Diabetes Mellitus Research Advances [1 ed.] 9781608766031, 9781600217111

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Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Diabetes Mellitus Research Advances, Nova Science Publishers, Incorporated, 2008. ProQuest Ebook Central,

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Diabetes Mellitus Research Advances, Nova Science Publishers, Incorporated, 2008. ProQuest Ebook Central,

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DIABETES MELLITUS RESEARCH ADVANCES

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Diabetes Mellitus Research Advances, Nova Science Publishers, Incorporated, 2008. ProQuest Ebook Central,

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Diabetes Mellitus Research Advances, Nova Science Publishers, Incorporated, 2008. ProQuest Ebook Central,

DIABETES MELLITUS RESEARCH ADVANCES

MAXIMILIAN N. HUBER

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

Editor

Nova Biomedical Books New York

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Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

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Contents

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Preface

vii

Chapter 1

The Metabolic Syndrome: Genetic Effects in Endocrine Pathways Santiago Rodríguez, Tom R. Gaunt, Mohammad Reza Abdollahi, Sabine Sonnenberg and Ian N.M. Day

Chapter 2

Reactive Oxygen Species and K+ Channel Function in Diabetes and Insulin Resistance Yanping Liu, Jefferson C. Frisbee and David D. Gutterman

Chapter 3

The Etiology of Obesity-Induced Insulin Resistance Kyle L. Hoehn, William L. Holland, Trina A. Knotts and Scott A. Summers

Chapter 4

Racial/Ethnic Disparities in Hypertension and Diabetes Ascribed to Differences in Obesity rate Ike S. Okosun and John M. Boltri

Chapter 5

Chosen Life Aspects of Diabetic Patients Ewa Otto-Buczkowska and Tomasz Dworzecki

Chapter 6

CNS Amyloidosis and Diabetes Mellitus: Vicious Circles of Misfolding Sergey V. Verevka

Chapter 7

Chapter 8

Erythrocyte Transplasma Membrane Electron Transport, Oxidative Stress, Body Mass and Lifestyle in Healthy and in Type 1 Diabetic Families E. Matteucci and O. Giampietro Impact of Oxidative Stress on Diabetes Mellitus and Inflammatory Bowel Diseases Jana Varvařovská, Rudolf Štětina, Josef Sýkora, Zdeněk Rušavý, Jaroslav Racek, Silva Lacigová and Konrad Siala

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1

63 99

133 153

169

179

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Contents

Chapter 9

Stem Cell Therapy for Type 1 Diabetes Mellitus Christopher J. Burns, Monica L. Courtney, Shanta J. Persaud and Peter M. Jones

Chapter 10

Explaining Type 2 Diabetes in Mexico: Patients’ and Physicians’ Perspectives Raminta Daniulaityte, Javier E. García de Alba García and Ana L. Salcedo Rocha

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Index

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287

301

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Preface Diabetes mellitus is a chronic disease of absolute or relative insulin deficiency or resistance characterized by disturbances in carbohydrate, protein and fat metabolism. It is estimated that between 5-10% of the population suffer from this disease. This syndrome is a contributing factor in a large percentage of deaths from heart attacks and strokes as well as renal failure and vascular disease. About 90% of the cases of diabetes mellitus fall into Type 2 where obesity plays a major role. Research in the field is wide-spread ranging from causes to treatment. This new book presents the lastest research in the field. Chapter 1 - Cardiovascular disease and mortality risk are significantly increased in people with metabolic syndrome, a cluster of interrelated metabolic disorders including obesity, insulin resistance, glucose intolerance, dyslipidemia and hypertension. A complex interplay between predisposing and protective factors ultimately determines whether an individual will develop this set of disorders or not. Genetic factors are one of the significant contributors that predispose to, or protect against, each component of the metabolic syndrome. As in other complex diseases and traits, such genetic factors are likely to be multiple and interacting, with individual polymorphisms producing only a moderate effect. The identification of genetic variants influencing the metabolic syndrome is of great importance to understanding pathogenesis, identifying groups of individuals with different relative risk, and developing or improving therapies against this cluster of metabolic disorders. This has greatly stimulated both theoretical and applied genetic research in recent years. A range of new analytical tools has been developed for the dissection of complex traits. Applied genetic analyses have identified large numbers of candidate markers and chromosomal regions (more than 600 for obesity, which represents only one of the disorders of this cluster). In this chapter we present a basic overview of the genetic approaches currently used for the identification of candidate genetic factors involved in the metabolic syndrome. We also summarise current evidence suggesting that genetic variants within elements of the endocrine system are directly involved in the risk of the metabolic syndrome. We focus our attention on endocrine pathways for which candidate genetic variants have been identified, and we introduce the foundations of a new hypothesis which postulates the involvement of a network of endocrine genetic setpoints as a combined contributor to the risk of the metabolic syndrome. Chapter 2 - Diabetes mellitus is associated with an increased prevalence of cardiovascular disease. Enhanced oxidative stress has been implicated in impaired vasomotor responsiveness in diabetes. Recent evidence suggests that reactive oxygen species (ROS) induced by

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hyperglycemia play an essential role in altered activity of potassium (K+) channels, a target of endothelium-dependent hyperpolarizing factors (EDHFs) . The enhanced oxidative stress may occur prior to the development of frank diabetes, such as insulin resistance. This review focuses on the mechanism of ROS production in hyperglycemia and insulin resistance and describes the consequences on hyperpolarization-mediated vasodilation. Several pathways have been proposed as mechanisms for hyperglycemia-inducedsuperoxide overproduction, including activation of polyol pathway, activation of the diacylglycerol-protein kinase C pathway, upregulation of NADPH oxidase and mitochondrial generation of ROS. The resulting excess production of superoxide has been implicated in the impaired dilator responses to KATP channel openers in aorta, mesenteric and cerebral arteries of streptozotosin-induced diabetic rats and in coronary arterioles from patients with diabetes. This may have important implications in ischemia-mediated vasodilation. The effect of ROS on Kv channel function has been examined in xenopus oocytes where shaker and shaw channel were expressed. Reduced K+ currents were observed in most shaker channels. Incubation of small rat coronary arteries in high glucose for 24 hours increases both superoxide and peroxynitrite generation, which greatly reduces Kv channel activity and functional responses, both of which can be partially restored by antioxidant treatment. In contrast to KATP and Kv channels, Ca2+-activated K+ channels are refractory to superoxide providing a compensatory mechanism for reduced dilator response to activation of other K+ channels. However, in conditions where peroxynitrite is increased, KCa channel function is inhibited. In this circumstance, compensatory dilator mechanism acting through hyperpolarization may fail. Insulin resistance, a condition where insulin-induced glucose uptake is reduced, is considered to be a precursor to the development of type II diabetes mellitus. In hyperinsulinemia, impaired vasodilator responses are observed in variety of vascular beds of obesity animals even in normoglycemia or mild hyperglycemia. Reduced K+ channel function, such as KCa and KATP channels, may be a mechanism responsible for the impaired vascular reactivity in insulin resistance. Treatment with antioxidants such as superoxide dismutase restores the dilator response in obese animals supporting an inhibitory role of ROS in vasomotor function in the insulin resistant state. In summary, determining the effect of ROS on K+ channel mediated dilation is important for understanding the pathophysiology of vascular dysfunction in diabetes and insulin resistance and may suggest new therapies to improve tissue perfusion. Chapter 3 - Insulin is an essential hormone with important roles in glucose homeostasis and anabolic metabolism. Cellular and/or molecular defects in insulin action result in a state of insulin resistance, which is an essential feature of the diseases type 2 diabetes and the metabolic syndrome. One of the largest correlations to insulin resistance is obesity; specifically the enlargement of adipose tissue in the abdomen. This correlation is unmistakably obvious today as the twin epidemics of obesity and type 2 diabetes have coemerged. One of the biggest challenges in the field of diabetes research is to determine how the increase in visceral adiposity leads to impaired insulin action. Recently, two hypothetical mechanisms have transpired: (a) obesity shifts the secretory profile of adipose tissue to favor a reduction in nutrient uptake by creating insulin resistance, and/or (b) overloaded fat cells and the increased caloric intake common in obesity leads to the inappropriate storage of lipids in non-adipose tissues where they antagonize insulin action. This review summarizes recent

