinflammatory adipokines, 128 mediators, 127
128 states, 365
366

548

INDEX

Proline-rich Akt substrate of 40 kDa (PRAS40), 140
141, 268
270 Prostaglandins, 319
320 PROT-AGE study group, 251
252 Protease inhibitors, 504 Protein kinase B (Akt), 129, 141, 300
301 Akt/mTOR signaling pathway, 58 phosphorylation, 494 Protein kinase C (PKC), 349
350 PKCε, 351 PKCζ, 351
352 Protein kinase RNA-like endoplasmic reticulum kinase—activating transcription factor 4 (PERKA
TF4), 321 Protein synthesis, 35
36, 55, 70
71, 73, 140, 300
302, 516 reduction, 516
517 resistance exercise and, 140
142 Protein
energy deficiency model, 310 restriction model, 309
310 Proteinogenic amino acid, 280 Proteins, 183, 272, 311
312 and amino acids acute supplementation of, 184
186 adaptations to endurance training with supplementation, 186 anabolism, 70
71 breakdown, 70
71, 517 degradation, 38, 140 intake, 77
78, 184 kinase, 140
141 metabolism, 109
110 nutrition, 251
253 “fast and slow” protein concept, 252
253 protein-calorie malnutrition, 512
513 protein-restricted diet, 76 requirements, 184 similar to βCGP, 478
479 skeletal muscle concentrations of sulfur amino acids, 324
325 sources, 20, 183 supplementation, 140, 188, 273
274 acute responses to resistance exercise with, 187 chronic adaptations to resistance exercise with, 188 turnover, 187 PROVIDE study, 21
22 Pseudo-Cushing syndrome, 514 PTA. See Percutaneous transluminal angioplasty (PTA) PUFAs. See Polyunsaturated fatty acids (PUFAs) Pulmonary diseases bronchial asthma, 225
227

cancer, 227
229 cerebral apoplexy, 210
212 COPD, 223
225 coronary heart disease, 215
217 heart failure, 218
221 hypertension, 212
215 intermittent claudication, 221
223 Pyruvate, 335
336

Q Qualitative requirements, 333
334 Quercetin, 486, 486f, 487t and glucose tolerance, 491
495 glucosides, 486 and physical endurance, 488
491 lipotoxicity, 492f mitochondriogenesis pathway, 489f

R Rag. See Ras-related GTPase (Rag) Ragulator, 39 Rancho Bernardo Study, 25 Randomized controlled trial (RCT), 22, 198, 200
201, 203, 209, 211, 213, 225, 393
394 RANTES. See Regulated on activation, normal T cell expressed and secreted (RANTES) Rapamycin, 37
38 insensitive
way, 142 Raptor. See Regulatory-associated protein of mTOR (Raptor) Ras homolog enriched in brain (Rheb), 39
40, 141, 300
301 Ras-related GTPase (Rag), 301 RCT. See Randomized controlled trial (RCT) RDA. See Recommended dietary allowance (RDA) rDNA. See Ribosomal DNA (rDNA) Reactive nitrogen species (RNS), 20
21 Reactive oxygen species (ROS), 20
21, 118
120, 153, 170
171, 284, 406, 420, 519 key role, 153
154 production, 120
121 ROS-induced damage, 423 Recommended dietary allowance (RDA), 272
273 Recovery from exercise, 189
191 Reduction in muscle strength, 224 Reference interval (RI), 157
158 Refractory cachexia, 67
68 Regeneration process, 125, 128
129 Regulated on activation, normal T cell expressed and secreted (RANTES), 126 Regulation of mTOR by growth factors, 39
40 of mTORC1 by energy, 40

