Cellular Physiology and Metabolism of Physical Exercise: Topical Clinical Issues [2 ed.] 3031271912, 9783031271915

Table of contents :
Contents
Human Evolution and Physical Exercise: The Concept of Being “Born to Run”
1 The Concept of Being Born to Run
2 From 5 Billion to 1 Million Years Ago
3 The Appearance of the Genus Homo
References
Cell Morphology and Function: The Specificities of Muscle Cells
1 Introduction
2 Striated Skeletal Muscles
3 Muscle Contraction
4 Muscle Fiber Classification
Further Reading
The Cell Membrane of the Contractile Unit
1 Cell Membranes
2 The Structure of the Cell Membrane
Lipids
Proteins
Carbohydrates
Membrane Asymmetry
3 Functions of the Cell Membrane
Transport
Diffusion
Facilitated Diffusion
Active Transport
Phagocytosis and Pinocytosis
Immune System
4 Membrane Receptors
5 The Sarcolemma
References
Gene Polymorphisms and Athletic Performance
1 Introduction
2 What Happens When the Balance in the Human Body Is Modified?
3 Human Performance Shows a Wide Variety of Responses
4 Can Genes Predict Athletic Performance?
5 Genetic Variability Between Individuals
6 Genetic Polymorphisms of the Enzymes Involved in DNA Methylation and Synthesis in Elite Athletes
Further Reading
Mitochondrial and Non-mitochondrial Studies of ATP Synthesis
1 Introduction
2 In Vivo Magnetic Resonance Spectroscopy
1H-MRS
13C-MRS
31P-MRS
3 Mitochondrial Function Assessed by 31P-MRS
Mitochondrial Function During Exercise as Assessed by 31P-MRS
4 Measurement of TCA Cycle Flux (VTCA)
5 Anaerobic Sources of ATP
Glycolytic Flux
PCr Breakdown
Glycogen
6 Integrative View
References
Excessive Nutrients and Regional Energy Metabolism
1 Introduction
2 Excessive Ectopic Fat Accumulation and Abnormal Regulation of Insulin-Dependent Metabolic Pathways
Skeletal Muscle
Heart
Liver
3 Excessive Ectopic Fat Accumulation as the Consequence of Increased Adipose-Derived FFA Flux
4 The Association of Excessive Ectopic Fat Accumulation and Abnormalities of Energy Metabolism
Skeletal Muscle
Heart
Liver
5 Conclusion
References
Muscle Biopsy to Investigate Mitochondrial Turnover
1 Skeletal Muscle Biopsy
2 Skeletal Muscle Function and Mitochondria
3 Mitochondrial Glucose and Fatty Acid Oxidation
4 Regulation of Mitochondrial Oxidative Metabolism
Energy Status
Exercise
Nutrition
Caloric Restriction
Fat and Glucose Substrates
Fatty Acids
Glucose
Substrate-Induced Metabolic Alterations with Mitochondrial Impact
Oxidative Stress
Inflammation
5 Mitochondrial Function and Turnover in Human Skeletal Muscle
Exercise
Obesity and Insulin Resistance
Mitochondrial Function in Obese and Insulin-Resistant Patients
Mitochondrial Effects of Diet and Exercise in Obese and Insulin-Resistant Patients
Substrate Availability: Acute
Dietary Treatment
Exercise
Mitochondrial Effects of Insulin
Mitochondria and Insulin Resistance: Cause or Effect?
Aging and Chronic Wasting Diseases
Exercise in Aging and Chronic Wasting Disease
6 Conclusions
References
Introduction to the Tracer-Based Study of Metabolism In Vivo
1 Introduction
2 Basic Concepts
3 Mass-Balance Principle
4 A Hydraulic Analogy
5 Steady State and Turnover
6 Clearance Rate
7 Measurement of Turnover: The Essential Role of Tracer Experiments
8 Characteristics and Properties of a Tracer
9 The Constant-Infusion Technique
10 The Single-Injection Technique
11 Concluding Remarks
Reference
Further Reading
Physical Activity and Inflammation
1 Inflammation Is an Important Feature of Metabolic Diseases and Diabetes
Peripheral and Adipose Inflammation
Islet Inflammation
2 Effect of Physical Activity on Inflammation
3 Molecular Effect of Physical Activity
4 Physical Activity and miRNA: A Unifying Hypothesis
5 Conclusion
References
The HPA Axis and the Regulation of Energy Balance
1 Introduction
2 Anatomy of the HPA Axis
The Hypothalamus
The Pituitary Gland
Hypothalamus–Pituitary Interaction
The Adrenal Cortex
3 Physiology of the HPA Axis
4 Molecular Mechanisms
5 HPA Axis and Energy Balance
Energy Intake
Glucocorticoids and Leptin
Glucocorticoids and Insulin
6 The HPA Axis and Non-homeostatic Energy Intake Regulation
7 The HPA Axis and Energy Expenditure
8 The Role of Glucocorticoids on Peripheral Organs
9 HPA Axis and Physical Activity
10 Glucocorticoids and Doping
References
Physical Exercise in Obesity and Anorexia Nervosa
1 Reduced Physical Activity in Industrialized Countries: A Potential Cause of the Obesity Pandemics?
2 Reduced Physical Activity: The Cause of Weight Gain in the Obese?
3 Can Humans Adapt Energy Expenditure to Energy Intake and Vice Versa?
4 Is Physical Activity a Meaningful Trait in Anorexia Nervosa?
5 Why Hyperactivity in Anorexia Nervosa?
6 Biological Basis of Activity-Based Anorexia
7 The Neuroendocrine Profile of AN Patients
8 Is Hyperactivity an Unfavorable Prognostic Behavior?
References
Physical Exercise and Transplantation
1 Introduction
2 Physical Work Capacity Before Transplantation
3 Physical Work Capacity After Transplantation
4 Exercise Therapy for Heart Transplant Recipients
5 Exercise Therapy for Lung-Transplant Recipients
6 Exercise Therapy for Kidney Transplant Recipients
7 Exercise Therapy for Liver Transplant Recipients
8 Exercise Therapy for Pancreas and Islet Transplant Recipients
Case Study: Exercise in an Islet-Transplanted Amateur Marathon Runner: Effects on Training, Autoimmunity, and Metabolic Profile
9 World Transplant Games
10 Conclusions
References
The Baboon as a Primate Model to Study the Physiology and Metabolic Effects of Exercise
1 Introduction: The Value of Nonhuman Primates in Biomedical Research
2 Nonhuman Primates in Biomedical Research
3 The Baboon as a New Model to Study Physical Activity and the Effects of Exercise
4 Summary
References
Specific Physical Exercises Adapt to Immune-Modulate the Nondiabetic and the Diabetic Individual and Reduce the Likelihood of Contagion by Respiratory Viruses-Like SARS-2 Coronavirus
1 Introduction
2 Effect of Exercise on the Immune System
3 Role of Physical Exercise-Induced Immunomodulation in Type 1 and 2 Diabetes
Diabetes and Oxidative Status
Diabetes and Inflammation
Impact of Exercise on Oxidative Stress in T2D
Impact of Exercise on Inflammation in T2D
Impact of Exercise on Cells of Immune System in Diabetes
4 SARS-CoV-2 Infection and Exercise-Induced Immunomodulation in Diabetes
Immune Function Impairments in Diabetes and Their Impact on COVID-19 Infection
The Role of Physical Activity in Diabetic Patients in Relation to COVID-19 Infection
References
Physical Exercise and Sexual Dysfunction
1 Physiology of Sexual Function in Man
2 Physiology of Sexual Function in Woman
3 Erectile Dysfunction
4 Physical Activity and Erectile Dysfunction
5 Mechanisms Linking Physical Activity and Male Sexual Function
6 Female Sexual Dysfunction
7 Physical Activity and Female Sexual Dysfunction
8 Mechanisms Linking Physical Activity and Female Sexual Function
References
Specific Physical Exercises Adapt to Patients with Obesity or with Diabetes Mellitus (Type 1 and Type 2)
1 Why We Should Keep Ourselves Physically Active
2 Exercise Can Positively Modulate Immunometabolism
Skeletal Muscle
Brain
Heart and Lungs
Gut
3 Exercise and Glucose Metabolism
Exercise in Type 2 Diabetes
Exercise in Type 1 Diabetes
4 Physical Activity in Obesity: What Can Really Be Done?
5 Concluding Remarks
References
Hypnosis and Sport
1 A Summary of Hypnosis’ History
2 Definition of Hypnosis
3 Hypnosis Can Help Athletes to Cope with Psychological Stress
4 Hypnosis to Reduce Stress and Mental Fatigue in Athletes
5 Conclusions
References
Physical Activity and Diabetic Retinopathy
1 Introduction
2 The Retinal Neurovascular Unit in DR
3 Multimodal Retinal Imaging for the Clinical Assessment of DR
4 Physical Activity and DR
5 From Clinical Aspects to Pathophysiology
Retinal Microvascular Function
Oxidative Stress
Neuronal Health
Inflammation
Other Possible Mechanisms
6 Conclusion
References

Citation preview

Cellular Physiology and Metabolism of Physical Exercise Topical Clinical Issues Livio Luzi Editor Second Edition

123

Cellular Physiology and Metabolism of Physical Exercise

Livio Luzi Editor

Cellular Physiology and Metabolism of Physical Exercise Topical Clinical Issues Second Edition

Editor Livio Luzi Department of Endocrinology Nutrition and Metabolic Diseases IRCCS MultiMedica Milan, Italy

ISBN 978-3-031-27191-5    ISBN 978-3-031-27192-2 (eBook) https://doi.org/10.1007/978-3-031-27192-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2012, 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Contents

Human Evolution and Physical Exercise: The Concept of Being “Born to Run” ��������������������������������������������������������������������������������������    1 Livio Luzi  Cell Morphology and Function: The Specificities of Muscle Cells��������������    9 Anna Maestroni  The Cell Membrane of the Contractile Unit��������������������������������������������������   17 Gianpaolo Zerbini  Gene Polymorphisms and Athletic Performance������������������������������������������   23 Ileana Terruzzi  Mitochondrial and Non-mitochondrial Studies of ATP Synthesis��������������   33 Roberto Codella  Excessive Nutrients and Regional Energy Metabolism��������������������������������   45 Gianluca Perseghin  Muscle Biopsy to Investigate Mitochondrial Turnover��������������������������������   57 Rocco Barazzoni  Introduction to the Tracer-Based Study of Metabolism In Vivo ����������������   75 Andrea Caumo and Livio Luzi Physical Activity and Inflammation ��������������������������������������������������������������   89 Cristian Loretelli, Francesca D’Addio, Moufida Ben Nasr, and Paolo Fiorina  The HPA Axis and the Regulation of Energy Balance����������������������������������  101 Francesca Frigerio  Physical Exercise in Obesity and Anorexia Nervosa������������������������������������  115 Alberto Battezzati and Simona Bertoli

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Physical Exercise and Transplantation����������������������������������������������������������  125 Valentina Delmonte, Vincenzo Lauriola, Rodolfo Alejandro, and Camillo Ricordi The Baboon as a Primate Model to Study the Physiology and Metabolic Effects of Exercise��������������������������������������������������������������������������  139 Francesca Casiraghi, Alberto Omar Chavez Velazquez, Nicolas Musi, and Franco Folli Specific Physical Exercises Adapt to Immune-Modulate the Nondiabetic and the Diabetic Individual and Reduce the Likelihood of Contagion by Respiratory Viruses-Like SARS-2 Coronavirus����������������������������������������������������������������������������������������  155 Anna Ferrulli, Daniele Cannavaro, and Livio Luzi  Physical Exercise and Sexual Dysfunction����������������������������������������������������  169 Carmine Gazzaruso and Adriana Coppola Specific Physical Exercises Adapt to Patients with Obesity or with Diabetes Mellitus (Type 1 and Type 2)��������������������������������������������������  181 Roberto Codella Hypnosis and Sport������������������������������������������������������������������������������������������  195 Livio Luzi and Luca Filipas  Physical Activity and Diabetic Retinopathy��������������������������������������������������  201 Stela Vujosevic

Human Evolution and Physical Exercise: The Concept of Being “Born to Run” Livio Luzi

1 The Concept of Being Born to Run Born to Run was the third album produced by the American singer-songwriter Bruce Springsteen. It was released by Columbia Records on August 25, 1975. The same title was used in the following decades for: at least one novel, an episode in the TV series Terminator, a book on a Mexican tribe of extreme runners, and it even appeared on the cover page of Nature, in November 2004. The common denominator of all the uses of Born to Run is the recognition of the need of humans to run in order to survive.

2 From 5 Billion to 1 Million Years Ago The present atmosphere of the Earth is composed of 21% oxygen. The remaining gases are nitrogen (78%), argon (0.9%), carbon dioxide, and other trace elements (0.012%). About 5 billion years ago, at the birth of our planet, the atmosphere contained virtually no oxygen. The advent of the first forms of life on Earth (prokaryotes, primordial unicellular bacteria) was crucial for the change in composition of the gas content of the atmosphere. Primordial bacteria were able to carry out photosynthesis, utilizing hydrogen, obtained from water, and CO2 to release oxygen. Therefore the development of life on Earth was determined by the appearance of organisms capable of surviving in the absence of oxygen, with their survival L. Luzi (*) Department of Biomedical Sciences for Health, University of Milan, Milan, Italy Department of Endocrinology, Nutrition and Metabolic Diseases, IRCCS MultiMedica, Milan, Italy e-mail: [email protected]; [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 L. Luzi (ed.), Cellular Physiology and Metabolism of Physical Exercise, https://doi.org/10.1007/978-3-031-27192-2_1

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exclusively founded on anaerobic metabolism. The increasing amount of oxygen released by prokaryotes into the primordial atmosphere favored the development of oxidative reactions to produce energy for life, a much more efficient method than anaerobic metabolism. Some 1500 million years ago, the first eukaryotes capable of producing energy with oxidative metabolism appeared on Earth. Millions of years were then necessary for the development of multicellular eukaryotes. It is relevant for evolution in general and for human evolution in particular that in parallel with the appearance of more complex multicellular organisms much of the Earth’s ecosystem was altered by dramatic geologic events [1, 2]. The volcanic eruptions, continent shifts, and meteoric collisions forced major evolutionary leaps, as only organisms capable of adapting to the new environment survived. One such adaptation is described by the endosymbiotic theory. Endosymbiosis means “cohabiting within” and in this case refers to the postulated collaboration/interaction between organisms with different metabolic capabilities and dimensions, both of which gain an evolutionary advantage by merging their living environments. As stated, not all organisms were able to tolerate an oxidant atmosphere (i.e., an atmosphere increasingly rich in oxygen produced by photosynthesis). According to endosymbiotic theory, primordial eukaryotes were able to survive due to their incorporation of prokaryotes bearing much-needed complementary skills. Peroxisomes and mitochondria are thought to be remnants of prokaryotes that eventually became eukaryotic organelles, conferring upon their hosts the cellular machinery needed for oxygen detoxification and energy production in aerobiosis [3].

3 The Appearance of the Genus Homo Roughly 1.5 million years ago, Homo erectus appeared on the Earth. Our present genes are similar to those of Homo erectus, Homo habilis, and the first Homo sapiens (200,000–100,000  years ago) [4]. Australopithecines were the ancestors of Homo erectus and their evolution was driven by an important change in the ecosystem: the replacement of woodlands by grasslands and savannas in Central Africa [5, 6]. The expansion of savannas caused a fundamental change in the way hominids foraged and, consequently, in the quality and caloric content of food as well as the amount of physical activity required to gather food. In fact, the disappearance of woodlands induced hominids to cover longer distances in savannas, prompting the natural selection of individuals with longer lower limbs, the ability to run, better thermoregulatory capacity, and with a higher resting and total daily energy expenditure. Evolutionarily, longer lower limbs and bipedalism facilitated foraging behavior in the new ecosystem, determining a strong association between changes in body size (and metabolism) and ranging/foraging patterns [7–9]. Therefore, the earliest representative of the human genus, considered to be the African Homo erectus, was indeed “born to run,” that is, to cope with an environment strikingly different from the woodlands where previous hominids had gathered food. Several musculoskeletal adaptations are representative of the genus Homo, including a large cranial vault,

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a prominent nose, a thin mandible, a chin, small teeth, a modified hip joint, and a light skeleton. These anatomic changes allowed our ancestors to walk and run for long distances and times, as their bodies were specialized for endurance and physical activity [10–14]. Indeed, humans are specifically adapted to engage in prolonged strenuous muscular activity, such as efficient long-distance bipedal running. This capacity evolved to allow the running down of game animals by persistent slow but constant chase over many hours. Central to the success of this strategy were at least four distinct factors [15, 16]: (1) energetics: the lower cost of running vs. walking (the other human gait) at speeds above ~2 m/s; (2) skeletal length: as long lower limbs gave Homo erectus greater speed in chasing and hunting; (3) the development of the central nervous system: with the differentiation of specific brain areas responsible for equilibrium, movement coordination, and postural stabilization; (4) thermoregulation: in which the human body, unlike that of animal prey, can effectively remove muscle heat waste. In most animals, a temporary increase in body temperature allows the storage of muscle heat waste. This enables them to escape from animal predators that quickly speed after them for a short duration (the method used by nearly all predators to catch their prey). Unlike other animals that hunt, humans remove body heat with a specialized thermoregulatory system based on sweat evaporation. One gram of sweat can remove 2598 J of heat energy. Another mechanism is increased skin blood flow during exercise, which allows for greater convective heat loss and is aided by humans’ upright posture. This skin-based cooling is a function of an increased number of sweat glands combined with a lack of body hair that would otherwise stop air circulation and efficient evaporation. Because humans can remove exercise-generated heat, they can avoid the heat exhaustion that affects animals chased in persistence hunting and so eventually catch their prey. The amount of food available was much greater in the savannas than in woodlands, mainly due to the higher caloric and protein content of the large herbivores hunted. This produced an increase in the body size of Homo erectus (∼65 kg males and 52 kg females) compared to previous hominids (e.g., Australopithecines, ∼44 kg males and 31  kg females). The increase in body weight, per se, determined a higher resting energy expenditure (REE: in Homo erectus, an average of 1565 kcal/day in males and 1361 kcal/day in females vs. 1130 and 902 kcal/day in males and females, respectively, of Australopithecus africanus). By adding the calories consumed by daily activities for Homo erectus to the REE, a total energy expenditure (TEE) of 3165 cal for males and 2141 cal for females can be estimated. These values are quite similar in each case to those of a 70 kg individual contemporary to us [15]. Did Homo habilis actually hunt quadrupeds, or did our earliest ancestors merely scavenge meat from lion and other predator kills? Many experts now believe that Homo habilis scavenged meat from nearby predator kills, chasing away lions with stones and loud calls. The hominids would then grab choice pieces of meat and retreat to a convenient place, far away from predators. There they would eat the fresh meat and break up the bones for their marrow. Once their hunger was satisfied, they would move off, leaving the crushed bones for other predators to scavenge. The hominids would return to the same place on several occasions. However, their visits were sufficiently infrequent so that carnivores did not hide in wait.