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findings implicating each mechanism, alone or in concert, in the etiology of obesity-induced insulin resistance. Chapter 4 - American ethnic minorities, particularly those of African and Hispanic descent have a greater risk of developing hypertension and type 2 diabetes compared to American Whites. Despite the consistency of the epidemiologic evidence of the racial/ethnic variation for these diseases, relatively little is known with confidence about the causes of the non-White dilemma. Objective - To determine how much of the relative difference in the rates of hypertension and type 2 diabetes between high-risk Blacks and Hispanics and lowrisk Whites is attributable to their differences in obesity. Methods - Data (n=5531) from the 1999-2002 U.S. National Health and Nutrition Examination Surveys were utilized for this analysis. Gender-specific proportions of White to non-White differences in odds of hypertension and diabetes that were due to their relative differences in the prevalence of obesity were estimated using relative attributable risk derived from multiple logistic regression modeling. Statistical adjustment was made for age, education, alcohol intake, education, and physical activity. Results - 50.2% and 30.6% of differences in odds of hypertension between White men and Black men and between White men and Hispanic men, respectively, are attributable to their differences in rates of obesity. The analogous values for diabetes were 70.7% and 57.4% for Black men and Hispanic men. Also, 30.6 % and 13.4% of differences in odds of hypertension between White women and Black women and between White women and Hispanic women, respectively, are associated with their differences in rates of obesity. The analogous values for diabetes are 62.2% and 83.7% for Black women and Hispanic women when compared with White women. Conclusion - The magnitude of racial/ethnic differences in hypertension and diabetes due to their differences in obesity provides an encouraging reason to continue to implement public health obesity prevention programs in the United States’ minority groups. Aggressive programs to reduce obesity and increase physical activity in Blacks and Hispanics may prove useful in reducing racial/ethnic disparities in hypertension and diabetes. Chapter 5 - Progress in diabetes treatment and in maintenance of good metabolic control that has been made during last decade caused a radical decrease in prevalence of diabetic acute complications and gave a chance to prevent its chronic complications. As a result, diabetic patients, with a bit of effort and strong will may substantially improve their ability to perform various activities including traveling, working and driving vehicles. Diabetic patient on a journey Diabetic patient can travel too, however such trip, especially when long and associated with time zones changes, requires very careful preparations. Those should concern both trip schedule and health problems. Medical dilemmas on eligibility for driving licenses Driving a vehicle requires a high psychomotor performance, keenness and quick responses in order to react appropriately to the situation on the road. These functions may be impaired in the course of diabetes. The course of diabetes may be different for each patient and depends not only on the type of the diabetes and the method of treatment but also, to a large extend, on patients’ training and motivation for most careful control. Since many years there have been attempts to determine whether the number of accidents among insulin treated diabetic patients is higher then among other drivers. The results are inconsistent. Serious changes of diabetes treatment methods and strictness of metabolic control that have been introduced during recent years substantially improved performance of insulin treated diabetic drivers. That is why results obtained many years ago cannot be used to evaluate the risk of accidents for people with diabetes nowadays.

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Access to education and employment for diabetic patient Majority of properly treated patients are able to lead almost normal life – study, work, set up families and participate in social life. Well controlled diabetes, without chronic complications does not disable the patient from working in most of the professions. People that developed diabetes during childhood or adolescence face different problems than group of patients who became sick later. Another group consists of patients who had to change the method of treatment due to progression of the disease or whose health was deteriorated by chronic complications. Chapter 6 - Molecular mechanisms of prion diseases possess an unusual place in a number of the mysteries composing a leading edge of the modern biochemistry. Indeed, the inconsistency between a huge number of experimental data and the absence of the proven explanation of the pathogenic action of prion protein seems surprising and unprecedented. The proof of infecting action of prions may serve as an axiom of bright verification of the inexplicable fact, however the absence of answers to key questions of prion-mediated pathogenesis derived with doubts in protein-only nature of the pathogenic agent. This work represents an attempt to explain major aspects of the prion-mediated diseases in terms of already known experimental data. It substantiates mechanism resulting in the self-sustaining and progressing cycle of disorders of well-known cellular processes. These processes include the disturbance of cellular folding system and the structuring of de novo synthesizing prion protein induced under the influence of cell’s membrane. The possible analogies between molecular mechanisms of prion-caused diseases and auto-immune ones are discussed. The question about the belonging of prion diseases to a large group of pathologies caused by damage of folding process is raised. Chapter 7 - Erythrocytes export electrons across the cell membrane to external oxidants (such as ferryicyanide) through a redox system that remains still unknown. The rate of ferricyanide reduction varies as a function of cytoplasmic electron donor concentration. Indeed, the pathway is linked to a set of intracellular redox couples including NADH/NAD+, GSSG/SGSH, and ascorbate/vitamin C. Since the activity of transplasma membrane electron transport (TPMET) systems appears to be closely related to the redox homeostasis and the metabolic state of the cell, our aim was to characterise the membrane reducing system in healthy and in type 1 diabetes (T1D) families and its relationship with nutritional indicators and oxidative biomarkers. Particularly, present research work aimed at evaluating in healthy and T1D families: (a) the rate of erythrocyte electron transport in relation with body mass and metabolic efficiency, (b) modulating effects of diet and lifestyle on erythrocyte electron transfer system. We measured the erythrocyte electron transport to extracellular ferricyanide (RBC vfcy) in 100 healthy controls and 99 non-diabetic relatives of type 1 diabetics. Erythrocyte Na/H exchange (RBC NHE), RBC glutathione (GSH), plasma thiols, plasma and RBC malondialdheyde were also determined, in addition to plasma glucose, insulin, lipids, HbA1c, creatinine clearance, and urinary albumin. Moreover, we assessed dietary habits and lifestyle of 76 relatives and 95 healthy subjects by using the European Prospective Investigation of Cancer and Nutrition questionnaires. a.

Among healthy controls, individuals with BMI≤25 kg/m2 had lower rates of electron transport in comparison with age-gender-matched subjects who were overweight or obese. Indeed, RBC vfcy correlated positively with two