INDEX

Regulatory-associated protein of mTOR (Raptor), 268
270 Renal failure, 376 Renin
angiotensin
aldosterone system, 127
128 1-Repetition maximum (1RM), 53, 140 Resistance, 153, 205 Resistance exercise, 53, 140, 361
362. See also Physical exercise performance, 187
189 acute responses to resistance exercise with protein supplementation, 187 chronic adaptations to resistance exercise with protein supplementation, 188 resistance exercise-induced adaptations, 189 resistance exercise-mediated muscle hypertrophy resistance exercise and protein synthesis, 140
142 resistance exercise and ribosomal biogenesis, 143
145 resistance exercise and satellite cells, 145
146 training, 366
367 Resistance training (RT), 18, 47, 207, 379
380 muscular adaptation to, 53
57 Resting blood pressure, effect on, 213
214 Resveratrol (RSV), 405, 405f, 446, 456 potential for RSV to reducing apoptosis and oxidative stress, 446
448 Retinopathy, 205
206 Retrospective CT studies, 70 Reverse mitochondrial death signaling, nutritional strategies to, 440
458 Rhabdomyolysis, 499
500, 509 Rhabdomyositis, 499
500, 502 Rheb. See Ras homolog enriched in brain (Rheb) RI. See Reference interval (RI) Ribonucleoproteins, 143
145 Ribosomal biogenesis, resistance exercise and, 143
145 Ribosomal content, 55 Ribosomal DNA (rDNA), 143
145 Ribosomal protein S6K1, 142 simplified model of mTORC1 signaling pathway, 56f synthesis control, 55
56 Ribosomal protein S6 (rpS6), 36 Ribosomal RNAs (rRNAs), 143
145 Ribosomes., 143
145 RNA polymerase I (RNA Pol I), 143
145 RNS. See Reactive nitrogen species (RNS) Rodent(s), 111 model, 380 1RM. See 1-Repetition maximum (1RM) ROS. See Reactive oxygen species (ROS) Rosuvastatin, 505
506 rpS6. See Ribosomal protein S6 (rpS6) rRNAs. See Ribosomal RNAs (rRNAs)

549

RSV. See Resveratrol (RSV) RT. See Resistance training (RT) Rumex japonicus (R. japonicus), 409

S 45S pre-rRNA, 145 S6 kinase-1 (S6K-1), 142, 268
270, 516
517 phosphorylation, 516
517 S6K1 Aly/REF-like target (SKAR), 36 S6K1. See p70 ribosomal protein S6 kinase 1 (S6K1) S-adenosylhomocysteine, 317
318 S-adenosylmethionine, 317
318 Salvage effect of MUFA on SFA-mediated lipotoxicity, 352
353 Saponin, 48 Sarcopenia, 17
19, 21
22, 25, 67
70, 83
85, 87, 93
94, 109, 130, 251, 376, 383, 393, 420 autophagy, 430
434 in chronic diseases, 67
68 impact on disease outcomes in chronic setting cancer, 69
70 chronic diseases, 68
69 linking autophagy and apoptosis, 434
435 localized apoptotic signaling spreads to remove entire fiber in, 438
440 loss of mitochondrial function, 420 mitochondria function in aging muscles and motor neurons, 421
423 initiators of cell death signaling in aging muscles, 427
430 mitophagy, and UPS, 437
438 nutritional targeting of mitochondrial initiated sarcopenia, 460f PGC-1α regulation of mitochondria in, 425
426 potential for nutritional strategies to reverse mitochondrial death signaling, 440
458 potential sources of mitochondrial dysfunction in aging, 423
425 syndrome, 6 UPS, 435
437 Sarcopenic obese group, 95 Sarcopenic obesity (SO), 83
85, 93
95 and all-cause mortality in older age, 96
100 and cardiovascular disease in older age, 96 and cardiovascular risk factors in older age, 95 clinical implications, 86
87 and dynapenic obesity, 84t examining associations between cardiovascular disease or mortality and, 97t nutritional recommendation, 89t pathogenesis, 85
86 treatment, 88
89