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Contemporary humans have a genetic background, body size, resting, and total energy expenditure comparable to Homo erectus. Nonetheless, the environment of Western countries in which many twenty-first century humans live has dramatically changed: (1) there is no longer a need to consume energy for food foraging and hunting; (2) many more calorie-rich and refined foods are available, in virtually unlimited supply; (3) food deprivation and starvation, except during religious fasts, are unknown (in contrast to the winters and other periods of food scarcity faced by Homo erectus). As a matter of fact, we are currently benefiting from a major ecosystem change that started 10,000  years ago, with the agricultural revolution (when populations of hunters/gatherers settled down and began to raise grains and conserve food for the winter) and reached its apex at the beginning of the twentieth century, with the industrial revolution and the introduction of machines to help humans perform labor-intensive and energy-demanding tasks. Therefore, due to the mismatch between our genetic background (what we are predisposed for) and our new environment (what we are actually doing), the incidence of diseases such as obesity, type 2 diabetes, metabolic syndrome, hypertension, cardiovascular events, and some forms of cancer has increased dramatically, especially in recent decades [17–20]. The metabolic mechanism utilized by our body to store rather than to burn calories is insulin resistance. Insulin sensitivity (the opposite of insulin resistance) is defined as the ability of insulin to metabolize a load of glucose (and other energy substrates such as free fatty acids). An impairment of the body’s capacity to metabolize a glucose load protects the individual from periods of food scarcity, starvation, or a deficit in carbohydrate or fat intake. Obviously, if evolution selected insulin-­ resistant humans based on their ability to survive periods of famine, the above-­ described changes in the twentieth century ecosystem have made modern humans susceptible to hyperglycemia, hyperlipidemia, and their pathological consequences, namely diabetes, obesity, and atherosclerotic disease. In principle, more insulin-­ sensitive individuals should be favored today, as they are able to dispose of regular, high-calorie loads in less time, whereas during life on the African savanna they would have been condemned to extinction [17]! The maintenance of normal glycemia is obtained by the balance between insulin secretion and insulin action, a relationship known as glucose tolerance. In normal individuals, there is a hyperbolic relationship between insulin secretion and insulin action (Fig. 1); accordingly, normal glucose tolerance can be obtained over a wide range of secretory capacity and insulin action. It is also well established that an imbalance between insulin secretion and insulin action causes hyperglycemia. The secretion of insulin must therefore be considered along with its action in order to determine “the metabolic wellness” of an individual. It is a common belief that today’s marathon runners are the closest modern humans come to Homo erectus in terms of lifestyle and metabolism. Marathon runners maintain a normal glucose tolerance by means of relatively efficient insulin action, tempered by relatively low levels of insulin secretion. In this scenario, hunters/gatherers should have benefited from a very high level of insulin action matched by a low secretory capacity of the hormone. There is an apparent discrepancy between the predisposition of our genes to store energy (the “thrifty

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Fig. 1  The relationship between insulin sensitivity and beta-cell secretion is well-described by a hyperbolic function, such that the product of insulin sensitivity times beta-cell secretion tends to remain constant. Physical exercise is known to enhance insulin sensitivity. Since less insulin is required to metabolize glucose, a concomitant reduction in beta-cell secretion takes place. The overall effect is as follows: a subject undertaking physical training slides along the hyperbola achieving a position characterized by elevated insulin sensitivity and low circulating insulin levels

genotype” hypothesis [17]) and the highly efficient insulin action of marathon runners (and, probably, of Homo erectus). Thus, an organism predisposed to saving and storing energy needs constant physical exercise to maintain normal insulin action and proper substrate utilization. Accordingly, a healthy lifestyle is defined by regular physical exercise along with appropriate dietary habits. In other words, the lack of a physical exercise program renders vain all dietary interventions (this is basically the clinical “on the field” experience of most physicians). It is worth noting that it is not only the total amount but also the pattern of insulin secretion that determines glucose disposal and the effective clearance a glucose load. First-phase insulin release has been shown to have a consistent role in inhibiting endogenous glucose production following a meal. Early stages of diabetes and obesity are characterized by a loss of first-phase insulin release and thus by postprandial hyperglycemia and a reduction of the thermogenic effect of food. The combination of the two defects leads to diabetes and obesity, respectively (or a combination thereof). Similar to insulin action, the “blindness” of the β-cell to glucose is overcome by amino acid administration via a high-protein diet, indicating that protein homeostasis is the metabolic domain best protected by evolution. In fact, on the one hand, in most conditions (with the notable exception of obesity) insulin’s action on protein metabolism is spared (despite a marked impairment of its action on carbohydrate and lipid metabolism). On the other hand, amino acids/high-­protein diets are able to restore a normal secretory pattern of insulin secretion, thus overcoming β-cell blindness to glucose during the early stages of type 2 diabetes mellitus. Based on these considerations there are two possibilities. One is that current evolutionary pressure will select one or a few protective genes/features of the

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sedentary Homo sapiens that will allow humans to evolve such that the insulin sensitivity of future generations is much higher than that conferred by our present genes. In other words, presumably, only individuals with a higher capacity to burn calories and dispose of nutrient loads (without needing to perform physical exercise) will be selected for survival by evolution. In this case, we have no choice but to passively wait for evolution to find a solution (as our ancestors did!). The other possibility is to change our behavior such that it mimics our ancestors’ way of life in terms of patterns of physical activity and the diet of hunters/gatherers. That lifestyle was characterized by three cornerstones. First, physical exercise was performed several hours a day, with different modalities and intensities. In Homo erectus, walking and running were frequent forms of physical exercise. The behavior of contemporary species of primates has been studied to deduce the physical exercise patterns and total daily energy expenditure of our ancestors. Although this kind of information is difficult to extrapolate, based on a total energy expenditure of 2500–3500 kcal/day, physical exercise, ranging from active to strenuous, was likely performed for between 1 and 4 h daily. Moreover, even during periods of daily rest and over the year, the average energy expenditure was higher than the present-day value, reflecting non-shivering thermogenesis secondary to cold-temperature exposure. Second, the diet of hunters/gatherers contained a much lower (up to 30% less) percentage of complex carbohydrates than is consumed today, a higher protein content (both vegetable and animal protein), and a total fat content similar to today’s level, with the notable prevalence of mono- and polyunsaturated fats over saturated fats. Third, of particular relevance was the modality of caloric intake of Homo erectus, characterized by periods of forced starvation (presumably ranging from 1 day to longer periods). Therefore, periodic fasting was a constant for hunters/gatherers, whereas, unless voluntarily performed, periods of food deprivation are for the most part completely unknown in modern Western societies. Interestingly, a metabolic model of fasting is provided by the initial stage of mental anorexia. Patients with this disease voluntarily reduce their caloric intake while engaging in physical exercise for several hours a day. Consequently, body weight, total daily energy expenditure, and blood concentrations of glucose, lipids, and amino acids (with respect to matched controls) are reduced, resulting in a clinical picture that is the mirror image of type 2 diabetes and metabolic syndrome. This clinical model suggests that our genes predispose us with the ability to well resist long periods of reduced caloric intake. If we succeed in changing our lifestyle accordingly, we will eradicate diabetes, obesity, hypertension, metabolic syndrome, cardiovascular disease, and even some forms of cancer.

References 1. Kasting JF, Siefert JL. Life and the evolution of Earth’s atmosphere. Science. 2002;10:1066–8. 2. Bosch TCG, Miller DJ. Major events in the evolution of planet Earth: some origin stories. In: The holobiont imperative. Vienna: Springer; 2016. https://doi.org/10.1007/978-­3-­7091-­1896-­2_2.

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3. Alberts B, Heald R, Johnson A, et al. Molecular biology of the cell. 7th ed. W. W. Norton & Company; 2022. 4. Wood B, Collar M. The human genus. Science. 1999;284:65–71. 5. Cerling TE.  Development of grasslands and savannas in East Africa during the neogene. Paleogeog Paleoclimatol Paleoecol. 1992;97:241–7. 6. Uno KT, Polissar PJ, Jackson KE, deMenocal PB. Neogene biomarker record of vegetation change in eastern Africa. Proc Natl Acad Sci U S A. 2016;113:6355–63. 7. Leonard WR, Robertson ML. Comparative primate energetics and hominid evolution. Am J Phys Anthropol. 1997;102:265–81. 8. Ulijaszek SJ. Human eating behaviour in an evolutionary ecological context. Proc Nutr Soc. 2002;61:517–26. 9. Luca F, Perry GH, Di Rienzo A. Evolutionary adaptations to dietary changes. Annu Rev Nutr. 2010;30:291–314. 10. Isbell LA, Pruetz JD, Lewis M, Young TP. Locomotor activity differences between sympatric patas monkeys (Erythrocebus patas) and vervet monkeys (Cercopithecus aethiops): implications for the evolution of long hindlimb length in Homo. Am J Phys Antropol. 1998;105:199–207. 11. Zihlman AL, Underwood CE.  Locomotor anatomy and behavior of patas monkeys (Erythrocebus patas) with comparison to vervet monkeys (Cercopithecus aethiops). Anat Res Int. 2013;2013(2013):409534. 12. Steudel-Numbers KL, Weaver TD, Wall-Scheffler CM.  The evolution of human running: effects of changes in lower-limb length on locomotor economy. J Hum Evol. 2007;53:191–6. 13. Polk JD. Influences of limb proportions and body size on locomotor kinematics in terrestrial primates and fossil hominins. J Hum Evol. 2004;47:237–52. 14. Steudel-Numbers KL. Energetics in Homo erectus and other early hominins: the consequences of increased lower-limb length. J Hum Evol. 2006;51:445–53. 15. Bramble DL, Lieberman DE.  Endurance running and the evolution of Homo. Nature. 2004;433:345–53. 16. Viranta-Kovanen S.  Ihmisen liikuntaelimistön evoluutiohistoria—kävelystä kestävyysjuoksuun [Evolutionary history of human locomotor system—from walking to long-distance running]. Duodecim. 2015;131:1995–2001. Finnish. 17. Luzi L, Pizzini G. Born to run: training our genes to cope with ecosystem changes in the twentieth century. Sport Sci Health. 2004;1:1–4. 18. Neel JV.  Diabetes mellitus: a “thrifty” genotype rendered detrimental by “progress”. Am J Hum Genet. 1962;14:353–62. 19. Prentice AM, Rayco-Solon P, Moore SE. Insights from the developing world: thrifty genotypes and thrifty phenotypes. Proc Nutr Soc. 2005;64:153–61. 20. Stöger R. The thrifty epigenotype: an acquired and heritable predisposition for obesity and diabetes? BioEssays. 2008;30:156–66.

Cell Morphology and Function: The Specificities of Muscle Cells Anna Maestroni

1 Introduction Muscles can be of different types. Based on morphology, we can distinguish several types: • Striated skeletal muscles have characteristic cross-striations which are due to the regular arrangement of contractile elements, the sarcomeres. Striated skeletal muscles contract in response to nerve impulses from the motor neurons of the central nervous system (CNS) or at the conscious level. They are related to skeletal segments. • The striated cardiac muscle of the heart is called the myocardium. Microscopically, cardiac muscle fibers are marked by transverse striae, which are also present on skeletal muscle fibers, as well as other transverse striations that make up the joint areas of the fibers. Cardiac muscle contracts independently of the will. • Smooth muscles, as their name implies, do not possess cross-striations. They are generally lighter in color than striated muscles and form the muscular component of the viscera. The walls of organs and structures such as the esophagus, stomach, intestines, bronchi, uterus, urethra, bladder, blood vessels, and the erector pili in the skin (which control the erection of body hair) all contain smooth muscle. The contractions of smooth muscles (with very few exceptions) are involuntary and occur under the control of hormones or external stimuli and in response to impulses from the autonomic nervous system.

A. Maestroni (*) International Center for T1D, Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Science, University of Milan, Milan, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 L. Luzi (ed.), Cellular Physiology and Metabolism of Physical Exercise, https://doi.org/10.1007/978-3-031-27192-2_2

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2 Striated Skeletal Muscles The muscles are covered externally by connective tissue referred to as the epimysium, which surrounds the entire muscle, holding it together. Inside the epimysium are fiber bundles, the fasciculi, wrapped in a sheath of connective tissue. The connective tissue sheath surrounding each fasciculus is called the perimysium. Finally, within the perimysium are the muscle fibers, which are the individual muscle cells. The endomysium, another sheath of connective tissue, surrounds each muscle fiber. The epimysium, perimysium, and endomysium are connective structures that together form the tendon (Fig. 1). Muscle fibers range in length from 1 mm to a maximum of 12 cm in the sartorius muscle. Their diameter ranges from a minimum of 10  μm to a maximum of 100–105  μm (average: 10–50  μm). These cellular elements are derived from the fusion of progenitor cells called myoblasts and thus form syncytia. Skeletal muscle fibers are cylindrical in shape and contain many nuclei (even hundreds) located near the sarcolemma (the cell membrane of muscle cells). However, the defining characteristic of muscle fibers is the cross-striations seen on light microscopy. A gelatin-like substance fills the spaces between the myofibrils. This is the sarcoplasm and it comprises the cytoplasm of muscle fiber. The sarcoplasm differs from true cytoplasm in that it contains a large quantity of stored glycogen as well as the oxygen-binding compound myoglobin, which is quite similar to hemoglobin (Fig. 2). Another special structure of the muscle fiber is the sarcoplasmic reticulum, which is the smooth endoplasmic reticulum. Its distinct shape can be recognized in every sarcomere. The sarcoplasmic reticulum is structured as follows: at the junction between the A and I bands are the terminal cisternae, along which branching tubules are arranged longitudinally, resulting in fenestrated central cisternae. At the

Fig. 1  Schematic structure of skeletal muscles

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Fig. 2  Representation of skeletal muscle fibers

confluence of two terminal cisternae is a tubular formation, the transverse tubules (T-tubules). This sarcolemmal invagination communicates with the extracellular environment but not with the lumen of the sarcoplasmic reticulum. The membranes of the two systems are coupled but they are separated by a gap. Together, these structures are referred to as the triad of the reticulum and they are involved in modulating the release of calcium ions, which are essential for muscle contraction. Each muscle fiber also contains several hundred to several thousand myofibrils: these are the contractile elements of skeletal muscle. Sarcomeres are the building blocks of myofibrils (Fig. 3). They are composed of thin actin filaments and thick myosin filaments. A sarcomere is defined as the segment between two neighboring Z-lines (or Z-discs, or Z bodies). In electron micrographs of cross-striated muscle, the Z-line (from the German Zwischenscheibe, the band in between the I-bands) appears as a series of dark lines. Surrounding the Z-line is the region of the I-band (I for isotropic). Following the I-band is the A-band (A for anisotropic). Within the A-band is a paler region called the H-band (from the German heller, bright). The names of these bands derive from their properties as seen on polarization microscopy. Finally, inside the H-zone is a thin M-line (from the German mittel, middle of the sarcomere) (Fig. 4).

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Fig. 3  Schematic structure of skeletal muscles Fig. 4 Schematic representation of contracted and relaxed sarcomeres

Each myosin filament typically comprises about 200 myosin molecules, lined up end to end and side by side. The myosin molecule is composed of two identical heavy (larger) chains and two pairs of light (smaller) chains. The heavy protein chains intertwine to form a tail end, a rigid spiral, and two globular heads. One of the two light protein chains is associated with one of the heavy-chain heads. The globular heads of the myosin cross-bridges mediate the interaction with thin actin filaments during muscle contraction. The myosin filaments are connected from the M-line to the Z-disc by tinin. Actin is a globular protein (G-actin) that combines to form long, thin chains (F-actin). Two F-actin strands form a helical twist, much like two strands of pearls twisted together. Each actin molecule has an active binding site that serves as the point of contact with the myosin filament.

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Fig. 5 Schematic representation of thick and thin filaments

In addition to actin, the thin filaments of the sarcomere are composed of tropomyosin and troponin. Tropomyosin is a tubular protein that twists the actin strands, while troponin is a more complex protein made up of three subunits (TnC, TnI, and TnT) and attached at regular intervals to both the actin strands and to tropomyosin. When calcium is bound to specific sites on TnC, tropomyosin rolls out of the way of the actin filament’s active sites, thus allowing myosin to attach to the thin filament and to subsequently produce force and/or movement. In the absence of calcium, tropomyosin interferes with the action of myosin such that the muscles remain relaxed. The individual subunits of troponin serve different functions in muscle contraction: troponin C binds to calcium ions to produce a conformational change in TnI; troponin T binds to tropomyosin to form a troponin–tropomyosin complex; and troponin I binds to actin in thin myofilaments to hold the troponin–tropomyosin complex in place. Tropomyosin and troponin require the presence of calcium ions to maintain relaxation or to initiate contraction of the myofibril, which we examine later in this chapter. In addition, actin and myosin interactions are regulated by another protein, nebulin, which serves as an anchoring protein for actin (Fig. 5).

3 Muscle Contraction The events that trigger a muscle fiber are complex. The process is initiated by a motor nerve impulse from the brain or spinal cord. An action potential originating in the CNS reaches an alpha motor neuron, which then transmits the action potential down its own axon. The action potential is propagated by the activation of sodium-­ dependent channels along the axon toward the synaptic cleft. An influx of Ca2+ causes vesicles containing the neurotransmitter acetylcholine to fuse with the plasma membrane, releasing acetylcholine into the extracellular space between the motor neuron terminal and the motor end plate of the skeletal muscle fiber. Acetylcholine diffuses across the synapse and binds to and activates acetylcholine receptors on the motor end plate of the muscle cell. Activation of the acetylcholine receptor opens its intrinsic sodium/potassium channel, causing sodium to rush in and potassium to trickle out. Since the channel is more permeable to sodium, the

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membranes of the muscle fibers become more positively charged, triggering an action potential. The action potential spreads through the muscle fiber’s network of T-tubules, depolarizing the inner portion of the muscle fiber. Depolarization activates voltage-dependent calcium channels in the T-tubule membrane, which are in close proximity to calcium-release channels in the adjacent sarcoplasmic reticulum. Activated voltage-gated calcium channels physically interact with and thereby activate calcium-release channels, causing the sarcoplasmic reticulum to release calcium. The released calcium binds to the troponin C present on the actin thin filaments of the myofibrils. Troponin then allosterically modulates tropomyosin. Normally, tropomyosin sterically obstructs myosin-binding sites on the thin filament; however, once calcium binds to troponin C and causes an allosteric change in the protein, troponin T allows tropomyosin to move, unblocking the binding sites. Myosin has ADP and inorganic phosphate bound to its nucleotide-binding pocket and is in an active state. In this form, it binds to the newly uncovered binding sites on the thin filament in a process very tightly coupled to inorganic phosphate release, in which actin serves as a cofactor. Myosin is now strongly bound to actin, with the release of ADP and inorganic phosphate tightly coupled to the power stroke. During the latter, the Z-bands are pulled toward each other, thus shortening the sarcomere and the I-band. Conversely, ATP binding to myosin allows it to release actin and to remain in a weak binding state (a lack of ATP makes this step impossible, resulting in the rigor state characteristic of rigor mortis). Myosin then hydrolyzes the ATP and uses the energy to move into the “cocked back” conformation. In vivo studies have confirmed model-based predictions regarding movement of the myosin head of skeletal muscle: during each power stroke the myosin head moves 10–12 nm; however, there is also in vitro evidence of variations (smaller and larger) in this range of movement that are specific to the myosin isoform (Fig. 6). Fig. 6  The mechanism of muscle contraction

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Sliding of the filaments occurs as long as ATP is available and calcium is present. During the above-described steps, calcium is actively pumped back into the sarcoplasmic reticulum, which creates a deficiency in the environment around the myofibrils. As a result, calcium ions are removed from troponin such that the tropomyosin reverts to its previous state, forming a complex with troponin and again blocking myosin-binding sites. Myosin is thus unable to bind to the thin filaments, and contraction ceases.

4 Muscle Fiber Classification The classification of muscle fibers is complex. Broadly we can distinguish three types of muscle fibers: type I or slow-twitch and type II or fast-twitch that can be further divided into type IIa (oxidative) and type IIx (glycolytic). The metabolic, contractile, and motor characteristics of the three types of fibers are summarized in the following table:

Type of motor unit

Type I fibers Slow oxidative

Motor neuron size Nerve conduction speed Contraction speed Relaxation speed Contraction force Fatigue resistance Glycogen level Capillary spraying Capillary density Myoglobin Red color Mitochondrial density Enzymatic oxidative capacity Width of the Z line Fiber diameter

Little Slow Slow Slow Low High Rare Rich High High Intense High High Medium Little

Type IIa fibers Fast oxidative/ glycolytic Big Rapid Rapid Rapid Medium Medium High Rich Medium High Intense High Medium-high Wide Medium

Type IIx fibers Fast glycolytic Big Rapid Rapid Rapid High Little High Poor Low Low Pale Low Low Tight Big

Traditionally fibers were classified according to their color, which depends on the myoglobin content. Type I fibers appear red due to high content of myoglobin, have more mitochondria and greater capillary density. Type II fibers are white, or in any case clear, due to the scarcity of myoglobin and the greater presence of glycolytic enzymes. Fibers can be classified according to their contractile speeds into fast and slow. These traits largely, but not completely, overlap with color-based classifications.

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Type I fibers (slow-twitch) are slower to shrink, but more suited to endurance because they use oxidative metabolism to generate ATP (adenosine triphosphate) from glucose and fatty acids over time. They tend to have low levels of ATPase activity, slower contraction rate with a less developed glycolytic capacity. Slow-twitch fibers have more mitochondria and capillaries, which makes them better for endurance work. Type II fibers (fast-twitch) are those where myosin splits ATP very quickly. These fibers also have greater electrochemical transmission capacity of action potentials and a rapid calcium release and absorption by the sarcoplasmic reticulum. They are based on a well-developed, anaerobic, and fast energy transfer glycolytic system and can contract 2–3 times faster than slow-twitch fibers. Fast-twitch muscles are better suited for generating short bursts of strength or speed than slow muscles and therefore fatigue more quickly.

Further Reading Biga LM, et al. Anatomy & physiology (chapter 10.2 Skeletal muscle). OpenStax & Oregon State University; 2020. Lieber RL. Skeletal muscle structure, function, and plasticity. Baltimore: Lippincott Williams & Wilkins; 2002. Macintosh BR, Gardiner PF, McComas AJ. Skeletal muscle: form and function. Leeds: Human Kinetics; 2006. Sakuma K. In: Sakuma K, editor. Muscle cell and tissue-current status of research field. London: IntechOpen; 2018. Schmalbruch H. Skeletal muscle. Berlin: Springer; 2012. Scott W, Stevens J, Binder-Macleod SA. Human skeletal muscle fiber type classifications. Phys Ther. 2001;81(11):1810–6.