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indices of fat body mass (BMI and circulating triglycerides), and negatively with an index of lean body mass (24-hour urinary creatinine excretion). Moreover, RBC vfcy showed a negative association with RBC NHE and plasma MDA. On the contrary, among relatives, RBC vfcy did not change significantly with BMI. It showed a positive association with RBC MDA, negative with RBC GSH. b. Both Spearman’s rank correlation and stepwise multiple regression analyses including lifestyle information found different independent variables to be positively associated with RBC vfcy: daily dietary intake of vitamin C among healthy controls, whereas time spent in regular exercise among relatives. In conclusion, (a) electron transfer catalysed by the transmembrane ferricyanide reductase activity may reflect the functional state of membrane proton pumps that modulate cellular metabolism by regulating the intracellular redox levels. In the case we should have a useful tool to indirectly evaluate some aspects of energy balance in human metabolic diseases by using easily accessible cells and simple laboratory procedure. Moreover, the transport system, that seems functionally normal, contributes to oxidation in T1D families, whereas in healthy people it protects from oxidation. Furthermore, (b) dietary intake of vitamin C and sporting activities modulate erythrocyte electron transfer efficiency. In the cytosol, ascorbic acid or vitamin C can donate electrons to trans-plasma membrane electron transfer activity in erythrocytes. Thus, intracellular electron donors available from dietary sources can be very important in maintaining the redox environment of a cell, i.e. the summation of the products of the reduction potential and reducing capacity of the linked redox couples present. Our data also support indirect evidence suggesting that regular exercise may improve electron transport efficiency. However, the reason why independent lifestyle variables associated with RBC vfcy markedly differed among population subgroups remains unknown. Chapter 8 - Formation of reactive oxygen species (ROS) is a natural process during oxidative metabolism. ROS play an important role not only in pathological processes of human organism as usually presented but less attention is paid to their proper important role in cell signaling, biosynthesis or non-specific antiinfectious defence. Overproduction of ROS during numerous pathological situations in presence of insufficient antioxidant protection leads to substantial oxidative changes of lipids, proteins, sugars, and also DNA. Protection against ROS is assured by different extracellular or intracellular antioxidant mechanisms as studied during last decades. Antioxidant enzymes rectifying the oxidative damage are studied with regard to their different activities and usefulness in body protection. Their genetic polymorphisms are certainly involved in different response to oxidative stress. Special attention should be devoted to the topic of oxidative nuclear and mitochondrial DNA damage and its restoring via DNA repair process, especially base excision repair (BER). A large scale of antioxidant enzymes is involved in correction of DNA oxidative damage. Natural trend of worsened DNA repair is usually associated with aging. Other pathologies related with deficient DNA repair are susceptibility to carcinogenesis (lack of apoptosis control) or degenerative diseases. Oxidative stress is involved in the pathophysiology of diabetes mellitus (DM – oxidative stress of mainly metabolic origin) and inflammatory bowel diseases (IBD – oxidative stress of mainly inflammatory origin). In spite of confirmed OS in DM or IBD, the substantial

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information about the intensity of DNA repair and its possible relationship to the disease course and development of chronic complications is missing. Our pilot studies completed both in adult and pediatric patients with DM or IBD confirmed an increased oxidative stress as well as oxidative DNA damage examined with comet assay. The surprising findings were ascertained in intensity of DNA repair (analysed with modified comet assay). DNA repair process was stimulated in Type 1 diabetes adults without diabetic microvascular complications and still more in diabetic children with short disease duration. On the other hand adults with Type 2 diabetes had substantially increased oxidative DNA damage and extremely low DNA repair. This finding could be the link to increased susceptibility of Type 2 DM patients to cancerogenesis. Patients with IBD (Crohn´s disease - both children and adults) had similar tendencies in OS intensity and oxidative DNA damage and repair but less intensive. Large population studies in DM or IBD studying intensity of OS and expression of DNA repair enzymes are needed in order to get the correlation between individual repair enzymes expression and the long-term course and occurence of complications in DM or IBD. Chapter 9 - Type 1 diabetes mellitus (T1DM) is a disorder of a single cell type, the insulin-secreting pancreatic β-cell, and as such is particularly amenable to treatment by cell replacement therapy. For this reason, T1DM has received much attention recently as a potential target for the emerging science of stem cell medicine. In this autoimmune disease, the pancreatic β-cells are selectively and irreversibly destroyed by autoimmune assault. Advances in islet transplantation procedures now mean that individuals with T1DM can be cured by human islet transplantation. A major drawback in this therapy is the limited availability of donor islets, and the search for substitute transplant tissues has intensified in the last few years. This review will summarise recent progress in using stem cell populations for generating substitute β-cells for transplantation therapy. The relative merits of stem cells from different sources will be considered, and the major technical obstacles to the production of reliable and reproducible stem cell to β-cell differentiation protocols will be addressed. Chapter 10 - Conducted in Guadalajara, Mexico, the study focuses on patients’ and physicians’ beliefs about diabetes causality. The study was conducted in two stages and used cultural consensus model. First, qualitative interviews were conducted with a convenience sample of 28 Type 2 diabetes patients. On the basis of the elicited themes, 21 scenarios on diabetes causes were developed. In the second stage, a convenience sample of 46 Type 2 diabetes patients and 25 physicians working at the primary care level was recruited. Participants were asked to rate each scenario on a three-point scale. Scenario-type interviews were consensus analyzed using ANTHROPAC. Patients and physicians shared very different cultural models of diabetes causality. The patient model included emotional, environmental, some behavioral, and hereditary causes of diabetes. The physician model emphasized heredity as a single most important cause of diabetes. Differences between patient and physician views of diabetes causality may contribute to mistrust and miscommunication in medical interactions. There is a need for clinical practice that would include psychosocial stress and environmental factors in diabetes prevention and care.

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In: Metabolic Syndrome Research Trends Editor: Maximilian N. Huber

ISBN: 978-1-60021-711-1 © 2009 Nova Science Publishers, Inc.

Chapter 1

The Metabolic Syndrome: Genetic Effects in Endocrine Pathways Santiago Rodríguez1*, Tom R. Gaunt1, Mohammad Reza Abdollahi2, Sabine Sonnenberg1 and Ian N.M. Day1 1

Human Genetics Division, University of Southampton, School of Medicine, Duthie Building (MP 808), Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK 2 Bristol Genetic Epidemiology Laboratory, University of Bristol, No. 24 Tyndall Avenue, Bristol, BS8 1TQ, UK

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Cardiovascular disease and mortality risk are significantly increased in people with metabolic syndrome, a cluster of interrelated metabolic disorders including obesity, insulin resistance, glucose intolerance, dyslipidemia and hypertension. A complex interplay between predisposing and protective factors ultimately determines whether an individual will develop this set of disorders or not. Genetic factors are one of the significant contributors that predispose to, or protect against, each component of the metabolic syndrome. As in other complex diseases and traits, such genetic factors are likely to be multiple and interacting, with individual polymorphisms producing only a moderate effect. The identification of genetic variants influencing the metabolic syndrome is of great importance to understanding pathogenesis, identifying groups of individuals with different relative risk, and developing or improving therapies against this cluster of metabolic disorders. This has greatly stimulated both theoretical and applied genetic research in recent years. A range of new analytical tools has been developed for the dissection of complex traits. Applied genetic analyses have identified large numbers of candidate markers and chromosomal regions (more than 600 for obesity, which represents only one of the disorders of this cluster). In this chapter we present a basic overview of the genetic approaches currently used for the identification of candidate genetic factors involved in the metabolic syndrome. We also summarise current evidence suggesting that genetic variants within elements of the endocrine system are directly involved in the risk of the metabolic syndrome. We focus our attention on endocrine pathways for *

E-mail address: [email protected]. Tel.: +44 (0) 23 8079 8731 Fax: +44 (0) 23 8079 4264; Correspondence: Dr Santiago Rodríguez, Human Genetics Division, University of Southampton, School of Medicine, Duthie Building (MP 808), Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK.

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which candidate genetic variants have been identified, and we introduce the foundations of a new hypothesis which postulates the involvement of a network of endocrine genetic setpoints as a combined contributor to the risk of the metabolic syndrome.

1. Introduction Unraveling the genetic basis of complex traits is one of the greatest challenges of science and medicine in the 21st century (Dean, 2003). Complex traits (also known as multifactorial or non-Mendelian traits) influence the normal and abnormal function of all body systems, and are directly involved in the diseases that account for most of the mortality and morbidity in humans (Sankaranarayanan et al., 1999). Most phenotypes for which there is a continuous variation at the population level are complex traits. Examples span all kinds of human features, including skin colour, weight, hormone levels, blood pressure, intelligence, etc. The genetic dissection of complex traits is relevant to improving our basic knowledge of mechanisms for life. In particular, it is key for understanding pathogenesis and potentially for disease prediction. It is hoped that advances in this field will fuel the development or improvement of therapeutic approaches, diagnostics and risk assessment for common diseases such as cardiovascular disease, diabetes and cancer. However, multifactorial traits are doubly complex. Firstly, they have a complex genetic basis. Their phenotypic expression is the result of the action and interaction of both genetic and non-genetic factors (Chakravarti and Little, 2003). A large number of genes are involved in the determination of complex traits, but none of them is necessary or sufficient to explain all phenotypic variation. On the contrary, most of these “polygenes” have a small effect and contribute in a quantitative manner (Sankaranarayanan et al., 1999). Polygenes may interact with each other and with multiple environmental factors in unpredictable ways. As a result, the mode of inheritance of complex traits is far more complex than that imposed by the laws of classical Mendelian inheritance. And secondly, at present, the analysis of the genetic basis of multifactorial traits is also complex. A number of approaches have been developed and implemented in recent years to identify and disentangle the role of such genetic factors. But the fact that the inheritance of multifactorial traits follows non-Mendelian patterns, together with the existence of genetic heterogeneity, polygenic effects and interactions, uncontrollable development-environmental variability, etc., (Risch, 2000) has resulted in a low rate of independently confirmed findings (Sillanpaa and Auranen, 2004). The metabolic syndrome is one of the paradigms in the genetic analysis of complex traits. In this chapter we summarise recent advances in the genetic analysis of the metabolic syndrome, focusing the emphasis on the endocrine factors involved.