550 Sarcopenic rodents, 446 Satellite cells activation, 56
57 depletion, 145
146 resistance exercise and, 145
146 Saturated fatty acid (SFA), 347
348, 359
360 de novo production of ceramides by, 351
352 of diacylglycerols by, 351 lifestyle interventions to alleviate SFA-mediated lipotoxicity, 354 mechanisms of action in SM, 349
353 salvage effect of MUFA on SFA-mediated lipotoxicity, 352
353 SFA-mediated lipotoxicity and inhibition of insulin action, 349
350, 350f SBP. See Systolic blood pressure (SBP) SCD1. See Stearoyl-CoA desaturase 1 (SCD1) Scoring system, 500 SDA. See Stearidonic acid (SDA) Second mitochondria-derived activator of caspase/ direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO), 428
429 Sepsis, 362 Serine palmitoyl-CoA transferase-I (SPT-I), 351
352 Serine/threonine kinase mTOR, 140
141 protein, 35
36 Sestrin2, 39, 268
270 Setting intervals, principle of, 157
158 SFA. See Saturated fatty acid (SFA) SGLT1, 162 Short-term interventions, 71
75 Side-chain amide group, 280
281 Signaling molecules, 153
154 pathway, 382
383 Sildenafil, 504 Simvastatin, 504
505 Single-nucleotide polymorphisms, 383 Sirtuin 1 (SIRT1), 286, 446, 456, 489
490 SKAR. See S6K1 Aly/REF-like target (SKAR) Skeletal muscle (SM), 3, 38
39, 67
68, 107, 117, 125
128, 139
141, 420 adaptations, 58
59 adaptive growth, 141
142 adhesion molecules involving in immune cell infiltration into, 126
128 aging, 86 atrophy, 139
140 βCGP influencing glucose metabolism in, 477
478 biological bases underlying indices, 4
6

INDEX

clinical implications of indiscriminate use, 10
11 concentrations plasma supply, and other compounds metabolically sulfur AAs, 329
332 and plasma supply of taurine, 329 of sulfur amino acids contained in proteins, 324
325 expression of genes, 491 fibers, 111 free sulfur amino acids, 325
328 function, 349 glutamine metabolic biochemistry in, 280
284 muscle glutamine stores, 282f synthesis and hydrolysis, 281f glutamine nutritional properties glutamine intertissue metabolic flux, immune system, and inflammation, 286
288 glutathione antioxidant system, 284 HS response, 284
286 glutamine-GSH axis, 284 glutathione homeostasis in skeletal muscle, 328 hypertrophy, 145
146 immune cell activation, 128 immune cell infiltration, 126 indices, 6
10 inflammation, 125 injury, 125, 128
129 insulin resistance, 125
126 loss, 251
253 “fast and slow” protein concept, 252
253 mass, 53, 95, 267
268 microvascular network, 51, 54 protein, 189 anabolism, 74 metabolism and obesity, 109
110 synthesis, 35 published indices and cutoff points, 7t, 9t recovery, 128
129 reducing skeletal muscle mass and lifestyle, 17 combining physical activity and nutrition, 22
25 nutrition, 19
22 physical activity, 18
19 smoking, alcohol use and socioeconomic status, 25
26 regeneration process, 129
130 regulation of skeletal muscle metabolism dietary fat and health, 347
348 fatty acid composition and skeletal muscle function, 349 lifestyle interventions to alleviating SFA-mediated lipotoxicity, 354

INDEX

mechanisms of action of SFA and MUFA in, 349
353 repair, 130
132 requirement in sulfur amino acids and metabolically related compounds, 333
336 qualitative requirements and interorgan fluxes, 333
334 specific situations and potential supplementations, 334 skeletal muscle-specific knockout of raptor, 140
141 sulfur amino acids and related compounds in, 324
332 tissue perfusion, 35 Skeletal muscle mitochondrial, obesity, and high-fat feeding, 117 factors involved in mitochondria dysfunction, 120
121 mitochondrial content, 117
118 muscle oxidative capacities, 118
120 Skeletal myoblasts, 349 Skeletal myofibers, 48
49 SLC15A1 gene, 328 SLC38A2 gene, 327 SLC6A8 gene, 331 membrane transporter, 298 SLCO1B1, 504 Slow-twitch fiber, 111 SM. See Skeletal muscle (SM) Smac/DIABLO. See Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) Smoking, 25
26 SNAT2. See Sodium-coupled neutral amino acid transporter 2 (SNAT2) SO. See Sarcopenic obesity (SO) SOCS. See Suppressor of cytokine signaling (SOCS) Sodium-coupled neutral amino acid transporter 2 (SNAT2), 327 Sodium-dependent organic cation transporter-2 (OCTN2), 329
331 Soleus muscle strips, 352
353 Soy isoflavones, 410 Soy protein isolate (SPI), 475
476 Soy proteins, 475
476 Soybean (Glycin max L.), 475 SPI. See Soy protein isolate (SPI) Splanchnic tissues, 271
272 Sport nutrition, 152 ergogenic aids and risks, 173
174 health claims and applications to, 172
174 SPT-I. See Serine palmitoyl-CoA transferase-I (SPT-I)