The Cell Membrane of the Contractile Unit Gianpaolo Zerbini

1 Cell Membranes The cell membrane was initially considered only as a barrier delimiting the cytoplasm from the extracellular environment but further research revealed that the cell membrane has a number of functions that are essential to the cell [1]. Structurally, the cell membrane is formed by a lipid bilayer (Fig. 1), with each of the two layers composed of molecules called phospholipids. The lipid component is, by definition, water-repellent, while the phosphate component is hydrophilic. The membrane is formed as the phosphate moves toward the outer surface of the cell, attracted by the aqueous environment, which the inward-oriented lipids seek to escape. An additional and very important component of this double lipid structure consists of the membrane proteins. The organization of the cell membrane is therefore referred to as a “fluid mosaic,” in which the hydrophobic and hydrophilic components interact with each other in such a way that membrane fragments are able to detach from the main structure without creating permanent holes. The membrane surrounding internal organelles, such as the endoplasmic reticulum, the Golgi apparatus, lysosomes, and vacuoles, interacts with these structures and is crucial to their function [2, 3].

G. Zerbini (*) Complications of Diabetes Unit, Diabetes Research Institute (DRI), Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 L. Luzi (ed.), Cellular Physiology and Metabolism of Physical Exercise, https://doi.org/10.1007/978-3-031-27192-2_3

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Fig. 1  Graphical representation of the cell membrane. The proteins are inserted inside the membrane. The fat content allows the membrane to repair itself in case of external injury

2 The Structure of the Cell Membrane Lipids Lipids are retained on the internal aspect of the cell membrane because of their water repellency. Although they may also bond with oxygen molecules, lipids mainly consist of hydrocarbons. The three main classes of lipids that make up the cell membrane are fats, phospholipids, and steroids. Fats (triacylglycerols) are not true polymers but are nonetheless composed of large molecules formed from a number of smaller molecules; these are held together due to their water-repellent properties. Fat consists essentially of two molecules: glycerol and fatty acids. Glycerol belongs to the class of alcohols, while fatty acids are composed of 16–18 carbon atoms. One end of the fatty acid is the carboxylic group, which is joined to a long hydrocarbon tail. The C-H bonds of the fatty-acid tail account for the hydrophobicity of fat. Fat is formed by the binding of three fatty acids to a glycerol molecule, giving rise to a bond between the hydroxyl group and the carboxylic group. The fat molecule thus generated is called triacylglycerol or triglyceride. The fatty acids comprising the fat molecule can be identical or different. The length, number, and location of the double bonds present in a fatty acid define its physical and chemical characteristics. Fat may be saturated or unsaturated depending on the structure of the fatty acids that make up the hydrocarbon tail. The fluidity of the membrane tends to change according to the prevalence of saturated or unsaturated fats within the cell membrane [4, 5]. Phospholipids are the major component of the cell membrane. They are structurally similar to fat but contain only two fatty acids instead of three. The third hydroxyl group of glycerol is in this case attached to a negatively charged phosphate group, which is usually linked to small hydrophilic molecules. Different types of phospholipids can be generated based on the nature of the molecule bound to the phosphate. Phospholipids contain both a hydrophobic and a hydrophilic region and are thus defined as amphipathic molecules. The hydrophobic component is the

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hydrocarbon tail, while the hydrophilic head is formed by the phosphate group and its attachments. The morphology of phospholipids is such that once they are in contact with water they organize themselves in clusters in which the hydrophilic side is exposed toward the aqueous extracellular milieu, while the hydrophobic part is aligned inward. This structure is called a micelle and it is the main structural component of the phospholipid bilayer, comprising the semi-permeable structure characteristic of any cell membrane. Steroids consist of cholesterol and include several hormones. The carbon skeleton of steroids is arranged in four concentric rings. Cholesterol in particular is a key element of animal cell membranes and is essential to their stability. All steroids are formed from a cholesterol precursor. In the cell membrane, cholesterol molecules are incorporated into the phospholipid bilayer [6].

Proteins Proteins alone account for >50% of a single cell’s dry weight. Although the cell membrane contains tens of thousands of proteins, each protein can be considered as a polymer organized from the different sequential arrangements of 20 amino acids. Membrane proteins may be integral or peripheral. Integral proteins are generally transmembrane proteins in which the hydrophobic part traverses the cell membrane between its extracellular and intracellular aspects, while the hydrophilic ends of the protein emerge on either side. Within the membrane, integral proteins are larger than lipids; some of them diffuse very slowly in this environment, while others are anchored to the cytoskeleton. Peripheral proteins, as their name implies, are not located inside the cell membrane but are instead weakly anchored to its outer surface, often in contact with the external portions of integral membrane proteins.

Carbohydrates Membrane carbohydrates are usually branched oligosaccharides. Those covalently bonded to lipids form glycolipids, while those covalently bonded to proteins form glycoproteins. The oligosaccharides on the cell surface differ between individuals but also from cell to cell; in the latter case, they can therefore be used as markers to distinguish one cell from the other.

Membrane Asymmetry Membranes are exposed to the extracellular milieu and to the cytoplasm; accordingly, they have different internal and external surfaces. Since the two lipid layers differ in their composition, membrane proteins also assume different spatial arrangements. However, carbohydrates are found only on the outer surface of the membrane.

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3 Functions of the Cell Membrane Transport The cell membrane allows the internal and external passage of material. Transport of the various molecules may be energy-independent or coupled to an energy-­ dependent reaction or process.

Diffusion Many small molecules are able to cross the cell membrane simply by moving across a gradient from an area of higher to one of lower concentration. Only molecules small enough to pass through the small pores within the membrane are transported by diffusion. Since no energy is involved to move these molecules, diffusion tends to be a slow process. The transition of the molecules through the membrane is also influenced by whether they are lipid-soluble or water-soluble.

Facilitated Diffusion Some membrane proteins can form channels that allow water-soluble molecules to pass through the hydrophobic lipid layer inside the membrane. This is the mechanism by which important molecules such as glucose, which supplies energy to the cell, pass through the membrane. The protein channels allow these molecules to pass from areas of higher to those of lower concentration.

Active Transport For some molecules, a higher concentration must be maintained on one side of the membrane than on the other. To maintain this concentration gradient requires energy. Perhaps the best studied model of active transport is the sodium-potassium pump, but minerals are also moved by this mechanism. Nerve cells use pumps to transport ions in order to ultimately transmit their chemical messages.

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Phagocytosis and Pinocytosis Sometimes the cell must allow the entry of molecules that are too large to pass through the normal channels of the plasma membrane. In this case, the membrane surrounds the molecule of interest, forming a vesicle that can be easily transported inside the cell. Phagocytosis and pinocytosis refer to the vesicle-mediated transport of solid and liquid molecules, respectively.

Immune System The proteins that make up the cell membrane are obviously very important for the immune system. Some of them form channels or transporters, but others are needed for the identification and characterization of the cell. The recognition of self relies on the presence of proteins and glycoproteins on the cell surface. An organ that is transplanted from one individual to another will be recognized as foreign if the membrane proteins differ from those of the recipient organism; in such cases, unless the so-called immunosuppressive drugs are administered to the host, the transplanted organ will be rejected. The same mechanism underlies autoimmune diseases such as rheumatoid arthritis and diseases of the thyroid; in both cases, membrane proteins of the human body are mistakenly recognized as foreign and then rejected.

4 Membrane Receptors Some transmembrane proteins form membrane receptors, in which case a portion of the protein is located on the outer surface of the membrane and, based on its highly specific structure, is recognized by its ligand. The ligand may be a specific substance, such as a hormone, or a protein present on the membrane of another cell, as occurs when a killer lymphocyte recognizes a foreign cell. The binding of a hormone to its specific membrane receptor results in aggregation of the receptor–ligand complex followed by either on-site degradation of the complex itself or its internalization with further activity inside the cell.

5 The Sarcolemma The sarcolemma is the cell membrane of a muscle cell (skeletal, cardiac, and smooth muscle). It consists of the typical plasma membrane but also an outer coat made up of a thin layer of polysaccharide material containing thin collagen fibrils. At each

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end of the muscle fiber, the surface layer of the sarcolemma combines with a tendon fiber. The tendon fibers finally collect into bundles to form the muscle tendon, which inserts into bones. The sarcolemma is specialized to receive and conduct stimuli. Dysfunctions in the stability of the sarcolemma membrane and its repair system underlie diseases such as muscular dystrophy [2, 3].

References 1. Hollán S. Membrane fluidity of blood cells. Haematologica. 1996;27:109–27. 2. Jacobson K, Sheets ED, Simson R. Revisiting the fluid mosaic model of membranes. Science. 1995;268:1441–2. 3. Engelman DM. Membranes are more mosaic than fluid. Nature. 2005;438:578–80. 4. Singer SJ. Some early history of membrane molecular biology. Annu Rev Physiol. 2004;66:1–27. 5. Kwan TOC, Axford D, Moraes I.  Membrane protein crystallography in the era of modern structural biology. Biochem Soc Trans. 2020;48:2505–24. 6. Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science. 1972;175:720–31.

Gene Polymorphisms and Athletic Performance Ileana Terruzzi

1 Introduction Researchers have long worked to identify and describe the morphologic, anthropometric, physiologic, and functional characteristics of athletes who have reached high levels in various sports. But year after year, athletic records are broken and the limits of human performance are continuously redefined. Despite these increasingly high performance levels, all living organisms are in a state of homeostasis, in which the body is maintained in a state of biochemical balance even when subjected to strong environmental stimuli. This adaptive ability is a primary defense mechanism that the body exploits to protect itself from changes in the external environment and/or from systematic repetition of stressful physical changes. Since training and exercise in general are stress factors with demands on the metabolism of protein and energy, the supply of oxygen in the blood, and all other homeostatic control systems, the body has evolved a state of readiness allowing it to react even to the demands of extreme physical performance. Physical effort, if sufficiently intense, causes a fatigue process that after an adequate and necessary recovery phase prevents the return of energy reserves, protein synthesis, and numerous regulatory mechanisms to their initial, pre-loading state but instead brings about a level that is significantly higher, resulting in greater performance capabilities (Fig.  1). In fact, during the mandatory recovery phase, not only is the energy consumed offset but reserves above the initial level are built up according to a mechanism called “super-compensation.” The ability to adapt to different situations and to different environmental circumstances related to physical I. Terruzzi (*) Department of Endocrinology, Nutrition and Metabolic Diseases, IRCCS MultiMedica, Milan, Italy Department of Biomedical Sciences for Health, University of Milan, Milan, Italy e-mail: [email protected]; [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 L. Luzi (ed.), Cellular Physiology and Metabolism of Physical Exercise, https://doi.org/10.1007/978-3-031-27192-2_4

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Fig. 1 Schematic representation of the fluctuation in athletic efficiency due to fatigue, compensation, and super-compensation

activity is an amazing feature typical of living beings. If the human body were not able to respond positively to all the demands it encounters, such as cold, heat, oxygen deprivation, manual work, inactivity, or disease—in other words, all those sometimes difficult circumstances that life entails—it would face certain death.

2 What Happens When the Balance in the Human Body Is Modified? The human body is a marvelous machine that will improve or worsen its performance depending on the type, amount, and frequency of the stimuli with which it is confronted and it will adapt its skills to cope with substantial workloads. Muscular work involves a coordinated series of intracellular changes that lead to movements of muscle fibers and, consequently, of the muscles themselves. The human body’s ability to adapt to muscular work means that its muscles can be trained to carry out this work and thus to reach a degree of contraction different from the resting state, resulting in improved neuromuscular response and increased resistance. In fact, muscle development is the natural adaptation of the body to increasing physical activity, with a very complex set of changes. Consequently, the body is equipped to deal with a stressful event of greater magnitude, as the duly stimulated muscles which periodically undergo effort become stronger each time. Therefore, systematic training induces the body to successfully confront increasingly higher levels of fatigue through the development of morphologic and functional changes that are stable over time and depend on the type, intensity, and duration of the exercise but also on the physiologic characteristics of the individual. Progress in the body’s performance occurs in response to a training stimulus that produces an improvement in the starting conditions. Moreover, improvement requires that the training stimulus consists of a steady and gradual increase based on a person’s individual organic capability and without interruptions, to avoid losing the adaptations thus far achieved. Accordingly, a new state of homeostasis is achieved. In contrast, low-intensity and inconsistent training do not alter either the quality or the metabolic performance of an athlete (Fig. 2).

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Fig. 2  Human body’s adaptations to different physical activities

In recent years, much attention has been paid to the type and amplitude of the changes that develop with physical exercise, at the cellular and molecular levels, in order to assess whether there is a correlation between them and the body’s adaptability and ability to perform. A series of tests can be used to investigate the physiologic factors that determine an athlete’s physical and sports performance. For example, the measurement of blood lactate is an indicator of lactic acid metabolism under stress, allowing training loads and recovery to then be modulated accordingly. The determination of maximal and submaximal O2 consumption is a good indicator of performance, while the evaluation of slow muscle fiber composition reflects the amount of muscle strength. All of these tests are very effective for periodic monitoring, which is extremely important for an athlete in order to assess the results of his or her training program. A thorough analysis of the results allows performance to be related to training strategies, thus creating a successful training program that provides optimal results. However, the chosen indicator serves only to measure that particular parameter, such that a related improvement or deterioration in performance can only be indirectly inferred. Instead, measurements of the complex processes of exercise-induced stress adaptation are necessary to make the appropriate choice of exercise and to decide upon the duration and characteristics of its execution, in order to provide the athlete with the right support and guarantee improved performance. But can these parameters, which show a significant correlation with performance and allow estimations of adaptability and performance capabilities, be used to identify an athlete a priori? These types of tests are able to measure retrospectively how an athlete responds to the training stimulus and to determine the effect of that training, but not to predict

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an individual’s response to the stimulus. Will the tested athlete have the talent to be among the elite? Will he have the skills to better respond to the kind of training in question? Will she merely be one of the many competitors or will she be a winner?

3 Human Performance Shows a Wide Variety of Responses Sports performance and motor ability have always shown a large degree of variation even between individuals who use the same training protocols. This variability can be seen in Fig. 3, which shows the running times of the athletes who participated in the Vancouver marathon in 1999 (Fig. 3, left panel). The distribution of the arrival times can be explained by a variety of factors—extrinsic and intrinsic—that affect the performance of each runner. Age (Fig. 3, center panel) and sex (Fig. 3 right panel) are certainly among the factors able to determine the different performance responses of each individual athlete. Figure 3 shows that, on average, women are slower than men, although it is not clear whether this reflects anatomic differences between the sexes or social and cultural influences. The environment is certainly one of the most relevant extrinsic factors influencing the development of athletic potential, but it is equally certain that potential is innate and determined by an individual’s genetic heritage. Each of us owes our uniqueness to the information contained in our DNA, the genetic code that we inherited from our parents and which we pass on to our children. What is written in that code determines not only phenotypic traits, such as hair, eyes, skin color and other physical features, but also our character, our susceptibility to disease, and our ability to react to stimuli. Of course, the environment and our life experiences greatly affect the manifestation of this information such that, depending on the type and amount of stimuli we receive, our response will reflect the adaptability with which our DNA has equipped us (Fig. 4). The way we progress as a result of training is certainly due to the presence of a stimulus that acts by placing our body under stress, but our response to that stimulus is dictated by the instructions written in our DNA and it is these instructions that generate different responses to equivalent stimuli. Physical activity induces a wide

Fig. 3  Graphical representation of the variability in athletic performance. (Modified from first edition of Cellular Physiology and Metabolism of Physical Exercise. I. Terruzzi (2012) Chap. 4, pag. 26)

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Fig. 4  Environmental and genetic factors affecting athletic performance. (From first edition of Cellular Physiology and Metabolism of Physical Exercise. I. Terruzzi (2012) Chap. 4, pag. 27)

variety of biochemical and biophysical responses that act on the organism and determine a broad range of phenotypic adaptations. The results in terms of performance vary and this variability is particularly observed in athletes, in whom almost no measurable differences in performance can distinguish the winner from his or her competitors. It is clear that some athletes possess an innate talent that distinguishes them from other competitors who show the same strength of will, the same effort, and the same perseverance in training: genetics provide the competitors with the opportunity to participate and the winner with the ability to excel. Sir Roger Bannister was the first person to run the mile in less than 4 min, but he was also the first to become aware of “the obvious but overlooked fact that black sprinters, and all black athletes in general, have natural anatomic advantages.” If we consider the 20 best runners of all time in the distances from 800 m to the marathon, more than half have been from Kenya. Does this depend on the high-altitude highlands of their country, on their nutrition, on their body structure, or on the fact that many Kenyan children run for miles every day? However, while East Africans reign over long distances, athletes with roots in West Africa, as is the case for most Afro-­Americans, dominate the sprint. This leads to the question: is there a genetic selection of talent?

4 Can Genes Predict Athletic Performance? Just what do genes really tell us about athletic ability? If genes determine the potential of each person and the environment acts on them by ensuring their optimal expression, then will identifying those genes and studying their function allow us to predict the performance of each athlete? What can our genes truly reveal about our athletic potential? Surely if we were able to distinguish the specific genes that contribute to performance, muscle strength, and maximal aerobic capacity, a genetic test would be able to reveal to a parent whether his or her child will excel in a

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particular sport. In actual fact, genetics shape us in many ways, including our potential to excel in sports. However, the relationship between genes and physical and sports performance is still an open field of investigation, although in recent years there have been significant advances. Searching for the effects of an individual genetic variant in a complex and environmentally influenced activity such as sports performance is extremely difficult. There is no direct relationship between a gene and the characteristics of performance; instead, multiple genes are responsible for defining a performance, even the simplest one imaginable. It is also very difficult to quantify the performance of a specific sport, for example, the marathon. Athletes who have reached high competitive levels have a combination of different genotypes favorable for physical performance. In fact, performance represents a trait controlled by multiple genes and a single gene cannot be responsible for performance; rather, it may only increase or decrease a person’s physical abilities. For this reason, modulating the expression of a single gene may not result in substantial changes in sports performance. In June 2001, the first human gene map linked to performance was described by Rankinen. Since then, the number of genes potentially associated with physical performance has increased yearly (Table 1). In the human gene map updated in 2005, the number of such genes had expanded to about 190.

Table 1  2012 London Olympic Games Qualification Standards Men A Standard 10.18 20.55 45.25 1:45.60 3:35.50 13:20.00 27:45.00 8:23.10 13.52 49.50 2.31 5.72 8.2 17.20 20.50 65.00 78.00 82.00 8200 Top 16 teams Top 16 teams

Event B Standard 10.24 20.65 45.70 1:46.30 3:38.00 13:27.00 28.05.00 8:32.00 13.60 49.8 2.28 5.60 8.10 16.85 20.00 63.00 74.00 79.50 7950

100 m 200 m 400 m 800 m 1500 m 5000 m 10,000 3000 m SC 110 m H/100 m H 400 m H High jump Pole vault Long jump Triple jump Shotput Discus Hammer throw Javelin Decathlon/heptathlon 4 × 100 m 4 × 400 m

Women A Standard 11.29 23.10 51.50 1:59.90 4:06.00 15:15.00 31.45.00 9:43.00 12.93 55.40 1.95 4.50 6.75 14.30 18.35 62.00 71.50 61.50 6150 Top 16 teams Top 16 teams

B Standard 11.38 23.30 52.30 2:01.30 4:08.90 15:25.00 32:10.00 9:48.00 13.15 56.55 1.92 4.40 6.65 14.10 17.30 59.50 69.00 59.00 5950

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5 Genetic Variability Between Individuals Approximately 20,000 genes make up the genetic heritage that defines each of us as human. However, despite our common heritage, substantial variations exist between individual human genomes, including alterations in gene sequences (copy number variation, tandem repeats) and changes in individual base pairs (mutations if 1% frequency). The genetic code written in our DNA is specified by four nucleotides referred to by their first letters: A (adenine), T (thymine), C (cytosine), and G (guanine). The particular sequence of these nucleotides is specific for every single gene and determines its specific function. A gene consists of a promoter, which indicates the starting point of transcription and determines when and how frequently a gene is expressed, as well as a specific coding sequence of nucleotides, which determines the amino acid sequence of the protein encoded by the mRNA transcript. Sometimes, variations occur within the original sequence of nucleotides such that, for example, an A replaces one of the other three nucleotides (C, G, or T). These small genetic changes are called single nucleotide polymorphisms (SNP). They occur once in every 300 nucleotides on average, which means that there are roughly ten million SNPs in the human genome. Most commonly, these variations are found in the intronic regions of the DNA (around 26% of the genome) and in regions that separate adjacent genes, i.e., stretches of non-coding DNA that, according to our current knowledge, are without function. When SNPs occur within a gene or in its regulatory region, they may directly influence its function. For example, the substitution of a base in the coding sequence of a gene can alter the corresponding protein, by the insertion of the wrong amino acid, or even cause its premature termination, by the insertion of a “stop” signal instead of an amino acid. Such proteins are very often non-functional. SNP is the most common type of “genetic variation” and each person’s genetic material contains a unique SNP pattern that is made up of many different genetic variations. Although more than 99% of human DNA sequences are the same, variations in DNA due to the presence of SNPs can have a major impact on how a person responds to environmental stimuli. In this light, SNP profile studies may help to predict an individual’s response to certain stimuli (such as physical exercise) in addition to being used to track the inheritance of predisposing genes within families. Because SNPs occur normally throughout DNA and are evolutionarily stable, with few changes from generation to generation, they represent excellent biological markers (segments of DNA with an identifiable physical location that can be easily tracked) in studies of the relationship between particular gene variations and their respective, potentially modified proteins. Given this crucial ability to use such genetic variations in gene identification, together with recent technological advances, the discovery and detection of SNPs has become an important field of genetic research.