2. The Metabolic Syndrome: Concept, Definition, Effect and Prevalence The scientific community has known for many decades that different (but interrelated) metabolic abnormalities (including glucose intolerance, insulin resistance, central obesity, dyslipidaemia and hypertension) occur simultaneously in some individuals with a frequency higher than might be expected by chance, and that this condition increases considerably the risk of cardiovascular disease and type 2 diabetes (Eckel et al., 2005). This concept has been recognised for many years and the syndrome was formally first labelled by Reaven in 1988 as

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The Metabolic Syndrome: Genetic Effects in Endocrine Pathways

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syndrome X (Reaven, 1988). Today, this syndrome is widely known as metabolic syndrome, but also called insulin resistance syndrome, dysmetabolic syndrome and Reaven syndrome. Recently, the International Diabetes Federation (IDF) has published a new definition of the metabolic syndrome aiming to provide a better prediction in the identification of individuals at high risk for cardiovascular disease and type 2 diabetes. This new definition considers that five complex traits can behave as risk factors for the metabolic syndrome, namely central obesity, triglyceride levels, cholesterol levels, blood pressure and plasma glucose levels. Each trait is considered to be a risk factor for the metabolic syndrome in a given individual according to established thresholds (Table 1). The metabolic syndrome is defined as the simultaneous ocurrence, in a given individual, of central obesity plus at least any two of the other risk factors (raised triglyceride levels, reduced HDL-cholesterol levels, raised blood pressure or raised fasting plasma glucose levels). Controversy exists, however, in the criteria followed to define individuals with the syndrome. Other definitions exist (Table 1), but given that all definitions to date are arbitrary, there can be no “correct” definition, merely good or bad predictors of outcome. The negative effect of the metabolic syndrome on human health and its prevalence [ranging from 22% to 39% of the adult population in developed countries (Khunti and Davies, 2005)] has fuelled and justified a huge effort by the scientific community in order to disentangle all the aspects of the syndrome.

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3. Pathophysiology of the Metabolic Syndrome The precise pathogenesis and the pathophysiological sequence of metabolic abnormalities resulting in metabolic syndrome remain uncertain, although it is widely accepted that both insulin resistance and obesity are central components. Some authors have suggested that insulin resistance, in both adipose tissue and muscle, is the main underlying factor leading to the metabolic syndrome (Eckel et al., 2005). Insulin resistance is an impaired metabolic response of the body to insulin action (Schinner et al., 2005). In a normal physiological condition, insulin exerts an anabolic role on glucose metabolism by lowering blood glucose levels, both by facilitating glucose uptake mainly into skeletal muscle and fat tissue and by inhibiting endogenous glucose production by the liver. In an insulin resistance state, the actions of insulin are impaired. In the initial stages of insulin resistance, there is a hypersecretion of insulin from pancreatic β cells. This compensates temporarily for insulin resistance, keeping normal glucose tolerance and glucose fasting levels. When insulin hypersecretion can not compensate for insulin resistance, glucose intolerance manifests, leading ultimately to β cell failure and type 2 diabetes. Insulin resistance is therefore causally related to each of glucose intolerance, dyslipidemia, high blood pressure and vascular dysfunction. However, it has been suggested that insulin resistance alone is insufficient to cause these anomalies (Ferrannini, 2006). In fact there is increased recognition that abdominal obesity is the most prevalent form of the metabolic syndrome, which has led some authors to suggest obesity as the primary cause of the metabolic syndrome (Despres, 2006). In an obesity state, free fatty acids (FFA) are released in abundance from the expanded adipose tissue mass. FFA stimulate the production

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Table 1. Definitions of the metabolic syndrome proposed by different organisations [adapted from (Magliano et al., 2006)] WHO (1999)

EGIR (1999)

ATPIII (2001)

IDF (2005)

Insulin resistance

Insulin resistance

Three or more of:

Central obesity

Plus two or more of:

Plus two or more of:

a) Central obesity: waist circumference>102 cm in M (>88 cm in F) b) Hypertriglyceridaemia: triglycerides≥1.7 mmol/L

Plus any two of:

a) Obesity: BMI>30 kg/m2 or WHR>0.9 in M (>0.85 in F)

a) Central obesity: waist circumference≥94 cm in M (≥80 cm in F)

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Chapter 2

Reactive Oxygen Species and K+ Channel Function in Diabetes and Insulin Resistance *

Yanping Liu,1 #Jefferson C. Frisbee and *David D. Gutterman

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*Cardiovascular Center, VA Medical Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA #Center for Interdisciplinary Research in Cardiovascular Sciences Department of Physiology and Pharmacology, West Virginia University Morgantown, West Virginia 26506, USA

Abstract Diabetes mellitus is associated with an increased prevalence of cardiovascular disease. Enhanced oxidative stress has been implicated in impaired vasomotor responsiveness in diabetes. Recent evidence suggests that reactive oxygen species (ROS) induced by hyperglycemia play an essential role in altered activity of potassium (K+) channels, a target of endothelium-dependent hyperpolarizing factors (EDHFs) . The enhanced oxidative stress may occur prior to the development of frank diabetes, such as insulin resistance. This review focuses on the mechanism of ROS production in hyperglycemia and insulin resistance and describes the consequences on hyperpolarization-mediated vasodilation. Several pathways have been proposed as mechanisms for hyperglycemia-induced-superoxide overproduction, including activation of polyol pathway, activation of the diacylglycerolprotein kinase C pathway, upregulation of NADPH oxidase and mitochondrial generation of ROS. The resulting excess production of superoxide has been implicated in the impaired dilator responses to KATP channel openers in aorta, mesenteric and cerebral arteries of streptozotosin-induced diabetic rats and in coronary arterioles from patients with diabetes. This may have important implications in ischemia-mediated vasodilation. The effect of ROS on Kv channel function has been examined in xenopus oocytes where shaker and shaw 1

Correspondence to: Yanping Liu, M.D., Ph.D.Cardiovascular Center, Medical College of Wisconsin 8701 Watertown Plank Road, Milwaukee, WI 53226, (414) 456-5633 Phone, (414) 456-6572 Fax, E-mail: [email protected].

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Yanping Liu, Jefferson C. Frisbee and David D. Gutterman channel were expressed. Reduced K+ currents were observed in most shaker channels. Incubation of small rat coronary arteries in high glucose for 24 hours increases both superoxide and peroxynitrite generation, which greatly reduces Kv channel activity and functional responses, both of which can be partially restored by antioxidant treatment. In contrast to KATP and Kv channels, Ca2+-activated K+ channels are refractory to superoxide providing a compensatory mechanism for reduced dilator response to activation of other K+ channels. However, in conditions where peroxynitrite is increased, KCa channel function is inhibited. In this circumstance, compensatory dilator mechanism acting through hyperpolarization may fail. Insulin resistance, a condition where insulin-induced glucose uptake is reduced, is considered to be a precursor to the development of type II diabetes mellitus. In hyperinsulinemia, impaired vasodilator responses are observed in variety of vascular beds of obesity animals even in normoglycemia or mild hyperglycemia. Reduced K+ channel function, such as KCa and KATP channels, may be a mechanism responsible for the impaired vascular reactivity in insulin resistance. Treatment with antioxidants such as superoxide dismutase restores the dilator response in obese animals supporting an inhibitory role of ROS in vasomotor function in the insulin resistant state. In summary, determining the effect of ROS on K+ channel mediated dilation is important for understanding the pathophysiology of vascular dysfunction in diabetes and insulin resistance and may suggest new therapies to improve tissue perfusion.