551

SREBP1. See Sterol regulatory-element binding protein 1 (SREBP1) SS mitochondria. See Subsarcolemmal mitochondria (SS mitochondria) SSC. See Stretch-shortening cycle (SSC) Starvation, 68, 456 Statins autoimmune-mediated necrotizing myositis, 503 definition of muscle toxicity, 499
500 management, 505
506 mechanism of muscle toxicity, 504
505 medications, 505 myalgias, 500
501 myopathy, 502 myositis and myonecrosis, 502 rhabdomyositis, 502 risk factors for muscle toxicity, 504 scope of problem, 499 statin-induced muscle toxicity, 499
500, 500t Statins on Skeletal Muscle Performance trial (STOMP trial), 500, 502 Stearate acid. See Stearic acid Stearic acid, 348 Stearidonic acid (SDA), 359
360 Stearoyl-CoA desaturase 1 (SCD1), 348, 353 Sterol regulatory-element binding protein 1 (SREBP1), 476 Stilbenoids, 405
406 STOMP trial. See Statins on Skeletal Muscle Performance trial (STOMP trial) Stress and adaptation, 152
157 training, 152
155 training periodization, 155
157 Stressor, 153 Stretch-shortening cycle (SSC), 190
191 Stroke, 210 patients, 211 Subsarcolemmal mitochondria (SS mitochondria), 117
118 Succinyl-CoA, 162, 266
267 Sulfate (SO422), 322
324 Sulfation. See Sulfonation Sulfide quinone oxidoreductase, 322
323 Sulfite (SO322), 322 Sulfonation, 323
324 Sulfur amino acids. See also Amino acids (AAs) general biochemical aspects on, 315
324 compounds metabolically linked to cysteine, 318
324 intrinsic chemical properties of sulfur amino acids, 315
316 metabolism and specific roles of methionine, 317
318

552

INDEX

Sulfur amino acids (Continued) and related compounds in skeletal muscle free sulfur amino acids, 325
328 glutathione homeostasis in skeletal muscle, 328 skeletal muscle concentration and plasma supply of taurine, 329 skeletal muscle concentrations, plasma supply, and compounds, 329
332 skeletal muscle concentrations contained in proteins, 324
325 requirements and supplementations skeletal muscle requirement and metabolically related compounds, 333
336 usual sulfur amino acid intakes, 332
333 whole body requirement, 332 Sulfur dioxygenase, 322
323 Supercompensation, principle of, 155
156 Superoxide anion (O22), 283 Superoxide radical (O2•2), 284 Supplementation, 24 side and adverse effects, 168
172 acute adverse effects, 168
169 hindrance of adaptive effects as side effect, 169
172 Suppressing protein catabolism, 38 Suppressor of cytokine signaling (SOCS), 349
350 Symporters, 326
327 Symptomatic ischemic cardiovascular disease, 199 Synthesis rates, 35 Systolic blood pressure (SBP), 201

T T helper type 1 (Th1), 127 T lymphocytes, 126 T2D. See Type 2 diabetes (T2D) Tacrolimus, 504 TAF1B expression, 145 TAG. See Triacylglycerols (TAG) Tasmanian Older Adult Cohort study (TASOAC study), 18
20 Taurine, 323
324, 333
336 skeletal muscle concentration and plasma supply, 329 TBARS. See Thiobarbituric acid reactive substances (TBARS) TBC1, 141 TCA cycle, 281
283 TEI. See Total energy intakes (TEI) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), 448
451 50 Terminal olygopyrimidine (50 TOP), 36
37, 143
145 TFAM. See Transcription factor A, mitochondrial (TFAM)