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6 Genetic Polymorphisms of the Enzymes Involved in DNA Methylation and Synthesis in Elite Athletes Athletes show an altered muscle phenotype and enhanced performance that are due to physical exercise, which is accompanied by a continuous and constant training stimulus leading to new metabolically and morphologically adaptive goals. The mechanism by which exercise stimulates these actions in athletes is poorly understood. While, as extensively detailed in this chapter, it is clear that environmental influences such as training and diet are important; nonetheless, genetic background is strongly related to performance. This aspect suggests that athletes possess a genetic advantage predisposing them to better sport performances than achieved by non-athletes. Hundreds of genes have been studied in relation to performance in an attempt to unravel the complex relationship between genetic expression and physical performance in athletes. Specific mechanisms (Fig. 5) take part in controlling gene expression, in particular the adequate supply of methyl groups to the DNA and therefore the specific enzymes responsible for the proper functioning of DNA methylation. The role of DNA methylation as a locking mechanism for an important event, such as tissue-­ specific gene expression during development, is well established. In particular, several studies on specific muscle genes have demonstrated a role of hypomethylation in the induction of muscle differentiation and hypertrophy. Polymorphic variants in the genes encoding DNA-methylating regulatory enzymes, due to alteration of nucleotides sequences, often result in enzymes with

Fig. 5  Simplified scheme of DNA methylation/synthesis cycle. (From first edition of Cellular Physiology and Metabolism of Physical Exercise. I. Terruzzi (2012) Chap. 4, pag. 30)

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reduced or otherwise abnormal activity. Such polymorphic variants include methylenetetrahydrofolate reductase (MTHFR), C677T and A1298C; cystathionine betasynthase (CBS), 844ins68; methionine synthase (MTR), A2756G; methionine synthase reductase (MTRR), A66G; betaine-homocysteine methyltransferase (BHMT), G742A; and cystathionine β-synthase (CBS), 68-bp ins, and they have been studied in the DNA of athletes. In vivo studies in a cohort of elite athletes have demonstrated the presence of polymorphic variants of three of these enzymes (MTHR, MTR, MTRR). As polymorphic forms of these genes result in a reduced function of the enzymes encoded, their presence in the DNA of the studied athletes suggests that elite athletes have a genetic predisposition to DNA hypomethylation. It can be speculated expected that in athletes, reduced enzyme activity due to genetic variants (namely MTHFR (AC), MTR (AG), and MTRR (AG) heterozygous genotypes) results in DNA hypomethylation and a consequent increase of muscle-specific gene expression. Likewise, the modifications caused by these polymorphisms might increase the functioning of genes responsible for the differentiation and growth of the muscle cell, with potential effects on athletic performance. The significant frequency of MTHFR A1298C, MTR A2756G, and MTRR A66G polymorphic variants in athletes adds these genes to a pool of genes directly associated with athletic ability, which could lead to a better understanding and recognition of the genetic basis of variation in performance.

Further Reading Bompa TO, Buzzichelli CA. Periodization: theory and methodology of training. 6th ed. Champaign, IL: Human Kinetics; 2019. Bray MS, Hagberg JM, Pérusse L, et al. The human gene map for performance and health-related fitness phenotypes: the 2006-2007 update. Med Sci Sports Exerc. 2009;41:35–73. Sarzynski MA, Loos RJ, Lucia A, et al. Advances in exercise, fitness, and performance genomics in 2015. Med Sci Sports Exerc. 2016;48:1906–16. Terruzzi I, Senesi P, Montesano A, et al. Genetic polymorphisms of the enzymes involved in DNA methylation and synthesis in elite athletes. Physiol Genomics. 2011;43:965–73. Watson JD, Baker TA, Bell SP, et al. Molecular biology of the gene. 7th ed. Boston, MA: Benjamin Cummings; 2013.

Mitochondrial and Non-mitochondrial Studies of ATP Synthesis Roberto Codella

1  Introduction Energy is required to perform any kind of mechanical work. In living organisms, the energy for all biological functions is provided chemically by the hydrolysis of adenosine triphosphate (ATP). ATP supplies the energy required to synthesize cellular components and to maintain cell viability, by donating one or two phosphate groups, leaving adenosine diphosphate (ADP) or adenosine monophosphate (AMP), respectively. However, energy storage in the form of ATP is limited such that ATP must be resynthesized continuously in order to meet cellular energy demands. The generation or replenishment of ATP depends upon key metabolic pathways, glycolysis, glycogenolysis, and oxidative phosphorylation, which interact to regulate the rate of ATP metabolism and to direct cellular bioenergetics toward a defined homeostasis. The different mechanisms involved in the breakdown and resynthesis of ATP may be summarized as follows: 1. ATP is broken down enzymatically to ADP and inorganic phosphate (Pi), yielding energy for muscle activity. 2. Phosphocreatine (PCr) is broken down enzymatically to creatine and phosphate, with the latter transferred to ADP, thereby yielding ATP. 3. Glucose 6-phosphate, derived from muscle glycogen or blood-borne glucose, is converted to lactate through anaerobic glycolysis and produces ATP by substrate-­ level phosphorylation reactions.

R. Codella (*) Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milan, Italy Department of Endocrinology, Nutrition and Metabolic Diseases, IRCCS MultiMedica, Milan, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 L. Luzi (ed.), Cellular Physiology and Metabolism of Physical Exercise, https://doi.org/10.1007/978-3-031-27192-2_5

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4. The products of carbohydrates, lipid, protein, and alcohol metabolism can enter the mitochondrial tricarboxylic acid (TCA or Krebs’) cycle, where they are oxidized to carbon dioxide and water. This process is known as oxidative phosphorylation and it produces energy for the synthesis of ATP. Some of this ATP is used for the resynthesis of PCr, which becomes depleted during exercise. Bioenergetics and metabolism are essential to maintaining health and may be disturbed in disease. Both can be studied with in vivo magnetic resonance spectroscopy (MRS), which is uniquely suited to quantitatively measure cellular ATP-­ generating activities in vivo. Intramuscular storage and the turnover of important nutrients (e.g., glycogen) can be monitored non-invasively by MRS as well. This technique has therefore made a substantial contribution to our understanding of mammalian cell energy metabolism, its control, and its alteration by disease. Thus, in vivo MRS represents a promising method to investigate human metabolism, with further developments and applications likely to ensure its continued use.

2 In Vivo Magnetic Resonance Spectroscopy In vivo MRS is a non-invasive, safe technique that enables a unique, innovative perspective on tissue biochemistry in that it allows: (a) assessment of cellular metabolite concentrations and their alterations; (b) monitoring of the intracellular fate of infused labeled substrates; (c) measurements of chemical exchange processes under steady state or equilibrium conditions. The most MR-sensitive nuclei and those most commonly used are 1H, 31P, and 13 C, each of which generates specific information on distinct metabolic and physiological processes and conditions.

 H-MRS 1

H is the most sensitive nucleus for MR studies because of its high relative sensitivity and natural abundance. However, while it produces a greater signal-to-noise ratio than any other nucleus, the generated 1H-MR spectra can be very complex owing to the ubiquity of hydrogen atoms in biological molecules. A major disadvantage of 1H for MRS is that it results in the presence of a large solvent peak (water) in spectra of aqueous solutions, although water suppression techniques have nowadays enabled the detection of metabolites present at low concentrations [1]. In MR imaging (MRI), the strong water signal, resulting from the large presence of water, can be exploited to generate images reflecting the anatomical shape of the organ [2]. Since 1H-MRS distinguishes muscle tissue from fat, bone, and connective tissue, its immediate application is to produce anatomical information and to estimate muscle volume. Other important applications of 1H-MRS to in  vivo research on skeletal 1

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muscle include the quantification of intramyocellular lipids [3], which have proven to be a surrogate marker for insulin sensitivity [4]; the detection of lactate formation [5]; and measurements of total muscle creatine content (especially relevant for bioenergetic studies) [6], metabolite diffusion in a single muscle cell [7], tissue deoxygenation based on the 1H-MRS signal of deoxymyoglobin [8], and blood flow [9].

 C-MRS 13

Although all biologically relevant metabolites contain carbon, 13C-MRS is still an intrinsically insensitive technique. Carbon 13 comprises only 1.1% of all naturally occurring carbon nuclei but the sensitivity of 13C-MR spectroscopy can be improved almost 100-fold by using 13C enriched isotopes, which are either infused intravenously or ingested. Moreover, the enrichment at one or two specific positions in the substrate chosen allows the fate of these carbons to be monitored such that fluxes through specific metabolic pathways can be quantified over time. In 13C-MRS studies, the signal is generated from carbon 1 of glycogen, with the signal size corresponding to the concentration of tissue glycogen. The incorporation of labeled [1-13C]-glucose into glycogen allows the rates of muscle glycogen synthesis to be measured [10]. However, peak assignment is sometimes difficult because these 13C spectra contain overlapping signals from numerous tissue compounds. 13 C-MRS has been used to quantify the flux through the TCA cycle, based on a non-invasive in  vivo assay of mitochondrial activity, which is vital for sustained skeletal muscle function. This technique requires the infusion of 13C-enriched glucose or acetate (see Sect. 4). The underlying principle is that ongoing TCA cycle activity is reflected in a very characteristic pattern of 13C-enrichment in the carbons of glutamate. Although glutamate itself is not an intermediate of the TCA cycle, its enrichment pattern is nevertheless representative of TCA turnover kinetics because the mitochondrial glutamate pool is in rapid equilibrium with α-ketoglutarate via a transaminase reaction. The 13C-label at the C-1 position of glucose will initially appear at the C-4 position of glutamate during the first turn of the TCA cycle and equally label the glutamate C-2 and C-3 positions on subsequent turns. Infusions with [2-13C]-acetate have been used to determine TCA cycle activity as a means of assessing mitochondrial coupling in  vivo in rat skeletal muscle [11] and human skeletal muscle [12] based on the concept that TCA cycle flux is a measure of the rate of mitochondrial oxygen consumption by the respiratory chain.

 P-MRS 31

Although the sensitivity of phosphorus is 7% that of proton, 31P-MRS is one of the most sensitive techniques used in MRS. 31P is 100% abundant, occurring naturally in all phosphate-containing compounds and thus obviating the need for isotopic

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a

R. Codella

b

Fig. 1 (a) 31P spectrum obtained from a hindlimb mouse muscle. (b) Principle underlying the measurement of ATP synthesis rate by 31P MR-saturation transfer as applied to the exchange between Pi and ATP. In blue, the symmetric saturation spectrum: the saturation pulse is applied symmetrically to γ-ATP, but on the downfield side of Pi. In red, the spectrum obtained with selective γ-ATP irradiation (NS = 128). Subtraction of the spectra yields the fraction of Pi involved in the synthesis of ATP by the reaction: ADP + Pi → ATP [13]

enrichment. The phosphorus spectra are usually simple and can be used to quantify high-energy phosphate intermediates such as ATP, Pi, and PCr (Fig.  1a). These phosphate compounds are found in living systems in concentrations high enough to be detectable [14]. A number of essential variables, obtained either directly or indirectly from dynamic 31P-MRS, can be used to quantitatively study the kinetics of energy metabolism in  vivo [15]. 31P-MRS has thus opened a window on bioenergetics during skeletal muscle exercise and recovery, allowing for detailed but non-invasive studies. The technique has made a significant contribution to our understanding of animal and human energy metabolism, its control, and its modulation by different factors, including mitochondrial dysfunction [16] and a number of chronic diseases. Several approaches to quantitatively analyze and interpret 31P-MRS measurements of energy balance in muscle during and after several types of exercise have been proposed [17]. For example, it is possible to estimate the rates of glycogenolytic and aerobic ATP synthesis, i.e., oxidative capacity, according to distinct protocols addressing various types of exercise, such as ischemic exercise, pure aerobic exercise under steady-state conditions or during work jumps, and mixed exercise. Chance et al. [18] carried out graded, steady-state, non-exhaustive metabolic investigations that identified ADP as a major control element of oxidative metabolism in human skeletal muscle under these conditions. The authors showed that maximum velocity could serve as a measure of oxidative capacity. Dynamic measurements of the initial rate of PCr depletion during pure aerobic work jumps [19] yielded an estimate of the total rate of ATP synthesis (typically, at low workloads

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glycogenolysis can be neglected). The recovery rate of PCr immediately after exercise appears to reflect mitochondrial capacity. Other protocols based on ischemic exercise were used to calculate glycogenolytic ATP production [20] and to measure the rate of oxidative metabolism under non-steady-state exercise conditions. Also, an additional and important piece of information retrievable from 31P spectra, albeit indirectly, is intracellular pH, as tissue pH can be deduced from the chemical shift of Pi. During mixed exercise, both glycogenolytic and oxidative ATP synthesis can be estimated by a calculation that includes total proton production and total ATP turnover; the latter can be determined in different ways: (a) by calibration from ischemic exercise at the same power; (b) from the non-oxidative ATP synthesis rate in the first exercise interval, when oxidative ATP production is still small; and (c) from very early changes in the PCr concentration alone (neglecting both glycogenolytic and oxidative ATP synthesis). In other words, during ischemic exercise glycogenolytic ATP production can be directly calculated from changes in pH, corrected for the number of protons consumed by PCr hydrolysis as calculated based on changes in the concentration of PCr and the rate at which the cell buffers protons, assuming the buffer capacity is known [18] (see section “Glycolytic Flux”). In another remarkable application of 31P-MRS, the measured signal strength can be sensitized to the rate of metabolite turnover even under steady-state conditions, making use of magnetization transfer between nuclei linked by chemical exchange [21]. Magnetization transfer can be studied by selectively perturbing the equilibrium magnetization of one of the nuclei involved in the exchange process and then measuring the effect on the signal strength from its exchange partner (see Sect. 3).

3 Mitochondrial Function Assessed by 31P-MRS P MRS offers a unique possibility to determine flux rates in biochemical pathways in vivo by magnetization transfer. This latter technique is based on MRS and has the capability to non-invasively measure the reaction kinetics of enzymes in situ when the reaction rates involved are relatively fast. It involves perturbing the magnetization of a nuclear spin system in a particular compound and then monitoring how this perturbation influences the nuclear magnetization of this spin system in another compound in chemical exchange with the first [21]. Saturation is a particular kind of magnetization transfer in which the compounds under discussion are, respectively, γ-ATP and Pi. In 31P-MRS saturation transfer studies, the rates of mitochondrial phosphorylation are assessed: the unidirectional rates of ATP synthesis are measured with the MR-saturation transfer method applied to the exchange between Pi and ATP (i.e., the kinetics of Pi → ATP). The steady-­ state intramyocellular Pi magnetization is determined during selective irradiation of the γ resonance of ATP and compared with the magnetization of Pi at equilibrium in a control spectrum (without saturation of the γ resonance of ATP). This reduction of Pi magnetization yields the fraction of Pi involved in ATP synthesis (VATP) (Fig. 1b). Hence, under appropriate conditions (i.e., resting, steady state) the magnetic 31

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transfer technique is capable of measuring mitochondrial oxidative phosphorylation catalyzed by mitochondrial ATPase.

 itochondrial Function During Exercise as Assessed M by 31P-MRS In addition to magnetization transfer under steady-state conditions, 31P MRS offers a variety of techniques for measuring mitochondrial function in exercising muscle. In MRS studies conducted on animal models in  vivo, muscular contraction was induced by electrical stimulation [22]. Using 31P MRS, the rate of oxidative phosphorylation was modeled from the recovery of PCr after exercise [23]. The PCr recovery rate can be expressed as a recovery-time constant and is thought to reflect maximal ATP generation from oxidative metabolism via the creatine kinase (CK) reaction (also called the Lohman reaction):

ATP + creatine  ADP + PCr + H +

The calculation assumes that the ATP concentration is constant during recovery and that ATP production from glycolysis is discontinued with the cessation of exercise [20]. The results are often described as being independent of work level providing that the acidic change in pH is not large [23]. This workload independence or insensitivity is assumed to have a practical advantage in that measurements of force or work are not required. This allows the evaluation of a broad range of subjects (including the elderly) without the need for them to perform sub-maximal exercise tests. Moreover, during pure aerobic work jumps, dynamic measurements of the initial rate of PCr depletion yield an estimate of the total rate of ATP synthesis. Pure aerobic exercise also allows the maximal mitochondrial ATP synthesis flux of muscle to be estimated, by following the methodology reported by Chance et al. [18] with MRS measurements of energy balance at multiple workloads in a ramp protocol. In fact, from this dataset, the kinetic transfer function of power-output and an index of the cytosolic ADP concentration can be derived. The 31P spectra do not yield a discernible ADP signal; thus, to assess ADP concentrations investigators have assumed CK to be at equilibrium and have used the observables in the 31P spectra (PCr, ATP, Pi, and pH) to calculate ADP activity, which has accordingly been found to significantly exceed the activity determined in biochemical assays. In other words, the ADP concentration can be calculated from the PCr/Pi value with appropriate assumptions [18].

4 Measurement of TCA Cycle Flux (VTCA) As discussed above, the administration of appropriate 13C-precursors yields specific and quantitative information about metabolic fluxes in vivo, for example, those of the TCA cycle [1, 11, 12]. 13C-enriched precursors in the form of [2-13C]-labeled

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Fig. 2  The fate of a labeled molecule through the TCA cycle. Infused plasma [2-13C]-acetate enters the TCA cycle after being converted into acetyl CoA, the primary fuel for the cycle. The 13C label is then incorporated into TCA cycle intermediates (citrate, α-ketoglutarate) and glutamate pools, forming [4-13C]-glutamate on the first turn of the TCA cycle. A second turn of the cycle yields [2-13C]- and [3-13C]-glutamate, which provide input for the metabolic model used to calculate the TCA cycle flux (VTCA)

acetate and [1-13C] or [6-13C]-glucose will enter the TCA cycle such that the 13C label appears as [2-13C]-acetyl-CoA. After intermediary metabolism the 13C label is transferred to metabolites that can be detected by MR, i.e., glutamate. In other words, the entry into the TCA cycle of a 13C label permits the calculation of TCA cycle flux rates (Fig. 2). For example, when the precursor [2-13C]-acetate is infused as an MR-visible label, it is converted into [2-13C]-acetyl-CoA, which enters the TCA cycle by condensing with oxaloacetate to form [4-13C]-citrate. The position of the 13C label is conserved through the initial steps of the TCA cycle, labeling α-ketoglutarate at the C-4 position. Since α-ketoglutarate is in relatively rapid exchange with glutamate, the latter will be likewise 13C-enriched at position C-4. Incorporation of the 13C label from plasma [2-13C]-acetate into the muscle [4-13C]-glutamate pool is essential for the determination of TCA cycle flux (VTCA). In fact, as the TCA cycle proceeds, in the second turn the labeling will involve the [2-13C]- and [3-13C]-glutamate pools. Sophisticated mathematical models can be used to infer the rates of TCA cycle flux from the time course of 13C isotopic enrichment of plasma acetate and muscle glutamate C-2 and C-4 by iterative fitting of metabolic simulations to the data using the program Cwave. The CWave model consists of isotopic mass-balance equations that describe the metabolic fate of plasma [2-13C]-acetate. The isotopic enrichment and concentrations are used as input drivers. Based on these input parameters, flux rates are determined that give the best fit to the observed time course of [2-13C]- and [4-13C]-glutamate enrichment (Fig. 2). Furthermore, the increased sensitivity offered by 13C-enriched isotopes in direct 13 C detection can be further improved by indirect 13C-MRS, during which the protons attached to 13C nuclei are detected (this MR technique is called POCE, “proton-­ observed carbon-edited”) [1].