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Introduction Diabetes mellitus is a major source of morbidity in the United States, almost exclusively due to concomitant vascular disease. Substantial evidence suggests that endothelial dependent dilation is reduced in diabetes largely due to reduced bioavailability of nitric oxide [ NO] [1,13,39,51] . Enhanced oxidative stress has been postulated as one of the mechanisms for the impaired NO-mediated dilation, since treatment with free radical scavengers provides protection against impaired dilation [ 35,56,73] . It is widely recognized that in certain vascular beds especially in microcirculation, a major class of compounds distinct from NO, endothelial derived hyperpolarization factors [ EDHF’s] , are released from the endothelium and are responsible for vasodilation by opening K+ channels. Although it has been suggested that EDHF may compensate for loss of NO in certain disease situations, such as hypercholesterolemia[ 141,142] , reactive oxygen species [ROS] may globally affect endothelium-dependent dilator mechanisms including EDHF. ROS interactions with EDHF may take place at the vascular smooth muscle cell [ VSMC] K+ channels, the target site of EDHF. However knowledge about redox modulation of K+channel activity is limited. Understanding these mechanisms is important for developing therapeutic strategies to reverse vascular dysfunction. This review will focus on the interaction of oxidative stress with K+ channel activity in the vasculature.

Physiological Importance and Basic Structure of K+ Channels There are many types of K+ channels have been identified in vasculature. Among them, there are three major types of potassium [ K+] channels, including ATP-sensitive K+ [ KATP] channels, voltage-gated K+ [ Kv] channels and calcium-activated K+ [ KCa] channels have

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Reactive Oxygen Species and K+ Channel Function in Diabetes and Insulin Resistance 65 been proposed playing critical role in regulating vasomotor function. Their physiological importance and basic structures are summarized below.

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KATP Channels KATP channels exist in pancreatic β cells, neurons, skeletal muscle, vascular and nonvascular smooth muscle [ 75] . The physiologic role of KATP channels is to couple the electrical activity of the cells to their metabolic rate by sensing changes in the concentrations of adenine nucleotides [ ATP and ADP] . In pancreatic β cells, ATP increases when the plasma glucose level rises. The closure of KATP channels by ATP induces depolarization of cell membrane, which triggers calcium entry and insulin release [ 75] . In cardiac myocytes, ATP falls as a result of hypoxia, opening of KATP channels shortens the cardiac action potential and reduces cardiac work [ 18,48] . KATP channel in vascular smooth muscle senses changes in oxygen [ 54,118,133] , pH [ 67] and metabolic products, such as adenosine [ 143] , and opening of these channels produces vasodilation. Growing evidence also suggests that KATP channels in the inner membrane of the mitochondria play an important role in cardiac ischemic preconditioning [ 86,120] . At the molecular level the channel is a complex of two subunits [ Fig. 1] , namely the sulphonylurea receptor [ SUR] , a member of the ATP binding cassette family of proteins, and a channel pore forming subunit [ Kir 6.x] , a member of the inwardly rectifying family of K+ channels. SUR is composed of two intracellular nucleotide binding domains [ NBD1 and NBD2] and three transmembrane domains [ TMD0, TMD1 and TMD2] that contain five, six and six transmembrane helix [ TMs] , respectively [ 75] . The TM 16 and 17, the intracellular loop between TM13 and TM14, and at the COOH- terminal are the binding areas for potassium channel openers. Two genes for SUR receptors have been identified, encoding the proteins SUR1 and SUR2 which are expressed in different tissues [ 3,93] . SUR1 is predominantly found in pancreatic β-cells and neurons. Alternative splicing of SUR2 produces a cardiac/skeletal muscle isoform [ SUR2A] [ 101] and smooth muscle isoform [ SUR2B] [ 101] . In addition to tissue specificity of SUR isoforms, the sensitivity to KATP channel openers between SUR1 and SUR2 type KATP channel are also different. For example, pinacidil, P1075, cromakalim and nicorandil, activate SUR2-type KATP channels, but have little effect on SUR1-type channels [ 162,163] . These properties are of therapeutic importance for selecting KATP channel opener therapy. For instance, nicroandil is used as a vasodilator for the treatment of angina, and does not impair insulin secretion from the pancreatic β-cells [ 17,162] . In contrast, diazoxide, has both vasodilatory and insulin inhibitory effects [ 17] . Two clones of the inwardly rectifying component, Kir 6.1 and Kir 6.2, have been isolated. Studies examining the distribution of Kir 6.x in variety of tissues indicate that Kir 6.2 is expressed in heart [ 92] , brain[ 91] , skeletal muscle[ 92] , smooth muscle[ 101] and pancreatic tissue[ 91] , but not in visceral smooth muscle or lung[ 16] , whereas Kir 6.1 is more widely expressed in these tissues as well as in visceral smooth muscle [ 191] .

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SUR NH3

TMD0

Inward rectifier channel Kir 6.x

TMD2

TMD1

P 1 2 34 5

6 7 8 9 1011

121314151617

L

L B A NBD1

Out

M1 M2

B A

NH3 COOH

66

COOH

In

C NBD2

Figure 1. KATP channels are composed of an ATP-binding cassette protein [ the sulfonylurea receptor, SUR] and Kir 6.X subunits. SUR is consisted of three transmembrane domains [ TMD0, TMD1 and TMD2] and two nucleotide-binding domains [ NBD1 and NBD2] . The NBDs are characterized by conserved sequence motifs involved in the binding and hydrolysis of ATP at sites labeled A and B and in the transmission of the signal to transmembrane domain through linker [ L] . The TM 16 and 17, the intracellular loop between TM13 and TM14, and the region termed C are the binging areas for K+ channel openers. In Kir 6.0, M1 and M2 represent the transmembrane domains, with a pore region [ P] . Adapted from reference [ 177] .

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Voltage-Gated K+ Channels Numerous studies have demonstrated that Kv channels contribute to the resting membrane potential in coronary [ 49,114,122] , cerebral [ 4] , and pulmonary [ 87,108,174] circulations, and that pharmacological inhibition of these channels depolarizes and constricts arteries and arterioles. Several studies have shown that Kv channels mediate pharmacological dilator responses. The β-adrenoceptor agonist isoproterenol, or the adenylate cyclase activator forskolin, increases 4-AP sensitive K+ currents in rabbit portal veins and coronary arteries [ 5,6] . Studies from our laboratory indicate that 4-aminopyridine [ 4-AP] , a Kv channel blocker, reduces dilation of rat small coronary arteries to isoproterenol and to forskolin [ 114] suggesting an important role of Kv channels in the coronary microcirculation. Kv channels are also involved in a variety of other vasomotor responses. Inhibition of Kv channels is a mechanism for hypoxic pulmonary vasoconstriction[ 87,159] . In the coronary circulation, activation of histamine H1-receptors inhibits 4-AP sensitive K+ channels consistent with a role for Kv channels in coronary vasospasm[ 100] . The vasoconstrictor effect of angiotensin II is, in part, mediated by a reduction in Kv current[ 47] . Kv channels also have been shown to regulate coronary vascular tone in response to changes in pH of rat coronary arterial myocytes[ 22] . Kv channels have been described in most types of vascular smooth muscle cells [ 12] . They emanate from at least 11 gene families [ i.e., Kv1 – Kv11] . Drosophila channels including Shaker [ Kv1] , Shab [ Kv2] , Shaw [ Kv3] and Shal [ Kv4] have been well characterized. Recent reports have document the expression of three Shaker K+ channel subtypes [ Kv 1.2, 1.4 1.5] in vascular smooth muscle cells from several critical regulatory

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Reactive Oxygen Species and K+ Channel Function in Diabetes and Insulin Resistance 67 beds, including the coronary, pulmonary and renal circulations [ 66,87,122,131,149,186] . As shown in Figure 2, Kv channels have four transmembrane domains. Each domain is composed of two subunits, α and β.. There are six transmembrane segments [ S1-S6] and a pore-forming region between S5 and S6 in the α subunit[ 44,158] . Three amino acid residues containing an aromatic ring located in the S5-S6 linker, namely, two tryptophans and one tyrosine, are common to the pore or P loop and are considered as a critical site for K+ ion selectivity and conductivity.