Th1. See T helper type 1 (Th1) Thermoregulation, 154 Thiobarbituric acid reactive substances (TBARS), 405 Thiosulfate (S2SO322), 322
323 3A4 isoenzyme, 504 Tibialis anterior muscle mass, 379 Timed-up-and-go test (TUAG test), 392 Tissue healing, Arg and, 303
304, 303f TNF. See Tumor necrosis factor (TNF) TNF-like weak inducer of apoptosis-Fn14 (TWEAKFn14), 425
426 50 TOP. See 50 Terminal olygopyrimidine (50 TOP) Total and specific amino acids in promoting muscle anabolism in cancer, 71
75 in chronic disease, 76
77 Total energy intakes (TEI), 157
158 Total parenteral nutrition (TPN), 288 Total skeletal muscle (TSM), 3
4 Total skeletal muscle indices (TSMI), 3
4 TPN. See Total parenteral nutrition (TPN) “Train-low” approach, 160
165 Training, 152
155, 161 adaptation to environmental stress, 154
155 based on adaptive theory, 153 interference, 141 mithormesis theory and key role of ROS, 153
154 periodization, 155
157 overreaching syndrome and OTS, 156
157 specificity of training cycles, principle of supercompensation, 155
156 specificity of training cycles, 155
156 training-induced increase in muscle mass, 53
54 Transamination, 318 Transcobalamin, 311
312 Transcription factor A, mitochondrial (TFAM), 37, 49
50, 311, 494 Transcription factor(s), 52
53 Transcription factor A, 456 Translational capacity of myofibers, 55 Transmembrane transporters, 326
328 Transmethylation, 317
318 Transsulfuration, 318 of methionine, 335 Triacylglycerols (TAG), 348 Tricarboxylic acid cycle, 280, 311 Tricyclic antidepressants, 504 Triglyceride, 200 depots, 125
126 Triglyceride accumulation. See Ectopic fat deposition TSC. See Tuberous sclerosis complex (TSC) TSM. See Total skeletal muscle (TSM) TSMI. See Total skeletal muscle indices (TSMI)

INDEX

Tti1/Tel2 complex, 268
270 T-tubule cholesterol, reduction in, 505 TUAG test. See Timed-up-and-go test (TUAG test) Tuberous sclerosis complex (TSC), 300
301 heterotrimeric complex, 141
142 TSC1, 39
40, 141 TSC2, 39
40, 141 Tumor necrosis factor (TNF), 126
127, 218 TNF-α, 349
350, 363
364, 406, 448
451 production, 518 secretion, 519 signaling, 423 TNF-α/SOCS3 pathway, 349
350 Tumor response, 70 TUNEL. See Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) TWEAK-Fn14. See TNF-like weak inducer of apoptosisFn14 (TWEAK-Fn14) Type 1 diabetes, 208
210 evidence-based physical exercise, 208
209 possible mechanisms, 209 type of training, 209
210 Type 1 fibers, 111 Type 2 diabetes (T2D), 108, 202
208, 351
353, 402, 491 effects on T2D model animals, 405
412 evidence-based physical exercise, 206 model mice, 405
406 aspalathin, muscle cell glucose uptake and glucose metabolism, 406
409 nepodin, gingerol, muscle cell glucose uptake, and glucose metabolism, 409
410 phytochemicals, microbial metabolites, muscle cell glucose uptake, and glucose metabolism, 410
412 stilbenoids, muscle cell glucose uptake, and glucose metabolism, 405
406 possible mechanisms, 207 type of training, 207
208 Type 2 diabetes mellitus (T2DM). See Type 2 diabetes (T2D) Type 2b fibers, 111 Type II fiber removal, mitochondria role in, 430 Type II muscle fibers, 378
379