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5 Anaerobic Sources of ATP Two separate systems are available that permit the generation of energy in muscle without oxygen, namely the phosphagen or “high-energy phosphate system” (intramuscular stores of ATP and PCr) and the glycolytic system (Fig. 3). The total capacity of the glycolytic system for producing energy in the form of ATP is larger than that of the phosphagen system, although the rate at which it can produce ATP is lower (Table 1). At the start of the energy challenge, hydrolyzed ATP is resynthesized from the breakdown of PCr and, depending on conditions, from anaerobic glycogenolysis. Anaerobic energy production is essential for the maintenance of high-intensity exercise, when the demand for ATP is greater than what can be provided aerobically. In fact, the approximate contribution of anaerobic and aerobic sources to overall ATP production during high-intensity exercising lasting ~3 min is 80/20% in the initial 30 s, 45/55% from 60 to 90 s, and 30/70% from 120 to 180 s [24]. To assess anaerobic ATP provision, such as during high-intensity exercise, key substrates and metabolites must be repeatedly measured. For years, this was accomplished through biopsy sampling of contracting muscle at frequent intervals during high-intensity exercise. The advent of MRS has several advantages over the former technique: (a) it is non-invasive, allowing repeated measurements of metabolite concentrations over time; (b) it involves stables isotopes (no ionizing radiation); and (c) it yields chemical information such that the intracellular fate of a labeled molecule can be monitored as it is metabolized. Pcr + ADP + H +

Glycogen

ATP + Cr

ADP ATP

Lactate + H +

ATP reserve

ATP

Fig. 3  Scheme of the anaerobic sources of energy Table 1  Capacity and power of anaerobic systems for the production of ATPa Phosphagen system Glycolytic system Combined

Capacity (mmol ATP kg dm−1) 55–95 190–300 250–370

Power (mmol ATP kg dm−1 s−1) 9 4.5 11

 Values are expressed per kg dry mass (dm) of muscle and are based on estimates of ATP provision during high-intensity exercise of human vastus lateralis muscle a

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Glycolytic Flux Localized 31P MRS can be used to calculate the glycolytic rate based on changes in pH during high-intensity exercise, after correcting for the buffering of protons by changes in PCr and ATP concentrations, the rate of aerobic ATP utilization, the apparent muscle buffer capacity, and proton efflux [25]. Conley et al. studied the regulation of glycolysis using 31P MRS during ischemic stimulation of the human forearm [26]. They showed that the glycolytic rate is proportional to the muscle stimulation frequency and does not depend on metabolite levels and intracellular pH. This result is consistent with the dominant control of glycolysis by factors other than the products of ATP hydrolysis that scale with nerve-firing frequency (e.g., the free calcium concentration).

PCr Breakdown The rate of ATP synthesis from the net breakdown of PCr (ATPCK) via the CK reaction (see section “Mitochondrial Function During Exercise as Assessed by 31P-­ MRS”) can be determined as during 31P MRS, i.e., from the change in PCr during each muscular contraction. Since the synthesis of ATP is stoichiometric with the hydrolysis of PCr in the CK reaction, calculation of the latter takes the simple form [15]:

ATPCK = dPCr / dt

Glycogen Glycogen is readily available in muscle and can quickly be used to fuel glycolysis. Following the infusion of [1-13C]-glucose, [1-13C]-glycogen can be detected in muscle. While the 13C MRS detection of glycogen is relatively straightforward, the detection of 1H glycogen remains elusive. During exercise, muscle glycogen levels decrease according to: (a) the intensity of exercise, reaching lower levels with increasing workloads and (b) the rate of muscle contraction. Muscle glycogen can be monitored during different types of exercise (also involving different and isolated muscle groups) by means of 13C MRS, which enables studies of muscle glucose metabolism, including the effects of acute exercise. For example, through MRS studies it has been possible to demonstrate that, when exercise ceases, the exercised muscles resynthesize glycogen at a rate that is influenced by the post-exercise concentration of intracellular glycogen. Also, between rapid muscle contractions, the PCr pool is replenished mainly by glycogenolysis after PCr breakdown [27]. However, the metabolic roles of muscle

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glycogen in exercise are currently not known. High glycogen levels improve endurance, whereas glycogen depletion is often associated with the onset of fatigue [27].

6 Integrative View With the advent of in vivo MRS, new techniques have emerged with which to investigate reaction kinetics directly. In probing energy regulation and assessing the above-described ATP-generating pathways, in vivo MRS might be preferred over biopsy measurements, with the advantages of greater accuracy, non-invasiveness, and complete biochemical information over time. The human musculature is well suited for MRS studies because: (a) it is the primary organ of carbohydrate uptake and storage; (b) it can be locally evaluated under controlled systemic metabolic and hormonal conditions; (c) it is the organ of physical work, and exercise is a natural metabolic stimulus that is non-invasive. For these reasons, a large number of measurements can be made over a short period of time and accurate metabolite rates and fluxes can be calculated. Furthermore, as discussed, a combination of 31P- and 13C MRS can be used to expand the measurable range of metabolic events. In conclusion, in  vivo MRS has enhanced the classical analysis of metabolic pathways, revolutionizing our understanding of human and animal metabolism in health as well as in disease. Acknowledgments  The preparation of this manuscript was supported by European grant: FP7-­ PEOPLE-­2009-RG (INMARESS project nr. 256506) to Roberto Codella.

References 1. De Graaf RA. In vivo NMR spectroscopy: principles and techniques. Chichester: Wiley; 2007. 2. Norris DG. The effects of microscopic tissue parameters on the diffusion weighted magnetic resonance imaging experiment. NMR Biomed. 2001;14:77–93. 3. Boesch C, Machann J, Vermathen P, Schick F. Role of proton MR for the study of muscle lipid metabolism. NMR Biomed. 2006;19:968–88. 4. Perseghin G, Lattuada G, Danna M, Sereni LP, Maffi P, De Cobelli F, Battezzati A, Secchi A, Del Maschio A, Luzi L. Insulin resistance, intramyocellular lipid content, and plasma adiponectin in patients with type 1 diabetes. Am J Physiol Endocrinol Metab. 2003;285(6):E1174–81. 5. Hsu AC, Dawson MJ. Accuracy of 1H and 31P MRS analyses of lactate in skeletal muscle. Magn Reson Med. 2000;44(3):418–26. 6. Kruiskamp MJ, de Graaf RA, van Vliet G, Nicolay K. Magnetic coupling of creatine/phosphocreatine protons in rat skeletal muscle, as studied by (1)H-magnetization transfer MRS. Magn Reson Med. 1999;42(4):665–72. 7. Nicolay K, Braun KP, Graaf RA, Dijkhuizen RM, Kruiskamp MJ. Diffusion NMR spectroscopy. NMR Biomed. 2001;14(2):94–111.

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8. Richardson RS, Duteil S, Wary C, Wray DW, Hoff J, Carlier PG.  Human skeletal muscle intracellular oxygenation: the impact of ambient oxygen availability. J Physiol. 2006;571(Pt 2):415–24. 9. Carlier PG, Bertoldi D, Baligand C, Wary C, Fromes Y. Muscle blood flow and oxygenation measured by NMR imaging and spectroscopy. NMR Biomed. 2006;19(7):954–67. 10. Jue T, Rothman DL, Shulman GI, Tavitian BA, DeFronzo RA, Shulman RG.  Direct observation of glycogen synthesis in human muscle with 13C NMR.  Proc Natl Acad Sci U S A. 1989;86(12):4489–91. 11. Cline GW, Vidal-Puig AJ, Dufour S, Cadman KS, Lowell BB, Shulman GI. In vivo effects of uncoupling protein-3 gene disruption on mitochondrial energy metabolism. J Biol Chem. 2001;276(23):20240–4. 12. Jucker BM, Dufour S, Ren J, Cao X, Previs SF, Underhill B, Cadman KS, Shulman GI. Assessment of mitochondrial energy coupling in vivo by 13C/31P NMR. Proc Natl Acad Sci U S A. 2000;97(12):6880–4. 13. Codella R, Alves TC, Befroy DE, Choi CS, Luzi L, Rothman DL, Kibbey RG, Shulman GI. Overexpression of UCP3 decreases mitochondrial efficiency in mouse skeletal muscle in vivo. FEBS Lett. 2023;597(2):309–19. 14. Hoult DI, Busby SJ, Gadian DG, Radda GK, Richards RE, Seeley PJ. Observation of tissue metabolites using 31P nuclear magnetic resonance. Nature. 1974;252(5481):285–7. 15. Kemp GJ, Radda GK. Quantitative interpretation of bioenergetic data from 31P and 1H magnetic resonance spectroscopic studies of skeletal muscle: an analytical review. Magn Reson Q. 1994;10(1):43–63. 16. Petersen KF, Befroy D, et al. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science. 2003;300:1140–2. 17. Kemp GJ, Taylor DJ, Thompson CH, Hands LJ, Rajagopalan B, Styles P, Radda GK. Quantitative analysis by 31P magnetic resonance spectroscopy of abnormal mitochondrial oxidation in skeletal muscle during recovery from exercise. NMR Biomed. 1993;6(5):302–10. 18. Chance B, Leigh JS, Clark BJ, Maris J, Kent J, Nioka S, Smith D. Control of oxidative metabolism and oxygen delivery in human skeletal muscle: a steady-state analysis of the work/energy cost transfer function. Proc Natl Acad Sci U S A. 1985;82(24):8384–8. 19. Jeneson JA, Wiseman RW, Kushmerick MJ.  Non-invasive quantitative 31P MRS assay of mitochondrial function in skeletal muscle in situ. Mol Cell Biochem. 1997;174(1–2):17–22. 20. Lanza IR, Wigmore DM, Befroy DE, Kent-Braun JA. In vivo ATP production during free-flow and ischaemic muscle contractions in humans. J Physiol. 2006;577(Pt 1):353–67. 21. Forsen S, Hoffman RA. A new method for study of moderately rapid chemical exchange rates employing nuclear magnetic double resonance. Acta Chem Scand. 1963;17:1787–8. 22. Drost MR, Heemskerk AM, Strijkers GJ, Dekkers EC, van Kranenburg G, Nicolay K.  An MR-compatible device for the in situ assessment of isometric contractile performance of mouse hind-limb ankle flexors. Pflugers Arch. 2003;447(3):371–5. 23. Thompson CH, Kemp GJ, Sanderson AL, Radda GK.  Skeletal muscle mitochondrial function studied by kinetic analysis of postexercise phosphocreatine resynthesis. J Appl Physiol. 1995;78(6):2131–9. 24. Bangsbo J, Gollnick PD, et al. Anaerobic energy production and O2 deficit-debt relationship during exhaustive exercise in humans. J Physiol. 1990;422:539–59. 25. Walter G, Vandenborne K, Elliott M, Leigh JS. In vivo ATP synthesis rates in single human muscles during high intensity exercise. J Physiol. 1999;519(Pt 3):901–10. 26. Conley KE, Blei ML, Richards TL, Kushmerick MJ, Jubrias SA. Activation of glycolysis in human muscle in vivo. Am J Phys. 1997;273(1 Pt 1):C306–15. 27. Shulman RG, Rothman DL. The glycogen shunt in exercising muscle: a role for glycogen in muscle energetics and fatigue. Proc Natl Acad Sci U S A. 2001;98(2):457–61.

Excessive Nutrients and Regional Energy Metabolism Gianluca Perseghin

1 Introduction There is general agreement that type 2 diabetes is the consequence of insulin resistance, defined as an impaired ability of insulin to control hepatic and peripheral glucose metabolism, and of compromised pancreatic β-cell function such that insulin secretion is insufficient to compensate the degree of insulin resistance [1]. The pivotal role of insulin resistance is confirmed by the fact that it is a consistent finding in patients with type 2 diabetes. Indeed, insulin resistance may be detected 10–20 years before the onset of overt hyperglycemia and prospective studies have demonstrated that it is the best predictor of whether an individual will later become diabetic [2]. However, while this “glucocentric” view represents the traditional explanation of the metabolic derangements of diabetes, a more “lipocentric” vision of the metabolic problem has been proposed to explain simultaneously (a) the impairment of insulin action in skeletal muscle, liver, heart, and adipose tissue and (b) the impaired β-cell function [3, 4] as a consequence of chronically high circulating free fatty acid (FFA) concentrations, i.e., lipotoxicity. Increased plasma FFA concentrations are associated with many insulin-resistant states, including obesity and type 2 diabetes mellitus [5]. In a cross-sectional study of the young, normal weight offspring of type 2 diabetic patients, we found an inverse relationship between fasting plasma FFA concentrations and insulin sensitivity, consistent with the hypothesis that altered FFA metabolism contributes to insulin resistance in patients with type 2 diabetes [6]. In addition, the deleterious effects of chronically elevated FFA on muscle glucose metabolism in healthy individuals [7] are similar to the established

G. Perseghin (*) Department of Medicine and Surgery, Università degli Studi di Milano Bicocca, Milan, Italy Department of Medicine and Rehabilitation, Policlinico di Monza, Monza, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 L. Luzi (ed.), Cellular Physiology and Metabolism of Physical Exercise, https://doi.org/10.1007/978-3-031-27192-2_6

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abnormality of glucose metabolism affecting the skeletal muscle of patients with overt diabetes [8] and of their non-diabetic insulin-resistant offspring [9]. In this chapter, we focus on some of the recent advances in our understanding of human insulin resistance with respect to its relationships to excessive ectopic fat accumulation in skeletal muscle, heart, and liver.

2 Excessive Ectopic Fat Accumulation and Abnormal Regulation of Insulin-Dependent Metabolic Pathways Skeletal Muscle Ectopic fat accumulation within skeletal muscle has been proposed as the pathogenic event in the development of peripheral insulin resistance [3]. An increased intramyocellular lipid content (IMCL) has been reported in association with insulin resistance in normal humans [10], individuals with an increased risk of developing type 2 diabetes [11, 12], and in patients with overt type 2 diabetes [13]. Convincing evidence of the association between IMCL content and whole-body insulin sensitivity also derives from an interventional study in which the effects of biliopancreatic diversion, which induces lipid malabsorption, and of a hypocaloric diet in the treatment of patients with morbid obesity were compared [14]. The surgical approach determined a drop of body weight in association with a selective depletion of the IMCL content (assessed by means of quantitative histochemistry of quadriceps-­ muscle biopsy specimens) paralleled by full reversion of the insulin resistance state. The same results were not obtained with the hypocaloric treatment, in which a less significant drop of body weight was associated with a smaller reduction of IMCL content and less improvement of insulin sensitivity. This work was of particular interest also because it showed that 6 months after surgical treatment a full normalization of whole-body insulin sensitivity and quadriceps IMCL content were achieved despite the persistence of a body mass index (BMI) still in the obese range. Although the association between insulin resistance and IMCL content may be considered a classical finding in humans, several discrepancies in the above-­ mentioned literature question this assumption. Specifically, when the anthropometric parameters of the study populations are more rigorously controlled there is less certainty of a causative association between increased IMCL content and the development of insulin resistance. For example, in a study of non-diabetic, normal weight men and women matched for peripheral insulin sensitivity as assessed by the clamp technique, the amount of total body fat and of soleus IMCL content was higher in women than in men [15], suggesting that the gender-dependent hormonal milieu modulates the interaction between IMCL content and insulin sensitivity. Moreover, obesity and aerobic fitness mutually interact regarding their impact on IMCL content: while IMCL content and insulin sensitivity were correlated in untrained subjects, in endurance-trained subjects, by contrast, high IMCL content predicted high insulin sensitivity [16]. Also, individuals with a primary muscular disease (myotonic dystrophy type 1) do not develop insulin resistance associated with glucose

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and lipid metabolism despite a markedly higher IMCL content in both the soleus and tibialis anterior muscles [17], suggesting a difference in the subcellular localization of fat in muscle and its metabolic influence. In fact, in contrast with other forms of muscular dystrophy, the replacement of muscle by fat and fibrous tissue is not typical of myotonic dystrophy type 1; rather, ultrastructural alterations are common and they may have an impact on fat localization around the mitochondria (rapid disposal pool) or within lysosomes.

Heart In addition to skeletal muscle, the heart is a site of ectopic fat accumulation. Unlike muscle, however, myocardial lipid is difficult to quantify because the heart is perpetually in motion and is surrounded by a large depot of adipocytes (epicardial fat pad) that interferes with measurements. For this reason few data in humans are available. Myocardial triglyceride content appears to increase progressively with BMI [18] and adiposity is not the sole determinant of lipid deposition in human myocardium. A single fatty meal does not change intramyocardial fat levels; however, a three-fold increase in myocardial lipid levels was shown in patients who fasted for 48  h [19]. It was speculated that this increase is not initially toxic but detrimental effects will occur after decades of sustained substrate excess [20]. More recently, the accumulation of triglyceride within the heart and in the epicardial fat pad of the myocardium was found to be significant already in individuals with moderately increased BMI and was related to FFA exposure, generalized ectopic fat excess, and peripheral vascular resistance. These alterations were noted in the absence of left ventricular (LV) overload and hypertrophy [21]. In a study of patients with heart failure who underwent cardiac biopsies, intramyocardial lipid levels were five to six times higher in obese individuals and in those with type 2 diabetes than in healthy controls. In addition, there was a negative association between the degree of lipid deposition and the genes that control the expression of proteins involved in contractile function [22]. Nevertheless, a direct association between myocardial lipid content and myocardial insulin-stimulated glucose metabolism has yet to be reported. Also, epicardial fat may be important; similar to other visceral fat depots, it is characterized by a high rate of FFA release [23], with no barriers or fascia to impede lipid transit toward myocardiocytes [24]. FFA levels were also reported to be predictive of LV mass, whereas myocardial and epicardial fat were more strongly related to LV work and mechanical load [21].

Liver An association between ectopic fat accumulation within the liver and impaired glucose metabolism has been reported. Both insulin-stimulated glucose metabolism and the suppression of endogenous glucose production were found to be impaired

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in individuals with non-alcoholic fatty liver disease (NAFLD) [24]. In addition, NAFLD is common in obese patients with type 2 diabetes [25] and is considered to be the hepatic component of the metabolic syndrome [26–29]. The development of fatty liver appears to be peculiarly associated with hepatic insulin resistance; in fact, moderate weight reduction in obese patients with type 2 diabetes is associated with reduced intra-hepatic fat content and improved hepatic insulin sensitivity although insulin-stimulated peripheral glucose metabolism remains unchanged [30]. Recently, intra-hepatic fat content was assessed as a continuous variable by means of 1H-magnetic resonance spectroscopy (MRS) and was reported to be associated with hepatic insulin resistance also in non-diabetic individuals [31] and obese adolescents [32].

3 Excessive Ectopic Fat Accumulation as the Consequence of Increased Adipose-Derived FFA Flux The hypothesis that excessive fat accumulation in skeletal muscle is determined by an increased adipose-derived FFA flux has received support from experiments in which circulating FFA availability was increased by the administration of a fat emulsion and i.v. heparin. This technique in combination with euglycemic hyperinsulinemia [1 mU/(kg min)] was reported to induce increments in the IMCL content to a much greater extent in the tibialis anterior (61%) than in the soleus (22%) in healthy humans [33]. This difference between the accumulations in soleus and tibialis anterior muscles was confirmed in healthy individuals who received a short-­ term (3  days) nutritional intervention consisting of either a high-fat (especially saturated fat) or a high-carbohydrate diet [34]. The high-fat diet clearly affected both the IMCL content and insulin sensitivity. In agreement with the infusion protocol, IMCL content in the tibialis anterior was much higher in individuals on the high-fat diet than in those on the high-carbohydrate diet, with only a non-significant increase in the soleus. The parallels between impaired insulin sensitivity and IMCL accumulation in the absence of a change in circulating plasma FFA concentration are interesting because they contradict the finding of another work in which nicotinic-­induced insulin resistance in humans was shown to be related to circulating FFA levels but not to IMCL content [35]. In the heart, cardiac steatosis may be the consequence of an increased flux of FFAs from adipose tissue toward the heart, as indicated by the above-described in  vivo 1H-MRS study in humans during conditions of prolonged fasting [19]. However, this relationship remains to be confirmed. Data on the liver are more consistent. In metabolic terms, ectopic fat accumulation by the liver is believed to be mainly sustained by an adipose-derived FFA flux. Consequently, therapeutic strategies have continued to focus on the reduction of FFA flux in adipose tissue (thiazolidinediones) [36]. Peripheral insulin resistance in patients with fatty liver appears to be associated not only with impaired

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insulin-­stimulated glucose metabolism but also with dysregulation of the FFA flux in the fasting state, during euglycemic-hyperinsulinemic clamps [25] and during oral glucose tolerance testing [37]. Insulin resistance with respect to lipolysis plays a relevant role in patients with fatty liver. Using tracer methodologies, it was found that in patients with NAFLD 60% of liver triglycerides arise from FFA in the fasting state [38]. Importantly, in the same study, 26% of liver triglycerides were shown to have been the product of de novo lipogenesis [38]. An apparent increased contribution of de novo lipogenesis vs. FFA reesterification was also reported in patients with NAFLD [39, 40].