α

1

2

3

+ + 4+ +

Out 5

6

In

β NH2

COOH NH2

Pore domain Essential for K+ selectivity and K+ channel blockers

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Figure 2. The putative membrane-spanning segments of the -subunit of Kv channel are designated as S1-S6. S4 segment is considered as a voltage sensor. P loop of -subunit is a critical portion for K+ ion selectivity and conductivity. The extracellular portion of P loop contains the binding sites for toxins. The intracellular side of P loop has the binding sites for compounds such as 4-AP, tetraethylammonium, and quinidine. The N-terminal domain of the -subunit contains the binding site for β-subunit [ black area] . Adapted from reference [ 193] .

The extracellular portion of the P loop contains the binding sites for toxins, such as iberiotoxin and charybdotoxin. The intracellular side of P loop has the binding sites for compounds such as 4-AP, tetraethylammonium, and quinidine [ 172] . S4 segment is considered a voltage sensor. This segment contains positively charged residues [ lysine, arginine and histidine] at approximately every third position. Membrane depolarization causes S4 conformational changes, which interact with S5 and S6 leading to channel opening. Kvβ subunits that are attached to NH3 terminals of α-subunits alter channel kinetics and modify cell surface expression. [ 32,61,140,161,164,170] . Recent studies suggest that Kv β may have NADPH oxidoreductase activity. Therefore the N-terminal domain of Kv channel may redox-sensitive [ 2] . Ca2+-Activated K+ Channels An important physiological role of KCa channels in vascular smooth muscle stems from the fact that these channels serve a negative feedback role, opposing depolarization, VSMC Ca2+ entry, and vasoconstriction. In the vasculature, KCa and Kv channels open at different stages of depolarization. In response to initial depolarizing stimuli, the opening of Kv channels provides the initial negative feedback to attenuate vasoconstriction. The subsequent increase in cytosolic free Ca2+, however, closes the Kv channels resulting in further depolarization. Both membrane depolarization and the rise in cytosolic Ca2+ act

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synergistically to enhance the opening of KCa channels and limit arterial constriction. KCa channels have also been postulated to respond directly to several endogenous vasodilators. For instance, nitric oxide induced relaxation are inhibited by KCa channel blockers such as tetraethylammonium [ 11,12,23] and iberiotoxin [ 23,58,146] in variety of vascular beds. Li et al demonstrated that KCa channels are involved in response to cytochrome P450 metabolites, such as epoxyeicosatrienoic acid [ 116,117] . In pathological conditions such as hypertension, overexpression of KCa channels is observed in cerebral [ 119] , coronary[ 76] , renal[ 127] and mesenteric microcirculation [ 14,52] and activation of these channels permits small arteries to maintain an optimal diameter for the perfusion of distal tissues. Most of molecular studies of KCa channel structure have focused on large conductance Ca2+-activated K+ channels. Unlike Kv channels that originate from multiple gene familes, KCa channel α-subunits appear to arise from a single gene family [ 132] . KCa channel shares partial homology with the shaker Kv channels, containing six transmembrane segments [ S1S6] and a highly conserved pore region between S5 and S6 [ 132] . Similar to Kv channels, the voltage sensor is located at S4. Ca2+ sensitivity of the KCa channels is conferred by an extra four transmembrane segments [ S7-S10] at the C-terminal region of the α-subunit, and by close association with a single regulatory β-subunit [ 187] . A Ca2+ binding motif composed of a string of aspartate residues may provide a site for Ca2+ binding on the Cterminal region. Additionally, interaction between the α-subunit and β-subunit greatly enhances Ca2+ sensitivity to the KCa channels. Studies by Hanner et al [ 78] suggest that coexpression of β-subunits also increases the binding of high-affinity peptidyl toxin blockers, such as iberiotoxin, to the mouth of the α-subunit of the KCa channels, thereby occluding the passage of ions through the pore.

Impaired Vasomotor Function in Diabetes and Insulin Resistance Vascular endothelial cells play an important role in maintaining cardiovascular homeostasis. In addition to providing a physical barrier between the vessel wall and circulating blood, the endothelium secretes mediators that regulate platelet aggregation [ 53,85,111] , coagulation [ 151,180] , fibrinolysis [ 41,168,175] and vessel tone [ 79,126] . Endothelial dysfunction results from an imbalance in the production of these mediators, which may reduce dilation, and promote vasoconstriction and thrombosis. The role of the endothelium in regulating vasomotor tone is one of the most widely studied aspects of vascular function. Endothelial cells release vasodilator substances including nitric oxide [ NO] , prostacyclin [ PGI2] and endothelium-derived hyperpolarizing factor [ EDHF] . NO is the major contributor to endothelium-dependent relaxation in large arteries with PGI2 and EDHF playing a less prominent role. In addition to regulating vessel tone, NO protects the blood vessel from atherosclerosis by stimulating molecular signals that prevent platelet and leukocyte interaction with the vascular wall and inhibit vascular smooth muscle proliferation. In small resistance arteries, EDHF predominates as a mediator of endothelium-dependent dilation by opening K+ channels in vascular smooth muscle membranes.

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Reactive Oxygen Species and K+ Channel Function in Diabetes and Insulin Resistance 69 In diabetes, including both type 1 and type 2, numerous studies have shown the impairment of endothelium-dependent relaxation to various receptor-mediated agonists, including acetylcholine [ 1,13,55] , bradykinin [ 74,155] , and flow-mediated dilation [ 13,71] in different vascular beds from human [ 45,153] or animal tissue [ 71,102,150] . Endothelial dysfunction in diabetes appears to be associated specifically with hyperglycemia rather than other metabolic disturbances. As gleaned from vitro studies, exposure of arteries to high glucose mimics impaired endothelium-dependent dilation, that occurs in diabetes. However altered vasomotor function in diabetes is not only due to endothelial dysfunction. Previous studies [ 114,122] from our laboratory suggest that vascular smooth muscle cell are also involved, particularly, K+ channel mechanisms as is discussed in detail below. Insulin resistance states typified by obesity, type 2 diabetes and polycystic ovarian syndrome are associated with impaired glucose uptake into skeletal muscle and fat cells, and inhibition of lipolysis in adipose tissue resulting in increase systemic free fatty acid release.. Insulin resistance has been extensively studied with respect to its role in the pathogenesis of type 2 diabetes. However, recent studies in animals and humans demonstrate that in addition to the impaired classic actions of insulin, dilations to endothelium-dependent pharmacological stimuli are also decreased in insulin resistance. The impaired vasomotor function involving both endothelium and smooth muscle cells is independent of hyperglycemia as discussed below.