U Ubiquitin ligases, 440 Ubiquitin proteasome pathway, 139
140 Ubiquitin proteasome system (UPS), 38, 435 disruption increases dysfunctional mitochondria, 438 mitochondria, mitophagy, and mitophagy regulation, 437
438 muscle wasting, 435
436

553

and neural dysfunction in aging, 437 and sarcopenia, 436 ucp3. See Uncoupling protein 3 (ucp3) UDP-GalNAc. See Uridine diphosphate-Nacetylgalactosamine (UDP-GalNAc) UDP-GlcNAc. See Uridine diphosphate-Nacetylglucosamine (UDP-GlcNAc) UL. See Upper limit (UL) ULK1. See Unc-51-likekinase 1 (ULK1) ULs. See Upper intake levels (ULs) Ultraviolet radiation (UVB), 375 Unc-51-likekinase 1 (ULK1), 38 Uncoupling protein 3 (ucp3), 494 Uniporters, 326
327 Unloading
reloading model of hindlimb suspension, 145
146 Upper intake levels (ULs), 165
166 Upper limit (UL), 158 right limit, 166 UPS. See Ubiquitin proteasome system (UPS) Upstream mTOR signaling, 38
40 Uridine diphosphate-N-acetylgalactosamine (UDPGalNAc), 286 Uridine diphosphate-N-acetylglucosamine (UDPGlcNAc), 286 US Health ABC Study, 20 US Health and Retirement Study, 26 US National Health and Nutrition Examination Survey, 22
23 US Preventative Task Force, 394 UVB. See Ultraviolet radiation (UVB)

V

Vacuolar H1 ATPase (v-ATPase), 39, 301 Valine, 263
267 catabolism, 266
267 Vascular endothelial growth factor (VEGF), 52, 222
223, 521 Vastus lateralis, 351 VDAC1. See Voltage-dependent anion selective channel (VDAC1) VDR. See Vitamin D receptor (VDR) Vegetable fats, 348 Vegetable oils, 348 “Vegetables
fruits” dietary pattern, 20
21 VEGF. See Vascular endothelial growth factor (VEGF) VEmax. See Minute ventilation at maximal exercise (VEmax) Visceral fat tissue, 204 Visual impairment, 393 Vitamin D deficiency, 21, 376, 380, 389
390, 513 falls and, 393
394

554 Vitamin D deficiency (Continued) muscle pain and, 391
392 muscle weakness and, 390
391 and physical performance measures, 392
393 vitamin D deficiency-associated myopathy, 376 Vitamin D receptor (VDR), 21, 376, 381, 389
390 polymorphisms, 21
22 Vitamin D signaling and skeletal muscle cells complex action of vitamin D, 383
384 function, 376
378 genetic contribution to vitamin D effects, 383 morphology, 378
379 development and regeneration, 379
380 vitamin D effect on skeletal muscle cell signaling, 381
383 on muscle cell metabolism, 382
383 on muscle cell proliferation and differentiation, 381
382 Vitamin(s), 165, 389
390 vitamin C, 452 vitamin D, 21
22, 255
256 infusion, 380 supplementation, 377
379, 389
391 vitamin E, 452 VLDL, 476 Voltage-dependent anion selective channel (VDAC1), 37
38

INDEX

W WADA. See World Anti-Doping Agency (WADA) Warfarin, 504 Watermelon (Citrullus vulgaris), 309 Wernicke’s encephalopathy, 512 Western blotting analysis, 409 Whey protein, 271
272, 335 “interest of fast” proteins for muscle anabolic response, 253
256 skeletal muscle loss and protein nutrition, 251
253 Whole protein(s), 191 supplementation, 189 Whole-body lean mass, 364
365 Whole-body protein turnover, 74 Wmax (W), 224 World Anti-Doping Agency (WADA), 174 Wound healing, 303
304

X XJB-5
131 (XJB), 455 X-linked inhibitor of apoptosis protein (XIAP), 428
429

Y Ying-Yang 1 transcription factor (YY1 transcription factor), 37 Young adult populations, 3
4, 10