4 The Association of Excessive Ectopic Fat Accumulation and Abnormalities of Energy Metabolism Skeletal Muscle Impairments of muscle or plasma FFA oxidation in obesity and type 2 diabetes have been reported by different groups [41–43]. A primary genetic defect has been hypothesized based on the observation that in obese women [42] and in type 2 diabetic patients [44] impaired fat oxidation was not reversed by a considerable body weight reduction and the same defect was detected in individuals with impaired glucose tolerance [45]. Altered fasting lipid oxidation in association with insulin resistance and IMCL accumulation is likewise observed as a secondary consequence of metabolic disturbances [46]. Our group reported the opposite metabolic picture in a group of healthy humans, who while moderately overweight still had normal insulin sensitivity and normal IMCL content in association with higher fasting lipid oxidation [47]. Moreover, using a longitudinal approach, it was shown that in obese individuals enhanced insulin sensitivity, achieved through physical activity, was associated with increased fat oxidation [48, 49]. More recently, improvements in insulin sensitivity across increasing quartiles of fasting lipid oxidation were demonstrated within a population comprising the offspring of type 2 diabetic parents, whereas insulin sensitivity remained constant in normal subjects without a family history, suggesting that impaired fat oxidation is a primary pathogenic factor of insulin resistance in people with a genetic background for type 2 diabetes [50]. In non-diabetic individuals with the same background these abnormalities at the whole-body level were associated with specific muscular defects of energy metabolism. 31P-MRS magnetization transfer experiments carried out in a cohort of offspring of type 2 diabetic parents previously known to be insulin-resistant showed that the abnormal IMCL content was associated with a skeletal muscle defect in mitochondrial oxidative phosphorylation and a reduced rate of ATP synthesis [51]. In vitro studies found evidence of disturbed oxidative enzyme activity in the skeletal muscle of type 2 diabetic and obese individuals [52, 53]. Mitochondrial dysfunction was proposed as the cause of the impaired lipid oxidation in the skeletal

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muscle of type 2 diabetic patients [54], and a specific age-associated decline in mitochondrial function has been suggested as a pathogenic factor in the development of insulin resistance in the elderly [55]. Indeed, primary mitochondrial dysfunction resulting in IMCL accumulation may constitute a self-perpetuating mechanism of mitochondrial damage, via the production of reactive oxygen species [56]. It has been suggested that the molecular mechanisms behind the abnormal pattern of lipid oxidation are linked to a common polymorphism of PPAR-γ coactivator 1 (a transcriptional regulator of genes responsible for mitochondrial biogenesis and fat oxidation), as shown in a Danish population [57] and in Pima Indians [58]. In addition, the expression of PPAR-γ coactivator 1 is reduced in the skeletal muscle of patients with type 2 diabetes [59, 60].

Heart P-MRS has proven to be an essential tool in the in  vivo study of cardiac high-­ energy phosphate (HEP) metabolism in humans. Image-guided spatially localized 31 P-MRS can now be routinely applied to examine anterior myocardial HEP metabolism in many stable patient populations, as recently reviewed by Neubauer [61] and as suggested by North American [62] and by European [63] authors. Phosphocreatine (PCr)/ATP, inorganic phosphorus (Pi)/ATP, and PCr/Pi ratios represent the phosphate potential (energy charge) of the myocardium and they are the most important indices of energy metabolism that can be detected with 31PMRS. In studies of the human heart, the PCr/ATP ratio is most often used as an indication of energy metabolism and of the phosphate potential (energy charge) of the myocardium [61, 64]. A reduced PCr/ATP ratio was found in vivo in humans with congenital cardiomegaly and other congenital cardiac muscular diseases, such as progressive muscular dystrophy, amyloidosis, and cardiac beriberi, as reviewed in [62]. Type 2 diabetes was shown to be associated with impaired LV energy metabolism, as was overweight/obesity. Cardiac energy metabolism is abnormal in patients with type 2 diabetes either without major cardiac dysfunction [65] or in the presence of diastolic dysfunction [66]. In addition, we reported that type 1 diabetic patients with end-stage renal failure had a similar pattern of abnormal LV energy metabolism; however, combined kidney and pancreas transplantation, curing both diabetes and renal failure, reverted this defect [67]. While these data support a major role of chronic hyperglycemia in inducing abnormal myocardial energy metabolism, the studies were performed in middle-age individuals (52–57 years old) in whom diabetes was diagnosed 1 [66] to 3 [65] years earlier or in patients with long-lasting type 1 diabetes [67]. Therefore, whether the alterations in cardiac energy metabolism were due to the hyperglycemic state itself or were secondary to the metabolic features characterizing the pre-diabetic state, before the onset of overt hyperglycemia, remains unresolved. In this respect, we recently showed that in non-diabetic, overweight/obese individuals cardiac HEP metabolism is depressed despite the lack of major cardiac dysfunction in the resting state [68], suggesting that the alteration 31

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is secondary not only to the effects of chronic hyperglycemia but also to other metabolic changes, such as insulin resistance. A confirmed link between abnormal cardiac energy metabolism in diabetes, on the one hand, and insulin resistance state and excessive FFA flow or excessive ectopic fat accumulation, on the other, has not, as stated above, been established. To pursue the hypothesis that the increased flow of FFA toward the heart has detrimental metabolic effects linked to their disposal via oxidative pathways we initiated a study of individuals with heart failure. The results showed that the prolonged (3 months) administration of a partial inhibitor of FFA oxidation (trimetazidine) improved the study patients’ functional class and their LV function. These effects were associated with an increase in the PCr/ATP ratio in the resting state, indicating the preservation of myocardial HEP levels [69]. Moreover, we were able to confirm the beneficial effects of inhibiting FFA oxidative disposal not only over the intermediate term but also within a few hours in patients with coronary disease. In these patients, the administration of trimetazidine 24 h before stress treadmill exercise testing (according to the Bruce protocol) after an 8-h fast, after a high-fat meal, or after a high-carbohydrate meal improved the time to 1-mm ST-segment depression (time to 1 mm) and the stress wall motion score index (WMSI) compared to patients taking placebo. Furthermore, this improvement was achieved regardless of the meal composition [70]. Taken together, these data suggest that the functional improvement and better LV energy homeostasis observed in these patients reflected the better glucose handling induced by the inhibition of FFA oxidative disposal. Also, the administration of perhexiline maleate, an antianginal drug that potently inhibits the mitochondrial FFA uptake enzymes carnitine palmitoyl transferase-1 (CPT-1) and CPT-2, thereby shifting substrate utilization from FFA toward glucose, improved VO2max, LV ejection fraction, symptoms, and resting and peak stress myocardial function in patients with heart failure [71]. A direct deleterious effect of FFA metabolism on cardiac energy metabolism is still controversial and deserves further investigation. For example, based on the same working hypothesis, other authors [72] performed the reverse experiment in patients with heart failure. In that study, acute administration of the lipolysis inhibitor Acipimox resulted in a depletion of circulating FFA.  Accordingly, an improvement in the parameters of myocardial efficiency (as measured by positron emission tomography and [15O]H2O, [11C]acetate, and [11C]palmitate administration) was expected. However, the myocardial efficiency of these patients deteriorated, suggesting that failing hearts are unexpectedly more dependent than healthy hearts on FFA availability. It was therefore proposed that both glucose and fatty acid oxidation are required for the optimal function of the failing heart.

Liver While defective FFA oxidative disposal may take place also in the liver, the data are controversial. We and others have recently shown that in patients with type 1 diabetes [73], in obese adolescents [32], and in lean patients with NAFLD [74] different

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patterns of whole-body lipid oxidation may be associated with the intra-hepatic fat content. We have also shown that habitual physical activity is associated with intra-­ hepatic fat content regardless of insulin resistance, suggesting an effect of exercise per se on hepatic lipid disposal [75]. These studies provide only indirect evidence because they were mostly performed using indirect calorimetry; thus, lipid oxidation reflected events taking place in the whole body rather than specifically in the liver. Liver-specific data were recently reported or are in the process of being evaluated and no conclusions have been drawn. Iozzo et al. reported that in patients with impaired glucose tolerance who have all the features of metabolic syndrome (overweight, high plasma triglyceride and low high-density lipoprotein cholesterol levels, hyperinsulinemia, insulin resistance, and a slight increase in blood pressure) the liver’s ability to extract FFA from the circulation is impaired [76]. Those authors speculated that in the fasting state beta-oxidation is the primary route for intracellular FFA utilization, with FFA uptake therefore dependent on the efficiency of this metabolic pathway. A consequence of this relationship is that defective liver FFA oxidative disposal is at the basis of the impaired liver FFA uptake in individuals with reduced glucose tolerance. Contrary to this finding, Misu et al. [77] recently reported that genes involved in mitochondrial oxidative phosphorylation (OXPHOS) are upregulated in the liver of patients with type 2 diabetes. This finding suggests that the regulation of OXPHOS genes in the liver of patients with type 2 diabetes, despite the presence of steatosis, is a mirror image of that in the skeletal muscle and heart of type 2 diabetics, in whom the genes involved in OXPHOS appear to be downregulated. It is possible that the liver compensates for steatosis by increasing fatty acid β-oxidation and activating OXPHOS even if the increased hepatic oxidative capacity is not enough to stop hepatic steatosis. Further studies are obviously necessary to elucidate the significance of oxidative fatty acids metabolism in the development of ectopic fat accumulation in the liver.

5 Conclusion Excessive ectopic fat accumulation may have direct relevance to the altered regulation of insulin-mediated metabolic pathways and the impaired function of different organs and tissues in diabetes and related diseases. An excessive FFA flux toward the peripheral tissues (skeletal muscle, heart, liver, and beta-cells) has been proposed as a pivotal event responsible for the high-level accumulation of intracellular triglycerides such as occurs in conditions of insulin resistance, i.e., obesity, type 2 diabetes, and metabolic syndrome. If this is indeed the case, then local acutely or chronically determined alterations of energy metabolism (oxidative phosphorylation and/or creatine phosphate and ATP metabolism) as well as oxidative FFA disposal also may be involved. So far, the spillover of FFA flux has yet to be confirmed; instead, efforts to treat fat-induced insulin resistance have focused on improving insulin sensitivity by reducing the levels of this hormone using, most commonly, thiazolidinediones, which are oral hypoglycemic agents. However, the

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demonstration of abnormalities of energy metabolism would offer new strategies to treat the deleterious metabolic effects of excessive ectopic fat accumulation. One approach would be to develop and implement novel therapeutic tools aimed at regulating the FFA oxidative potential of organs and tissues before the development of overt diabetes or functional alterations. Clearly, extensive research is still required in this field to fully understand what appears to be the heterogeneous behavior of the different organs and tissues under conditions of overt “lipotoxicity.”

References 1. Weyer C, Bogardus C, Mott DM, Pratley RE.  The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest. 1999;104:787–94. 2. De Fronzo RA.  The triumvirate beta-cell, muscle, live. A collusion responsible for NIDDM. Diabetes. 1988;37:667–87. 3. McGarry JD. What if Minkowski had been ageusic? An alternative angle on diabetes. Science. 1992;258:766–70. 4. McGarry JD.  Dysregulation of fatty acids metabolism in the etiology of type 2 diabetes. Banting lecture 2001. Diabetes. 2002;51:7–18. 5. Reaven GM. The fourth musketeer—from Alexandre Dumas to Claude Bernard. Diabetologia. 1995;38:3–13. 6. Perseghin G, Ghosh S, Gerow K, Shulman GI. Metabolic defects in lean nondiabetic offspring of NIDDM parents. A cross-sectional study. Diabetes. 1997;46:1001–9. 7. Roden M, Price TB, Perseghin G, et al. Mechanism of free fatty acid induced insulin resistance in humans. J Clin Invest. 1996;97:2859–286. 8. Shulman GI, Rothman DL, Jue T, Stein P, DeFronzo RA, Shulman RG. Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13C nuclear magnetic resonance spectroscopy. N Engl J Med. 1990;322:223–8. 9. Perseghin G, Price TB, Petersen KF, et al. Increased glucose transport/phosphorylation and muscle glycogen synthesis after exercise training in insulin resistant subjects. N Engl J Med. 1996;335:1357–62. 10. Krssak M, Falk Petersen K, Dresner A, et al. Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study. Diabetologia. 1999;42:113–6. 11. Perseghin G, Scifo P, De Cobelli F, et al. Intramyocellular triglyceride content is a determinant of in vivo insulin resistance in humans: a 1H-13C NMR spectroscopy assessment in offspring of type 2 diabetic parents. Diabetes. 1999;48:1600–6. 12. Jacob S, Machann J, Rett K, et  al. Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetic offspring of type 2 diabetic subjects. Diabetes. 1999;48:1113–9. 13. Perseghin G, Lattuada G, Danna M, et al. Insulin resistance, intramyocellular lipid content and plasma adiponectin concentrations in patients with type 1 diabetes. Am J Physiol Endocrinol Metab. 2003;285:E1174–81. 14. Greco AV, Mingrone G, Giancaterini A, et al. Insulin resistance in morbid obesità. Reversal with intramyocellular fat depletion. Diabetes. 2002;51:144–51. 15. Perseghin G, Scifo P, Pagliato E, et al. Gender factors affect fatty acids-induced insulin resistance in nonobese humans: effects of oral steroidal contraception. J Clin Endocrinol Metab. 2001;86:3188–96.

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16. Thamer C, Machann J, Bachmann O, et al. Intramyocellular lipids: anthropometric determinants and relationships with maximal aerobic capacity and insulin sensitivity. J Clin Endocrinol Metab. 2003;88:1785–91. 17. Perseghin G, Comola M, Scifo P, et al. Postabsorptive and insulin-stimulated energy and protein metabolism in patients with myotonic dystrophy type 1. Am J Clin Nutr. 2004;80:357–64. 18. Szczepaniak LS, Dobbins RL, Metzger GJ, et al. Myocardial triglycerides and systolic function in humans: in  vivo evaluation by localized proton spectroscopy and cardiac imaging. Magn Reson Med. 2003;49:417–23. 19. Reingold JS, McGavock JM, Kaka S, Tillery T, Victor RG, Szczepaniak LS. Determination of triglyceride in the human myocardium using magnetic resonance spectroscopy: reproducibility and sensitivity of the method. Am J Physiol Endocrinol Metab. 2005;289:E935–9. 20. McGavock JM, Victor RG, Unger RH, Szczepaniak LS. Adiposity of the heart, revisited. Ann Intern Med. 2006;144:517–24. 21. Kankaanpaa M, Lehto H-R, Parkka JP, et al. Myocardial triglyceride content and epicardial fat mass in human obesity: relationship to left ventricular function and serum free fatty acid levels. J Clin Endocrinol Metab. 2006;91:4689–95. 22. Sharma S, Adrogue JV, Golfman L, et  al. Intramyocardial lipid accumulation in the failing human heart resembles the lipotoxic rat heart. FASEB J. 2004;18:1692–700. 23. Marchington JM, Mattacks CA, Pond CM. Adipose tissue in the mammalian heart and pericardium: structure, foetal development and biochemical properties. Comp Biochem Physiol B. 1989;94:225–32. 24. Iacobellis G, Corradi D, Sharma AM. Epicardial adipose tissue: anatomic, biomolecular and clinical relationships with the heart. Nat Clin Pract Cardiovasc Med. 2005;2:536–43. 25. Marchesini G, Brizi M, Bianchi G, et  al. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes. 2001;50:1844–50. 26. Kelley DE, McKolanis TM, Hegazi RA, Kuller LH, Kalhan SC. Fatty liver in type 2 diabetes mellitus: relation to regional adiposity, fatty acids, and insulin resistance. Am J Physiol Endocrinol Metab. 2003;285:E906–16. 27. Marchesini G, Bugianesi E, Forlani G, et al. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology. 2003;37:917–23. 28. Dietrich P, Hellerbrand C. Non-alcoholic fatty liver disease, obesity and the metabolic syndrome. Best Pract Res Clin Gastroenterol. 2014;28:637–53. 29. Sheka AC, Adeyi O, Thompson J, et  al. Nonalcoholic steatohepatitis: a review. JAMA. 2020;2020(323):1175–83. 30. Petersen KF, Dufour S, Befroy D, Lehrke M, Hendler RE, Shulman GI. Reversal of nonalcoholic hepatic steatosis, hepatic insulin resistance, and hyperglycaemia by moderate weight reduction in patients with type 2 diabetes. Diabetes. 2005;54:603–8. 31. Seppala-Lindroos A, Vehkavaara S, Hakkinen A-M, et al. Fat accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free fatty acids independent of obesity an normal men. J Clin Endocrinol Metab. 2002;87:3023–8. 32. Perseghin G, Bonfanti R, Magni S, et al. Insulin resistance and whole body energy homeostasis in obese adolescents with fatty liver disease. Am J Physiol Endocrinol Metab. 2006;291:E697–703. 33. Brechtel K, Dahl DB, Machann J, et al. Fast elevation of the intramyocellular lipid content in the presence of circulating free fatty acids and hyperinsulinemia: a dynamic 1HMRS study. Magn Reson Med. 2001;45:179–83. 34. Bachmann OP, Dahl DB, Brechtel K, et al. Effects of intravenous and dietary lipid challenge on intramyocellular lipid content and the relation with insulin sensitivity in humans. Diabetes. 2001;50:2579–84. 35. Poynten AM, Gan SK, Kriketos AD, et al. Nicotinic acid-induced insulin resistance is related to increased circulating fatty acids and fat oxidation but not muscle lipid content. Metabolism. 2003;52:699–704.

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36. Belfort R, Harrison SA, Brown K, et al. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med. 2006;355:2297–307. 37. Holt HB, Wild SH, Wood PJ, et al. Non-esterified fatty acid concentrations are independently associated with hepatic steatosis in obese subjects. Diabetologia. 2006;49:141–8. 38. Donnelly KL, Smith CI, Schwarzberg SJ, et  al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 2005;115:1343–61. 39. Diraison F, Moulin P, Beylot M. Contribution of hepatic de novo lipogenesis and reesterification of plasma non esterified fatty acids to plasma triglyceride synthesis during nonalcoholic fatty liver disease. Diabetes Metab. 2003;29:478–85. 40. Utzschneider KM, Kahn SE. The role of insulin resistance in nonalcoholic fatty liver disease. J Clin Endocrinol Metab. 2006;91:4753–61. 41. Coldberg SR, Simoneau JA, Thaete FL, Kelley DE. Skeletal muscle utilization of free fatty acids in women with visceral obesity. J Clin Invest. 1995;95:1846–53. 42. Kelley DE, Goodpaster B, Wing RR, Simoneau J-A. Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity and weight loss. Am J Physiol Endocrinol Metab. 1999;277:E1130–41. 43. Blaak EE, Wagenmakers AJM, Glatz JFC, et al. Plasma FFA utilization and fatty acid binding protein content are diminished in type 2 diabetic muscle. Am J Physiol Endocrinol Metab. 2000;279:E146–54. 44. Blaak EE, Wolffenbuttel BH, Saris WH, Pelsers MM, Wagenmakers AJ.  Weight reduction and the impaired plasma-derived free fatty acid oxidation in type 2 diabetic subjects. J Clin Endocrinol Metab. 2001;86:1638–44. 45. Mensink M, Blaak EE, van Baak MA, Wagenmakers AJ, Saris WH.  Plasma free fatty acid uptake and oxidation are already diminished in subjects at high risk for developing type 2 diabetes. Diabetes. 2001;50:2548–54. 46. Luzi L, Perseghin G, Tambussi G, et al. Intramyocellular lipid accumulation and reduced whole body lipid oxidation in HIV infected patients with lipodystrophy. Am J Physiol Endocrinol Metab. 2003;284:E274–80. 47. Perseghin G, Scifo P, Danna M, et al. Normal insulin sensitivity and IMCL content in overweight humans are associated with higher fasting lipid oxidation. Am J Physiol Endocrinol Metab. 2002;283:E556–64. 48. Goodpaster BH, Katsiaras A, Kelley DE.  Enhanced fat oxidation through physical activity is associated with improvements in insulin sensitivity in obesity. Diabetes. 2003;52:2191–7. 49. Gan SK, Kriketos AD, Ellis BA, Thompson CH, Kraegen EW, Chisholm DJ. Changes in aerobic capacity and visceral fat but not myocyte lipid levels predict increased insulin action after exercise in overweight and obese men. Diabetes Care. 2003;26:1706–13. 50. Lattuada G, Costantino F, Caumo A, et al. Reduced whole body lipid oxidation is associated with insulin resistance but not with intramyocellular lipid content in offspring of type 2 diabetic patients. Diabetologia. 2005;48:741–7. 51. Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI. Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med. 2004;350:664–7. 52. He J, Watkins S, Kelley DE. Skeletal muscle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity. Diabetes. 2001;50:817–23. 53. Gaster M, Rustan AC, Aas V, Beck-Nielsen H. Reduced lipid oxidation in skeletal muscle from type 2 diabetic subjects may be of genetic origin. Evidence from cultured myotubes. Diabetes. 2004;53:542–8. 54. Kelley DE, He J, Menshikova EV, Ritov VB. Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes. 2002;51:2944–50. 55. Petersen KF, Befroy D, Dufour S, et al. Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science. 2003;300:1140–2. 56. Schrauwen P, Hesselink MKC. Oxidative capacity, lipotoxicity, and mitochondrial damage in type 2 diabetes. Diabetes. 2004;53:1412–7.