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Hyperglycemia, Oxidative Stress and Sources for Generating Reactive Oxygen Species The Enhanced oxidative stress of hyperglycemia has been linked to the vascular complications of diabetes[ 88] . Several lines of evidence suggest that the production of superoxide is increased when levels of glucose are elevated. Tesfamarian and Cohen showed that incubation of rabbit aorta in 44 mM glucose impaired NO-mediated dilation by an SODinhibitable mechanism [ 178] . Similarly, studies performed in our laboratory demonstrated increased superoxide production by fluorescence microscopy with hydroethidine as a probe, in rat coronary resistance arteries exposed to 23 mM glucose for 24 hours [ 122] . The enhanced fluorescence intensity can be reversed by superoxide dismutase indicating the contribution of superoxide. As a consequence of the quenching of superoxide by NO, a potent antioxidant byproduct is formed, peroxynitrite. Peroxynitrite [ ONOO-] is a strong and relatively stable oxidant species capable of causing lipid peroxidation and nitration of tyrosine residues on key cellular proteins[ 125,165,194,195] . An elevated level of peroxynitrite has been reported in diabetic patients [ 34,36,37] and in rat small coronary arteries [ 115] incubated in vitro in high glucose , and in aortic endothelial cells [ 195] . Since glucose and its metabolites can affect multiple pathways, there are several biochemical mechanisms that appear to explain the sources for generating reactive oxygen species in hyperglycemia. For instance, glucose is transported into the vascular cells mostly by GLUT-1 transporters, which can be regulated by extracellular glucose concentration. Once glucose is transported, it is metabolized to alter signal transduction pathways, such as activation of diacylglycerol and protein kinase C, or to increase flux through the mitochondria

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to change the redox states. The common pathways of sources for generating reactive oxygen species are described below.

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Activation of Polyol Pathway The polyol pathway consists of two enzymes, aldose reductase which reduces glucose to sorbitol in the presence of NADPH, and sorbitol dehydrogenase which converts sorbitol to fructose with its co-factor NAD. Under conditions of euglycemia, the intracellular concentration of sorbitol is very low due to high enzyme kinetics of aldose reductase. However, when glucose level is elevated, more glucose is channeled into polyol pathway. The conversion of glucose to sorbitol in cells by aldose reductase has been associated with microvascular complications and enhanced oxidative stress in diabetes [31,43,60] . In animal models, treatment with aldose reductase inhibitors effectively prevents the development of various diabetic complications, including cataract [68] , neuropathy [40,60,192] , and nephropathy [ 20] . There are three potential mechanisms by which the polyol pathway can contribute to oxidative stress. First, activation of the polyol pathway depletes the co-factor NADPH, which is required for generating glutathione, a key intracellular antioxidant. Second, the production of NADH is increased in the process of oxidation of sorbitol to fructose. NADH is a substrate for NADPH oxidase for generating reactive oxygen species [ ROS] . Finally, fructose, the product of polyol pathway and its metabolites, fructose 3-phosphate and 3-deoxyglucosone are glycation agents, which increase in the formation of advanced glycation end products [ AGE] and cause oxidative stress. However, enhanced oxidative stress in diabetes can not be fully explained by activation of the polyol pathway. For example, aldose reductase inhibitor, sobinil or tolrestat, caused only a modest improvement in the impaired endothelium-dependent dilation by glucose exposed platelets in rabbit carotid arteries [148] . In sciatic nerve, the level of sorbitol does not correspond to the severity of neural dysfunction [145] . These observations suggest that other mechanisms may also be involved in the generation of ROS in diabetes.

Activation of Diacylglycerol-Protein Kinase C Pathway In animals with chemically or genetically induced diabetes, enhanced protein kinase C [ PKC] activity has been observed in many vascular tissues such as the aorta, heart, retina, and renal glomeruli [ 27,72,82,95,97,97,99,112] . The diacylglycerol [ DAG] level has also been shown to be increased parallel with PKC. Increasing glucose levels from 5 to 22 mmol/L in the culture media elevated cellular DAG levels in aortic endothelial and smooth muscle cells [94] , retinal endothelial cells [ 171] and renal mesangial cells [ 15,176] suggesting the important contribution of DAG in elevated level of PKC in diabetes. DAG can be generated from multiple pathways. Several studies examining the source of generation of DAG associated with high glucose indicate that increased level of DAG by high glucose are derived partly from the de novo pathway. For example, in labeling studies using [6-3H]- or [U-14C]glucose, elevated glucose increased the incorporation of glucose into the glycerol backbone of

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Reactive Oxygen Species and K+ Channel Function in Diabetes and Insulin Resistance 71 DAG in aortic endothelial cells, aortic smooth muscle cells and renal glomeruli. Another pathway for excess DAG production in high glucose is the result of glycol-oxidation induced activation of the DAG pathway, since oxidants such as H2O2 are known to activate the DAGPKC pathway. One of the actions of PKC is to activate NAD[ P] H oxidase, an enzyme for generation of superoxide. Exposure of cultured bovine aortic endothelial and smooth muscle cells in glucose [ 400 mg/dl for 72 hours] significantly increased free radical generation measured by electron spin resonance, compared to the cells incubated in 100 mg/dl glucose. A similar increment in free radical formation was also observed in cells treated with phorbol myristic acid [ PMA] , a PKC activator. The enhanced free radical production by high glucose or by PMC can be restored by diphenylene iodonium [ DPI] , a NADPH oxidase inhibitor and PKC inhibitor, calphosin C or GF109203X indicating that high glucose stimulates ROS generation via a PKC-dependent activation of NADPH oxidases in vascular cells. The activation of PKC by hyperglycemia appears to be tissue specific, since it has been noted in the retina, aorta, heart and glomeruli but not in the brain and peripheral nerves in diabetic animals. Therefore other pathways may also be involved in the production of ROS in diabetes.

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Upregulation of NADPH Oxidase NADPH oxidases of the cardiovascular system are membrane-associated enzymes that catalyze the production of superoxide from molecular oxygen and NADPH. This enzymatic activity has been shown to be the major oxidase in vascular tissue [ 139,160,189] and in cardiac cells [ 138] as compared with production of ROS from xanthine oxidase, arachidonic acid, and mitochondrial oxidases. The vascular form of the NADPH oxidase consists of two membrane-bound elements, gp91phox and p22phox, two cytosolic components, p67phox and p47phox, and a low molecular weight G protein, either rac 2 or rac 1. The essential subunit of the NADPH oxidase is gp91phox, to which are bound the electron carrying components of the oxidase. p22phox has a tail in the cytosol. When p47phox is phosphorylated, it binds to p67phox and brings the entire cytosolic oxidase complex to the membrane to assemble the active oxidase. p67phox is generally thought of as an accessory protein whose exact function is unclear, although it is required for the activity of the oxidase. p47phox is the protein that carries the cytosolic proteins to the membrane proteins as described above. Several reports have recently shown that the expression of NADPH oxidase subunit proteins, including p22phox, p47phox, or p67phox, is upregulated in aorta from animal models of diabetes [ 42,82,96,106] and in saphenous vein and internal mammary arteries from patients with diabetes and coronary artery disease [ 77] . Studies by Hink et al [ 82] demonstrated a 7-fold increase in gp91phox mRNA accompanied by an increase in superoxide in aorta from chemically-induced diabetic rats. Studies from our laboratory showed an elevated level of superoxide production in rat small coronary arteries exposed to 23 mmol/l glucose for 24 hours [ 122] . The enhanced superoxide production was reduced by co-incubation of arteries with DPI, an NADPH oxidase inhibitor. DPI also restored the whole cell K+ current density in cells from arteries incubated with high glucose [ unpublished data] . These results support the idea that vascular NADPH oxidase may play a role in generation of superoxide in hyperglycemia.