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57. Ek J, Andersen G, Urhammer SA, et al. Mutation analysis of peroxisome proliferator activated receptor-gamma coactivator-1 (PGC-1) and relationships of identified amino acid polymorphisms to type II diabetes mellitus. Diabetologia. 2001;4:2220–6. 58. Muller YL, Bogardus C, Beamer BA, Shuldiner AR, Baier LJ.  A functional variant in the peroxisome proliferator-activated receptor gamma2 promoter is associated with predictors of obesity and type 2 diabetes in Pima Indians. Diabetes. 2003;52:1864–71. 59. Mootha VK, Lindgren CM, Eriksson KF, et  al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34:267–73. 60. Patti ME, Butte AJ, Crunkhorn S, et al. Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: potential role of PGC1 and NRF1. Proc Natl Acad Sci U S A. 2003;100:8466–71. 61. Neubauer S. Mechanisms of disease: the failing heart—an engine out of fuel. N Engl J Med. 2007;356:1140–51. 62. Bottomley PA. MR spectroscopy or the human heart: the status and the challenges. Radiology. 1994;191:593–612. 63. Beyerbacht HP, Vliegen HV, Lamb HJ, et  al. Phosphorus magnetic resonance spectroscopy of the human heart: current status and clinical implications. Eur Heart J. 1996;17:1158–66. 64. Forder JR, Pohost GM. Cardiovascular nuclear magnetic resonance: basic and clinical applications. J Clin Invest. 2003;111:1630–9. 65. Scheuermann-Freestone M, Madsen PL, Manners D, et  al. Abnormal cardiac and skeletal muscle energy metabolism in patients with type 2 diabetes. Circulation. 2003;107:3040–6. 66. Diamant M, Lamb HJ, Groeneveld Y, et al. Diastolic dysfunction is associated with altered myocardial metabolism in asymptomatic normotensive patients with well-controlled type 2 diabetes mellitus. J Am Coll Cardiol. 2003;42:328–35. 67. Perseghin G, Fiorina P, De Cobelli F, et al. Cross-sectional assessment of the effect of kidney and kidney-pancreas transplantation on resting left ventricular energy metabolism in type 1 diabetic-uremic patients: a 31P-MRS study. J Am Coll Cardiol. 2005;46:1085–92. 68. Perseghin G, Ntali G, De Cobelli F, et  al. Abnormal left ventricular energy metabolism in obese men with preserved systolic and diastolic functions is associated with insulin resistance. Diabetes Care. 2007;30:1520–7. 69. Fragasso G, Perseghin G, De Cobelli F, et al. Effects of metabolic modulation by trimetazidine on left ventricular function and phosphocreatine/adenosine triphosphate ratio in patients with heart failure. Eur Heart J. 2006;27:942–8. 70. Fragasso G, Montano C, Perseghin G, et al. Reduction of ischemic threshold in patients with stable coronary disease after meals of different composition: effects of partial inhibition of fatty acids oxidation. Am Heart J. 2006;151:1238.e1–8. 71. Lee L, Campbell R, Scheuermann-Freestone M, et al. Metabolic modulation with perhexiline in chronic heart failure. A randomized, controlled trial of short-term use of a novel treatment. Circulation. 2005;112:3280–8. 72. Tuunanen H, Engblom E, Naum A, et al. Free fatty acid depletion acutely decreases cardiac work and efficiency in cardiomyopathic heart failure. Circulation. 2006;114:2130–7. 73. Perseghin G, Lattuada G, De Cobelli F, et  al. Reduced intra-hepatic fat content is associated with increased whole body lipid oxidation in patients with type 1 diabetes. Diabetologia. 2005;48:2615–21. 74. Bugianesi E, Gastaldelli A, Vanni E, et al. Insulin resistance in non-diabetic patients with non-­ alcoholic fatty liver disease: sites and mechanisms. Diabetologia. 2005;48:634–42. 75. Perseghin G, Lattuada G, De Cobelli F, Ragogna F, Ntali G, Esposito A, Belloni E, Canu T, Terruzzi I, Scifo P, Del Maschio A, Luzi L. Habitual physical activity is associated with the intra-hepatic fat content in humans. Diabetes Care. 2007;30:683–8. 76. Iozzo P, Turpeinen AK, Takala T, et al. Defective liver disposal of free fatty acids in patients with impaired glucose tolerance. J Clin Endocrinol Metab. 2004;89:3496–502. 77. Misu H, Takamura T, Matsuzawa N, et al. Genes involved in oxidative phosphorylation are coordinately upregulated with fasting hyperglycaemia in livers of patients with type 2 diabetes. Diabetologia. 2007;50:268–77.

Muscle Biopsy to Investigate Mitochondrial Turnover Rocco Barazzoni

1 Skeletal Muscle Biopsy Skeletal muscle biopsy is a long-established diagnostic used primarily as a diagnostic tool for neuromuscular disorders characterized by reduced muscle function and strength. For anatomical and functional characteristics, leg muscles and especially the vastus lateralis have been most commonly investigated. Percutaneous needles, which overcame the more invasive open biopsies, were introduced more than 50 years ago, with the original instruments named after Bergstrom [1], in honor of his pioneering work in their development (Fig. 1). When adequate suction is applied and a sufficient amount of muscle tissue is recovered, muscle biopsy allows for multiple measurements as well as the assessment of different anatomical or physiological parameters. Fiber and cell isolation, incubation, or culture are also possible and enable additional ex vivo studies. Muscular dystrophies, mitochondrial myopathies, and conditions often characterized by impaired muscle strength and function were early and obvious targets for diagnostic and research applications of muscle biopsy. Needle biopsy has been further extensively applied in the study of exercise physiology and pathophysiology, with the goal of investigating the regulation of mitochondrial function and substrate oxidation. In recent years, studies in the fields of obesity and diabetes have also focused on muscle mitochondrial function, and muscle biopsies have become increasingly common in human metabolic assessments. This chapter provides an overview of mitochondrial function and substrate utilization while addressing the major concepts emerging from basic studies on mitochondrial regulation. Available data from human skeletal muscle biopsy studies are reviewed, with particular emphasis placed on the effects of nutritional state, diet, and exercise and their potential interactions with insulin resistance and disease state. R. Barazzoni (*) Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy e-mail: [email protected]; [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 L. Luzi (ed.), Cellular Physiology and Metabolism of Physical Exercise, https://doi.org/10.1007/978-3-031-27192-2_7

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Fig. 1  Skeletal muscle biopsy needles

2 Skeletal Muscle Function and Mitochondria Skeletal muscle is an essential component of the locomotive apparatus. An adequate energy supply in the form of adenosine triphosphate (ATP) is required for exercise performance as well as the maintenance and renewal of tissue contractile proteins. Indeed, skeletal muscle is a prominent contributor to basal metabolic rate, which reflects the resting metabolic needs of the body, and its contribution to energy expenditure rises in proportion to physical activity. Skeletal muscle also represents the major protein reservoir, with tissue protein balance regulated by nutritional and endocrine signals that maintain body protein and amino acid homeostasis [2–4]. Besides contraction and exercise, amino acid and protein turnover (i.e., the continuing processes of protein renewal through breakdown and synthesis) also represent major components of muscle energy requirements. ATP cannot be stored in tissues; rather, muscle energy stores are limited to high-­ energy bonds in phosphocreatine, which, however, cannot sustain prolonged exercise without continuous ATP production in the contracting muscle. ATP is provided by anaerobic glycolysis in relatively small amounts and mostly by oxidative phosphorylation in tissue mitochondria, the key site of tissue oxygen consumption for energy production. The concept that mitochondrial function is crucial for muscle contraction is supported by the observation that differences in mitochondrial density largely determine the ability of different muscle groups to sustain exercise and prevent fatigue. Mitochondria contain DNA molecules encoding a minority of mitochondrial genes that are transcribed and translated into proteins in the organelle. Coordinated mitochondrial and nuclear DNA gene expression is therefore necessary and crucial for mitochondrial biogenesis, i.e., the synthesis of new organelles, whose regulation is critical for energy supply. Studies of muscle mitochondrial function and turnover have traditionally focused on the pathophysiology of exercise and on neuromuscular congenital and acquired diseases involving the loss of muscle function and strength, including the aging process. In the last 15  years there has been increasing awareness that alterations in mitochondrial function and substrate oxidation are associated with metabolic disturbances in obesity, insulin resistance, and type 2 diabetes, thereby opening novel and exciting fields for mitochondrial research.

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3 Mitochondrial Glucose and Fatty Acid Oxidation Exercising and resting skeletal muscle may utilize both glucose and lipid substrates for energy production. Indeed, the energy demands of skeletal muscle are an important component of both glucose and lipid whole-body metabolism and disposal. Glucose as well as fatty acid catabolism leads to the synthesis of acetyl-coenzyme A (CoA), which enters the mitochondrial tricarboxylic acid (TCA) or Krebs cycle. Glucose utilization begins with anaerobic glycolysis, whereby pyruvate formation is associated with the production of limited amounts of ATP. Glucose-derived pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenase (PDH), thereby linking anaerobic and aerobic glucose metabolism. Free fatty acids are transported by carnitine palmitoyl transferase-I (CPT-I) to the mitochondria, where they are entirely catabolized to acetylCoA. Mitochondrial regulation of the balance between glucose and fatty acid utilization is a key process involving substrate availability and hormonal modulation [5, 6]. High glucose availability and PDH activation result in higher glucose utilization, with the relative suppression of fat oxidation [5, 6]. Insulin elevation as observed following a glucose meal contributes to this process by stimulating the PDH inactivator pyruvate dehydrogenase kinase (PDK) [6], with further stimulation of oxidative glucose disposal. By contrast, an enhanced fatty acid supply (as observed following a fatty meal) may result in PDH suppression [5]. Consequently, fatty acid elevation has been reported to induce resistance to insulin-mediated PDK activation [6], thereby shifting energy metabolism toward fat oxidation. Acetyl-CoA and oxaloacetate are used to synthesize citrate, the first substrate of the TCA cycle in the mitochondrial matrix. TCA cycle reactions provide reduced FAD (flavin adenine dinucleotide) and NAD (nicotinamide adenine dinucleotide) for electron flux through the respiratory chain. Respiratory chain enzymes (complexes I–IV) are located in the inner mitochondrial membrane, where they transport electrons to oxygen as the final acceptor while creating an electrochemical transmembrane proton gradient. The gradient is utilized by ATP synthase (complex V of the respiratory chain) to synthesize ATP from ADP and phosphate, providing chemical energy in the form of high-energy bonds.

4 Regulation of Mitochondrial Oxidative Metabolism Energy Status ATP consumption leads to adenosine di- and mono-phosphate (ADP and AMP) production. The ratios between ADP and AMP concentrations and the ATP concentration are sensitive markers of tissue energy state and they play an important role in the regulation of mitochondrial oxidative metabolism (Fig. 2). In the presence of low energy availability, a higher ADP and AMP/ATP ratio enhances mitochondrial function and ultimately potentially restores energy homeostasis. In full agreement with this concept, AMP-activated protein kinase (AMPK) has emerged in the last decade as a paradigm for energy sensing, mediating multiple responses aimed at

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Fig. 2  AMP-activated protein kinase (AMPK) activation favors changes in energy metabolism involving mitochondrial biogenesis and enhanced ATP production

lowering energy expenditure while enhancing ATP production to restore energy balance. An increase in tissue AMP concentration and the AMP/ATP ratio leads to AMPK activation through mechanisms that include direct allosteric modification by AMP, which favors protein kinase-mediated activating phosphorylation [7, 8]. AMPK-activated mitochondrial biogenesis involves phosphorylation of transcription factors that coordinate the expression of mitochondrial and nuclear genes such as peroxisome proliferator-activated receptor gamma-coactivator 1α (PGC-1α) [7, 8]. In recent years, the network of signaling molecules involved in AMPK-mediated activation of mitochondrial biogenesis has extended to sirtuins, especially SIRT1 [9–11]. These deacetylating enzymes were originally described as mediators of the positive effects of caloric restriction on energy metabolism (see below), and acetylated PGC1α is also a key SIRT1 target [9–11]. Coordinated AMPK effects are also aimed at enhancing substrate availability through stimulation of glucose uptake, fatty acid uptake, and fatty acid oxidative metabolism through the inhibition of acetyl-­CoA utilization for lipogenesis [7, 8]. Physical exercise and altered nutrient availability are among the major conditions involving physiological adaptive changes in AMPK activation to modulate mitochondrial energy production.

Exercise Exercise is one key area for the study of skeletal muscle mitochondria. Metabolic changes induced by exercise are discussed in detail elsewhere in this textbook. Overall, contraction-induced phosphocreatine and ATP consumption is a major cause for the activation of coordinated signals to enhance mitochondrial function. As outlined above,

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one metabolic signal of substrate and energy depletion is indeed muscle AMPK activation, and its coordinated effects on SIRT1 and PGC1α have been confirmed also following exercise in human and experimental models [12, 13]. A specific association with tissue metabolic needs is supported by elegant studies in which AMPK activation was measured during exercise protocols performed at different muscle glycogen levels [14]. Under the above conditions, AMPK activation was strongly enhanced in the presence of low glycogen stores while maintenance of an adequate glycogen supply prevented substantial AMPK phosphorylation [14]. At variance with AMPK, calcium-dependent signaling represents a paradigm of contraction-­associated signaling for mitochondrial biogenesis. Following contraction-­induced calcium release from the sarcoplasmic reticulum (12), calcium increments activate mitochondrial biogenesis through pathways involving protein kinase C and Ca-calmodulin-activated protein kinase [15] while also leading to transcriptional activation of PGC-1α, playing a pivotal role in orchestrating coordinated nuclear and mitochondrial gene expression [9, 16].

Nutrition Caloric Restriction A moderate balanced lowering of caloric intake leads to major health benefits and is associated with prolonged lifespan in several animal species, with recent reports supporting this effect also in non-human primates [17, 18]. From a metabolic standpoint, fasting is associated with a shift toward muscle fatty acid oxidative utilization [5], which is in part mediated by reduced PDH activity. Caloric restriction may enhance muscle mitochondrial oxidative capacity in experimental models in young-­ adult and in aging rodents, particularly in the presence of selective dietary protein supplementation [19–21]. Indeed, enhanced mitochondrial function through the prevention of oxidative damage also has been suggested as a key mediator of the life-prolonging effects of caloric restriction in non-obese animals [22]. In recent years, sirtuins including SIRT1 have rapidly been linked to the life-prolonging effects of caloric restriction in mammals; their effects involve PGC1α activating deacetylation [9–11, 23]. SIRT1 overexpression in non-calorie restricted models is also associated with enhanced muscle mitochondrial oxidative capacity and lower tissue lipid content, with an improved metabolic profile also in the presence of high fat-calorie intake [9, 10]. These combined findings indicate that changes in muscle energy metabolism are pivotal components of the metabolic response to moderate caloric restriction and likely major contributors to the health beneficial effects. Fat and Glucose Substrates The consequences of high glucose and fat availability on muscle mitochondrial function and turnover are controversial and some of the existing discrepancies could be due to experimental design in different investigations (in vitro compared to in  vivo studies, duration of nutritional treatment, diet type and composition).

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High-­fat and high-calorie feeding are indeed well-established models to study nutritional regulation of mitochondrial function, but it should be pointed out that in vivo studies of chronic dietary manipulation lead to major changes in body weight, body fat, metabolic and hormonal patterns that parallel the changes in substrate availability per se and could thus contribute to the observed alterations. Fatty Acids Several in vitro studies using muscle cell preparations including C2C12 myotubes or primary cultures from skeletal muscle cells have found a negative impact of free fatty acids on the expression levels and function of several mitochondrial genes, as reflected by mitochondrial DNA, enzyme activities, and ATP production [24–26]. The saturated fatty acid palmitate has been mostly, although not exclusively, used in experimental protocols and it appears that differential, less negative effects may be observed with unsaturated fatty acids [27]. In one study, however, the enhancement of AMPK activation despite the concomitant impairment of measured mitochondrial parameters was reported [25]. In addition, fatty acids were shown to enhance transcriptional expression of the muscle isoform of the rate-limiting enzyme for fatty acid oxidation (CPT-I) in cultured cardiomyocytes [28]. The above observations are consistent with potential stimulatory effects by fatty acids on muscle mitochondrial biogenesis and their own oxidative metabolism. In vivo reports on the impact of substrate availability on muscle mitochondrial function in experimental models provide the most controversial results. Studies on excess dietary fat are mostly based on high-fat feeding for several weeks, leading to diet-induced obesity. Short-term studies are also available that avoid the impact of substantial changes in body weight and fat content, although they also prevent the onset of potential adaptive metabolic muscle changes. A negative impact of high-fat feeding on several mitochondrial parameters, with particular regard to mitochondrial enzyme activities and oxygen consumption, has been observed in some studies [29, 30]. Additional investigations in rodent models, however, reported mitochondrial stimulatory effects of dietary fat at the level of protein content and oxidative capacity [31, 32]. A positive role of free fatty acids was specifically confirmed in one paper in which free fatty acid elevation was induced by heparin treatment in the context of fat-induced obesity [32]. A potential stimulatory effect of fatty acids on mitochondrial biogenesis could also be mediated through activation of peroxisome proliferator-activated receptor δ (PPARδ) which can in turn enhance PGC1α expression at a posttranscriptional level [12]. Short-term treatment with high-fat diets, also in humans, leads to a negative modulation of muscle PDH activity through stimulation of PDH kinase [33]. This effect indicates one potential mechanism whereby enhancement of fat oxidative utilization may be associated with insulin resistance through substrate competition and impaired glucose oxidation.

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Glucose The direct impact of hyperglycemia on mitochondrial parameters in muscle cell preparations has been less extensively studied. Available reports suggest a potential negative impact of sustained glucose elevations on substrate oxidation and mitochondrial functions in both skeletal and cardiac muscle preparations [34, 35]. With regard to glucose levels, diabetic models are often associated with skeletal muscle mitochondrial dysfunction. As stated above, it is difficult to dissect the potential role of glucose per se from that of concomitant profound hormonal and metabolic disturbances. One interesting observation from the insulin-deprived, markedly hyperglycemic streptozotocin-diabetic rodent model reported a lack of muscle mitochondrial dysfunction in untreated insulinopenic animals [36]. A seemingly paradoxical impairment of mitochondrial function following glucose lowering by insulin replacement was in turn reported, thereby confirming the complexity of metabolic interactions in these models [36]. Substrate-Induced Metabolic Alterations with Mitochondrial Impact Altered nutritional state from imbalanced dietary substrate intake is associated with profound metabolic alterations that could contribute to modulate skeletal muscle mitochondrial function. Excess substrate availability may enhance systemic and muscle oxidative stress and inflammation. Their potential interactions with muscle mitochondria are outlined below. Oxidative Stress Production of reactive oxygen species (ROS) from incompletely reduced oxygen molecules is inevitably associated with oxidative substrate metabolism. Antioxidant systems eliminate ROS and maintain their tissue concentrations within physiological levels. Excess ROS production may however overcome antioxidant capacity, thereby leading to oxidative stress, with damage to cell and tissue molecules, and potential disease. Importantly, oxidative stress has been long postulated to cause aging through mitochondrial damage, in the mitochondrial or oxidative theory of aging [22]. Recent studies have demonstrated that exposure to high levels of fat substrates (fatty acids in vitro and high-fat or high-calorie feeding in vivo) enhances ROS production in muscle cell preparations [37] or in muscle tissue [38, 39]. In one paper [38] diet-induced oxidative stress in skeletal muscle was directly reported to cause tissue mitochondrial dysfunction, suggesting that excess lipid substrates induce mitochondrial alterations at least in part by altering tissue redox state. Chronic and acute glucose elevation has been also reported to induce oxidative stress, with a relevant role in the onset of diabetic complications [40] and a potential negative impact on tissue energy metabolism.

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Inflammation Both local and systemic inflammation result from imbalanced production of proinflammatory and anti-inflammatory cytokines. While acute inflammation represents an adaptive mechanism contributing to limit and reverse specific infectious or traumatic insults, sustained activation of a systemic or local inflammatory response is associated with negative metabolic consequences. Negative effects of chronic proinflammatory changes in skeletal muscle include insulin resistance through IKKβ activation and direct inhibition of insulin signaling as well as activation of NFkB nuclear translocation [41]. Importantly, oxidative stress may also amplify inflammation by enhancing proinflammatory cytokine production and by activating NFkB in peripheral tissues, including skeletal muscle [42, 43]. Fatty acids have been shown to enhance proinflammatory cytokine production in muscle cell preparations in vitro [44]. In addition, fat-induced insulin resistance at the whole-body and skeletal muscle levels is acutely prevented by the IKKβ inhibitor salicylate [45]. Direct effects of glucose on skeletal muscle inflammation remain less completely defined, although glucose-induced pro-oxidant changes have the potential to activate inflammation also in muscle tissue. Recent studies have directly shown the potential for muscle inflammation and proinflammatory cytokine elevation to induce tissue mitochondrial dysfunction [46]. Based on the above observations, it appears plausible that muscle mitochondrial effects of glucose and fatty acids are both direct and indirect. Indirect effects may include a negative impact on mitochondrial function through enhanced oxidative stress and inflammation. Different combinations of direct and indirect effects related to treatment duration, as well as experimental model and design may explain, at least in part, the controversial results in available literature. Also, importantly, it cannot be excluded that adaptive changes in vivo, possibly involving activation of antioxidant defense systems, may prevent or delay the onset of detrimental mitochondrial effects of diet and substrates in selected experimental settings.

5 Mitochondrial Function and Turnover in Human Skeletal Muscle Exercise Aerobic exercise has been almost invariably reported to enhance muscle mitochondrial oxidative capacity and to activate mitochondrial biogenesis in humans. Several more recent studies have confirmed the role of PDH kinase in the regulation of substrate selection and fat in glucose-derived acetyl-CoA utilization [47]. The regulatory network of AMPK and SIRT1 activation has been fully confirmed also in human skeletal muscle as a key regulator of exercise-induced mitochondrial biogenesis involving PGC1α activation [13, 48–50]. Besides the sustained effect of regular

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training, acute bouts of exercise have been also shown to enhance PGC1α expression and activity [49, 50], indicating its involvement also in the short-term regulation of mitochondrial energy metabolism. It is important to point out that the positive impact of aerobic exercise on muscle mitochondrial biogenesis and oxidative capacity has been confirmed under several pathophysiological conditions characterized by altered aerobic capacity and impaired mitochondrial function. These groups include obese, insulin-resistant, and type 2 diabetic patients, aging subjects and patients with chronic wasting diseases and loss of lean muscle mass. These effects will be discussed below.

Obesity and Insulin Resistance In the last decade, it has become clear that altered muscle lipid metabolism plays a key role in the onset of insulin resistance in obesity and type 2 diabetes [51]. Muscle lipid accumulation may lead to impaired insulin signaling, with reported involvement of diglycerides and ceramides [52], whereas the potential direct role of triglyceride accumulation is still under debate [53]. It has been hypothesized that altered mitochondrial function contributes to lipid accumulation and thereby to insulin resistance in obese and diabetic skeletal muscle [54]. The hypothesis is plausible also in the light of the favorable mitochondrial effects reported from experimental studies for caloric restriction and exercise training, i.e., the two cornerstones of lifestyle changes recommended as treatment of obesity and type 2 diabetes. In the following sections, the in vivo evidence obtained through muscle biopsy studies in favor and against a causal role of muscle mitochondrial dysfunction in the onset of insulin resistance are summarized. Mitochondrial Function in Obese and Insulin-Resistant Patients An association between obesity, insulin resistance, and skeletal muscle mitochondrial dysfunction has been frequently reported in the last decade. Several muscle biopsy studies in Caucasian obese, insulin-resistant or type 2 diabetic patients have reported several abnormalities of skeletal muscle mitochondria ranging from mitochondrial DNA reduction to low transcript and protein levels, low enzyme activities, and altered ATP production [55–61]. Impaired mitochondrial oxidative capacity is often associated with triglyceride accumulation [53, 54]. Relevant discrepancies persist on the presence of intrinsic mitochondrial alterations as opposed to reduction of mitochondrial content with little or no impairment of mitochondrial function per se, reported in several papers [55–61]. Reduced expression of muscle mitochondrial biogenesis regulators such as PGC1α has been also reported along with functional abnormalities in first degree relatives and offspring of diabetic individuals [62].

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 itochondrial Effects of Diet and Exercise in Obese M and Insulin-Resistant Patients Substrate Availability: Acute Not many studies of acute nutritional intervention with changes in substrate availability are available for obese or insulin-resistant humans. Acute infusion of lipids to enhance free fatty acid concentration has been shown to impair the transcriptional expression of mitochondrial genes in healthy volunteers [63], and this effect was interestingly associated with the stimulation of inflammatory gene expression [63]. Conversely, short-term reduction of circulating free fatty acid concentration following administration of the inhibitor of lipolysis acipimox resulted in a paradoxical decrease in PGC1α and energy metabolism gene expression in insulin-resistant subjects [64]. In agreement with these findings, lack of negative effects on muscle mitochondrial ATP production and membrane potential was reported in another study in healthy humans following acute fatty acid elevation [65]. Negative effects have been reported for acute marked hyperglycemia on mitochondrial respiration in type 2 diabetic patients [66], although sustained changes in glucose control did not appear to affect muscle mitochondrial function in the same report and in other studies on type 2 diabetic muscle [66]. Dietary Treatment Long-term studies of controlled dietary manipulation are difficult in humans and muscle biopsy studies of mitochondrial function following sustained low-calorie dietary treatment in obese and insulin-resistant patients are scarce. Data from selected groups of healthy individuals voluntarily undergoing long-term strict hypocaloric dietary regimens have confirmed its substantial positive impact on several cardiometabolic risk factors including circulating lipids and systemic inflammation markers [18]. The potential positive impact on muscle energy metabolism has however not been measured under these conditions. Overall, evidence indicates that weight loss obtained in the absence of exercise training over a period of several weeks to a few months does not substantially stimulate muscle mitochondrial energy metabolism [67] despite concomitant increments in insulin sensitivity. Exercise Muscle mitochondrial effects of aerobic exercise training for usually 12–16 weeks have been extensively investigated in humans and the beneficial impact has been confirmed by most studies also in human obesity and insulin resistance [68–71]. Aerobic training was indeed reported to enhance mitochondrial enzyme activities, respiration, and lipid oxidative capacity [68–71] also in obese insulin-resistant

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patients, and studies further demonstrated a positive impact of exercise training with dietary intervention on fatigue and aerobic performance in diabetic patients [72, 73]. Mitochondrial Effects of Insulin A breakthrough in the understanding of the association between muscle mitochondrial dysfunction and insulin resistance came with the demonstration of a stimulatory effect of insulin on muscle mitochondrial gene expression, protein synthesis, and ATP production in healthy humans [74]. The above report introduced the concept that the association between muscle mitochondrial dysfunction and insulin resistance could be bidirectional, and insulin resistance could primarily contribute to impair mitochondrial function. This hypothesis was initially supported by the concomitant observation that acute insulin effects on mitochondria were abolished in insulin-resistant type 2 diabetic patients [74] and by additional studies in type 2 diabetic and non-diabetic patients following acute changes in plasma insulin concentration [75]. A recent study in healthy subjects reported muscle mitochondrial dysfunction after 3 days of fasting that was attributed by the authors to acute muscle insulin resistance due to high fatty acid mobilization and availability [76]. Mitochondria and Insulin Resistance: Cause or Effect? The association between low muscle mitochondrial oxidative capacity and obesity insulin resistance is well established in humans. The potential underlying cause– effect relationships remain however to be completely understood. A role of mitochondrial dysfunction to impair insulin signaling could be postulated through reduced lipid oxidation and the negative metabolic impact of tissue lipid accumulation. This hypothesis is appealing but it has been seriously challenged by the lack of parallel changes in mitochondrial function and insulin sensitivity under several acute and chronic experimental conditions. In particular, enhancement of mitochondrial function may not be associated with improved insulin sensitivity following aerobic exercise training [71, 77]. Lack of mitochondrial stimulation was conversely reported in the presence of higher insulin action following diet-induced weight loss in obese patients [67]. Acute insulin resistance following systemic free fatty acid elevation was, conversely, not associated with muscle mitochondrial changes in oxidative capacity and ATP production [65]. One important observation came from the study of type 2 diabetic patients of Indian origin, who exhibit insulin resistance in the presence of preserved or even enhanced muscle mitochondrial function, DNA copy number, and protein levels [78]. The above study introduced the concept that genetic background may profoundly alter the interaction between mitochondrial function and insulin action in humans, and it directly argued against a primary role of muscle mitochondrial dysfunction in the onset of insulin resistance. Also, importantly, overexpression of PGC1α in skeletal muscle in genetic models resulted in enhanced tissue mitochondrial density but was associated with a negative rather

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Fig. 3 Potential interactions between skeletal muscle mitochondrial dysfunction and insulin resistance and the putative role of oxidative stress and inflammation

than a positive impact on glucose metabolism particularly during high-fat feeding [79]. Based on available knowledge, it is therefore highly unlikely that mitochondrial dysfunction per se primarily and independently causes insulin resistance. Its onset could nonetheless cause a metabolic vicious cycle by worsening lipid utilization, and improvement of mitochondrial lipid oxidative capacity remains a potential target for insulin-sensitizing therapeutic strategies. The possibility that insulin resistance contributes directly to impair muscle mitochondrial oxidative capacity also needs to be considered and may at least in part explain their association. Finally, it must be pointed out that obese and insulin-resistant patients often exhibit systemic and muscle oxidative stress and inflammation [80, 81]. Since both alterations have been reported to cause insulin resistance and mitochondrial dysfunction, it is well possible that their association in skeletal muscle reflects a common pro-oxidant and proinflammatory metabolic milieu (Fig. 3).

Aging and Chronic Wasting Diseases Aging is characterized by a progressive decline in several body functions, and loss of skeletal muscle mass and strength are also important aging-associated alterations. The oxidative or mitochondrial theory of aging postulated decades ago that age-related tissue dysfunctions are due to progressive accumulation of oxidative damage specifically to mitochondria, where oxidative reactions generate high levels of reactive oxygen species [22]. A high prevalence of mitochondrial DNA mutations and deletions has been reported also in human tissues, providing general support for the hypothesis [82]. Importantly, biopsy studies have confirmed a general decline in all steps of mitochondrial gene expression in skeletal muscle in otherwise healthy aging humans, including low mitochondrial transcript levels, protein synthetic rate, enzyme activities, and ATP production [83, 84]. Based on current knowledge and the above consideration, the pathogenesis of mitochondrial dysfunction in aging muscle is multifactorial and likely due to a combination of oxidative stress, inflammation as well as sedentary lifestyle. Indeed studies in which aging and young participants were matched for sedentary or active lifestyle indicate that a lack of physical activity rather than aging per se is a major determinant of mitochondrial

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dysfunction [13]. Since insulin resistance may impair muscle mitochondrial anabolism, it is plausible that age-related insulin resistance also contributes to these changes. Similar to aging, several chronic and acute wasting conditions characterized by loss of muscle mass are often also characterized by skeletal muscle mitochondrial alterations. Impaired mitochondrial oxidative capacity in terms of citrate synthase and respiratory chain enzyme activities has been reported in muscle biopsies from chronic kidney disease, chronic heart failure, and chronic obstructive pulmonary disease patients [85– 88]. Critically ill patients with multiple organ failure also show an impairment of skeletal muscle mitochondrial enzyme activities [89]. It is possible to hypothesize that these alterations are due at least in part to a combination of low grade inflammation, oxidative stress, insulin resistance, and low physical activity which often characterize the disease condition. In chronic kidney disease patients undergoing conservative treatment, a decline in muscle mitochondrial protein synthesis has been specifically reported, associated with declining synthesis rates of mixed proteins, mainly contractile ones [90]. It is important to point out that ATP is required for the maintenance of muscle proteins mass, such that impaired muscle mitochondrial turnover and function may contribute to muscle wasting under several chronic disease conditions. Exercise in Aging and Chronic Wasting Disease Aerobic exercise treatment has been repeatedly demonstrated to improve skeletal muscle mitochondrial oxidative capacity also in aging individuals and chronic disease conditions [71, 85–88], with recent studies confirming the involvement of SIRT1 and PGC1 also in this setting [13]. The above observations further support the potential for beneficial effects of exercise training to involve mitochondrial changes in wasting disease states with low muscle mitochondrial oxidative capacity.

6 Conclusions Skeletal muscle strongly relies on a constant, adequate energy supply. Mitochondrial oxidative phosphorylation provides adequate amounts of ATP under physiological conditions, playing a major role in glucose and lipid substrate utilization and contributing to preserve muscle protein anabolism. Muscle mitochondrial dysfunction occurs in obesity, insulin resistance, and chronic diseases associated with metabolic abnormalities as well as impaired muscle mass and strength. Inflammation, oxidative stress, and insulin resistance likely contribute to disease-associated mitochondrial changes, and they can be induced and modulated by changes in nutrient intake and nutritional status. Exercise training represents a major stimulator of mitochondrial biogenesis and a powerful therapeutic tool for disease conditions involving mitochondrial-related alterations.

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Introduction to the Tracer-Based Study of Metabolism In Vivo Andrea Caumo and Livio Luzi

1 Introduction The term homeostasis was coined around 1930 by the American physiologist Walter Bradford Cannon and popularized in his book The Wisdom of the Body. Homeostasis refers to the attempts of living organisms to maintain certain physiological variables (temperature, acid–base balance, blood glucose, etc.) within narrow margins of variation. Cannon developed the concept of homeostasis, expanding on Claude Bernard’s idea of the milieu interieur, that is, the body’s internal environment, whose stability is a prerequisite for the maintenance of life. Around 1935, Rudolph Schoenheimer refined this insight in a theoretical framework that quantitatively described the dynamic state of body constituents. The main idea is that the concentration of a substance in the body is a function of three processes that occur simultaneously: production/secretion, distribution/ exchange between the blood and other body fluids, and utilization/disposal. The continuous renewal of the circulating levels of a substance is called turnover. Schoenheimer was a pioneer in the use of radioactive and stable isotopes to study the turnover of proteins and lipids in animals, and his work exerted tremendous influence on subsequent generations of biochemists.

A. Caumo (*) Department of Biomedical Sciences for Health, University of Milan, Milan, Italy e-mail: [email protected] L. Luzi Department of Biomedical Sciences for Health, University of Milan, Milan, Italy Department of Endocrinology, Nutrition and Metabolic Diseases, IRCCS, MultiMedica, Milan, Italy e-mail: [email protected]; [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 L. Luzi (ed.), Cellular Physiology and Metabolism of Physical Exercise, https://doi.org/10.1007/978-3-031-27192-2_8

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Indeed, the dynamic state of body constituents has become a paradigm of biomedical research. In this chapter, we introduce the reader to the fundamental tracer-­ based methods used to measure the turnover of a particular substance.

2 Basic Concepts The fundamental concepts underlying tracer-based methods can be more easily understood by describing the metabolic system under study with a compartmental model in which there are a finite number of compartments with specified interconnections among them. Each compartment is an idealized store of the substance of interest that behaves like a distinct, homogenous, well-mixed amount of material. Each interconnection represents the flux of the substance of interest, which in physiological terms represents transport from one location to another, chemical transformation, or both. In the model, we must distinguish between compartments that are accessible for measurement and those that are nonaccessible. Usually, there is only one accessible compartment (the blood) where one can measure the concentration of the substance, while the other, nonaccessible compartments represent the organs and tissues in which the substance is distributed. In the example shown in Fig. 1, the accessible compartment is indicated by a dashed line and a bullet, while the nonaccessible portion of the system is denoted by a gray area. The nonaccessible portion consists of three interconnected compartments, two of which exchange with the accessible pool. Arrows connecting the compartments represent fluxes of the substance from one compartment to another. It can be seen that the production of the substance, denoted as P, is a flux that enters the accessible compartment directly. This corresponds to the very common situation in which the substance, once produced or secreted, is released directly into the bloodstream. We can also observe that there are fluxes leaving the system not only from the accessible compartment but also from two nonaccessible compartments. Such outfluxes may represent utilization, elimination, or degradation. The presence of Fig. 1 Mathematical model of a hypothetical metabolic system describing the distribution of a substance within the body. The compartment that is accessible to measurement exchanges with the nonaccessible portion of the system. The arrows represent fluxes of material going from one compartment to another

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outfluxes in nonaccessible compartments is common and is well-exemplified by glucose turnover. Glucose is produced endogenously in the liver and enters the blood circulation. Two sites of glucose utilization are, for example, the brain and the muscular system, which are distinct from the blood.

3 Mass-Balance Principle The fluxes of the substance between one compartment and another, as well as the masses of the substance in various compartments of the metabolic system, are governed by the mass-balance principle. Consider a generic compartment within the body. The mass-balance principle states that at any point in time the rate at which the substance’s mass changes within the compartment is the difference between the mass entering the compartment and the mass leaving the compartment. The intuitive explanation is that each molecule of the substance entering the compartment has only two options: either leave the compartment or contribute to increasing the mass within the compartment. Let us call Fin the sum of the fluxes entering the compartment and Fout the sum of all fluxes leaving the compartment (Fig. 2). The relationship between input and output fluxes and the mass of the substance in the compartment, q(t), is governed by the mass-balance equation: dq ( t ) Fig. 2  The mass-balance principle as applied to a generic compartment. The principle describes the relationship among three components: the fluxes going into the compartment, the fluxes going out of the compartment, the mass of the substance within the compartment. The mass-balance principle describes the conservation of the mass across the compartment: the substance entering the compartment may either go out of the compartment or increase the mass within the compartment

dt

= Fin ( t ) − Fout ( t )



(1)

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At Fin > Fout the mass in the compartment increases; at Fin 30 kg/m2, systolic blood pressure >160 mmHg, insulin requirement >1 IU/kg/day, and hyperlipidemia (despite medical therapy) are among the exclusion criteria for islet transplantation. Physical activity could be a fundamental instrument to enable patients to qualify for transplantation. BMI, lipid profile, and blood pressure are perfect examples of outcomes positively modifiable by regular exercise. Furthermore, it ensures that the patient arrives at surgery in better physical condition, resulting in improved outcome and a faster recovery from the transplant procedure. Moderate physical activity in patients waiting for a transplant is also crucial in order to maintain health markers such as VO2max, muscle strength, lipid profile, and insulin sensitivity within their required ranges. In addition, cachexia, body-fat percentage, and hypertension diminish with exercise, while oxygen uptake increases and fitness level may surpass the age-related expected values. In addition, regular exercise can help the patient to attain a more favorable psychological profile. It can therefore be concluded that before transplantation physical training improves both exercise capacity and psychological state, while afterward it increases the likelihood of a better surgical outcome [4].

3 Physical Work Capacity After Transplantation The quality of life, defined as the ability to enjoy normal life activities, improves enormously after transplantation. Despite a marked reduction in physical work capacity in the immediate post-operative stage, the majority of transplanted patients successfully return to their jobs. However, both transplant- and organ-specific factors can trigger a chain reaction leading to an impaired level of physical fitness. Primary among the factors common to all transplants is the effects of immunosuppressive therapy, administered in order to prevent rejection. These drugs negatively influence the cardiovascular, muscular, and skeletal systems, compromising exercise capacity. Arterial hypertension, renal insufficiency, dyslipidemia, diabetes, hepatic gluconeogenesis, myopathy, a decrease in capillary number, and osteoporosis have been reported as possible side effects of the triple-drug immunosuppressive regimen cyclosporine, prednisone, and azathioprine.

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According to the literature, an association between cyclosporine and hypertension is seen in 50–80% of renal and heart transplant recipients. Cyclosporine impairs the vasodilator function of the endothelium and modifies vascular smooth muscle function. Reductions in renal blood flow and glomerular filtration rate have also been reported in up to 38% of transplanted patients taking cyclosporine. Nephrotoxicity is caused by vasoconstriction of the afferent arterioles and mucoid intimal thickening of the arterial walls in the kidney; similar modifications are seen in the coronary vessels [5–7]. Immunosuppressants also exert their effects on muscle tissue. For example, myopathy is another side effect of cyclosporine. Drummond et al. [5] showed that the immunosuppressant rapamycin is a potent inhibitor of skeletal muscle hypertrophy, i.e., protein synthesis, after acute effort. Alterations such as muscular fiber atrophy, myofibrillar disruptions, Z band streaming, mitochondrial damage, and lipid vacuoles have been seen on biopsies of patients treated with the drug. In vitro and in vivo studies have evidenced a reduction in mitochondrial respiration. Muscle pain and loss of muscular strength have been reported as major clinical symptoms. Painter et al. [6] showed a slower improvement in exercise capacity (VO2max, peak torque p