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Mitochondrial Generation of ROS The mitochondrial respiratory chain consists of four inner membrane associated enzyme complexes, with cytochrome c and the mobile carrier ubiquinone,[ 38,113,183,185] by which electrons donated by NADH derived from both cytosol and mitochondrial TCA cycle are transported to NADH:ubiquinone oxidoreductase [ complex I] . Complex I ultimately transfers its electrons to succinate:ubiquinone oxidoreductase [ complex II] . Electrons from reduced ubiquinone are then transferred to ubiquinol:cytochrome c oxidoreductase [ complex III] by the ubisemiquinone radical-generating Q cycle[ 184] . Electron transport proceeds through cytochrome c, cytochrome c oxidase [ complex IV] and, finally, molecular oxygen. The mitochondrial respiratory chain is the major site of ROS production within the cell. Superoxide is produced continually as byproduct of normal respiration. The main source of superoxide in mitochondria is from ubisemiquinone formed during the Q cycle of complex III. Complex I is also a source for ROS. Evidence that supports mitochondrial ROS production during hyperglycemia is based on experiments using bovine aortic endothelial cells exposed to 5 mmol/l or 30 mmol/l glucose. Increased ROS generation was observed in cells incubated with 30 mmol/l glucose. The enhanced ROS by high glucose can be normalized by co-incubation of cells with thenoyltrifluoroacetone [TTFA] , an inhibitor of complex II or carbonyl cyanide m-chlorophenylhydrazone [CCCP] , an uncoupler of oxidative phosphorylation that abolishes the mitochondrial membrane proton gradient. The data suggest that mitochondria play an important role in generating superoxide during hyperglycemia. In summary, multiple pathways have been proposed as responsible for generating superoxide during hyperglycemia, including increased polyol pathway flux, activation of DAG-PKC pathway, upregulation of NADPH oxidase and mitochondrial electron transport chain. The contribution of each pathway to the generation of ROS in hyperglycemia varies between tissue type, animal species and experimental conditions. A unifying linking mechanism of these pathways in hyperglycemia remains to be further investigated.

Hyperglycemia, Reactive Oxygen Species and K+ Channel Function Potassium efflux through K+ channels regulates membrane potential and vascular tone in response to a variety of pharmacological dilator agents including EDHF, and in response to hypoxia and ischemia. Alterations in the activity of these K+ channels by ROS during the enhanced oxidative stress of diabetes are likely to modulate vascular resistance. Understanding the mechanisms by which ROS affects K+ channels may suggest new therapeutic approaches for the treatment of the vascular dysfunction associated with this disease. The effect of hyperglycemia on three major types of K+ channels, including KATP, Kv and KCa, should be considered.

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Reactive Oxygen Species and K+ Channel Function in Diabetes and Insulin Resistance 73 KATP Channels In diabetes, the role of the KATP channel in vasodilation has been studied by several laboratories [ 103,129,129,166,166] . Impaired dilation to the KATP channel openers aprikalim, levcromakalim and cromakalim has been observed in aorta [ 103] , mesenteric [ 25] and cerebral arteries [ 129,130,130] of streptozotocin-induced diabetic rats. In human coronary arteriolar dilation to aprikalim, a selective KATP opener, is reduced in subjects with type 1 or type 2 diabetes and coronary artery disease [ CAD] compared to those with CAD but without diabetes. KATP channels contribute substantially to the dilation to hypoxia in these patients. Consistent with this finding, hypoxic dilation is reduced in subjects with diabetes. Surprisingly, enhanced vasodilator responses to aprikalim have been observed in coronary microvessels of diabetic and hyperglycemic dogs, and these enhanced dilator responses persist during coronary occlusion [ 169] . It remains unclear whether the expression or properties of the KATP channel are altered in diabetes, and how this would affect KATP channel-mediated vasodilator properties when diabetes co-exists with ischemic heart disease. In contrast to the vascular response, the effect of ROS on KATP channel activity in cardiac myocytes is different. Studies by Tokube et al [ 181] reported that O2•- increases the open state probability of KATP channels in ventricular cells from guinea pigs in cell-attached and inside out patches. This effect is enhanced by ADP, and abolished by either radical scavengers or glibenclamide suggesting the contribution of O2•- in mediating KATP channel function. It has been also reported that hydroxyl radicals and hydrogen peroxide activate cardiac KATP channels [ 182] , which could be one of the mechanisms contributing to the phenomenon of ischemic preconditioning [ 89] . The increased KATP channel activity by O2•may involves modulation of the ATP binding site of the SUR subunit of the channel [ 188] . NH3+

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Out

α

0

1

2

+ +

3

4+

5

β

6

+

NH3+

Pore domain

In COO-

COO10

9

8

7

Ca2+

Figure 3. α-subunit of KCa channel consists of ten transmembrane segments [ S1-S10] . Voltage sensor is located at S4. The region between S5 and S6 is the pore forming region. The Ca2+ sensitivity of the KCa channels is conferred by transmembrane segments, S7-S10 at the C-terminal region of the αsubunit, and by close association with a single regulatory β-subunit. Adapted from reference [ 107]

Data showing O2•- -induced activation of cardiac KATP channels contrast with the inhibitory effect of ROS on KATP channels in the vasculature, suggesting that major differences in the regulation or subunit composition of KATP channels exist between cardiac and vascular channels.

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Since vascular KATP channels, in contrast to cardiac KATP channels, demonstrate reduced activity when exposed to O2•-, it may be expected that the interaction between ROS and KATP channels during diabetes will be a complex relationship with qualitatively different effects on vascular vs myocardial KATP channels. Additional studies involving electrophysiological, molecular and functional approaches will be needed to determine the mechanisms by which hyperglycemia influences KATP channel -induced vasodilation.

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Kv Channels The effect of oxidative stress on Kv channel activity has been examined in Xenopus oocytes where shaker channel [ Kv1.3, Kv1.4 and Kv1.5] and shaw channel [ Kv4.3] were expressed[ 59] . Exogenous generation of ROS markedly inhibited Kv1.3, Kv1.4, and Kv1.5, but not Kv1.2, Kv2.1, and Kv4.1. It is likely that ROS other than superoxide were responsible for these effects since exposure to xanthine plus xanthine oxidase had no effect on any of the channels studied[ 59] . We have examined the effect of redox stress on Kv channels in rat coronary small arteries. 4-aminopyridine, a broad spectrum inhibitor of the Kv channels, produces a dosedependent constriction [ 21,24,122] . When vessels are incubated in elevated levels of glucose, an SOD-inhibitable reduction in constriction to 4-AP is observed [ 122] . This effect of oxidative stress on Kv channel function was confirmed in separate studies. Dilation to the beta-adrenergic agonist, isoproterenol which increases production of cAMP, is mediated in part by Kv channels since the dilation is blocked by 4-AP[ 114] . Exposure to elevated levels of glucose inhibits coronary dilation to both isoproterenol and forskolin, a selective activator of adenylate cyclase. Interestingly the production of cAMP was not affected by high glucose[ 114] suggesting that the inhibitory effect occurs distal in the signaling pathway. The 4-AP inhibitable component of the dilation was reduced by incubation in high glucose, supporting the idea that ROS inhibit dilation through an effect on Kv channels. We examined more directly the effect of superoxide generated by the reaction of xanthine and xanthine oxidase on Kv channel activity in freshly isolated rat coronary smooth muscle cells using patch clamping whole-cell configuration [ 122] . Superoxide reduced 4-AP sensitive K+ current density. A similar reduction in 4-AP sensitive current was also observed in cells from arteries exposed to high glucose [ figure 4] . The reduced whole cell current is not due to changes in osmolarity since increasing osmolarity with L-glucose [ not metabolized] had no effect on either the dilator response or current density. The role of ROS in this response to high glucose is evident by the fact that SOD and catalase partially restored the voltage-dependent potassium channel current density [ figure 5] . Catalase alone had little effect on Kv channel currents. Thus ROS, probably superoxide, impair Kv channel function in the coronary vasculature either directly or through the effects of elevations in glucose. Several studies have shown an increased level of peroxynitrite [ ONOO-] , a product formed by reaction of O2•- and NO in diabetes and in hyperglycemia [ 194,195] . One of the detrimental effects of ONOO- is nitration of tyrosine residues that alters the function of proteins, as occurs with superoxide dismutase [ 125] , cytochrome P450 [ 165] and prostacyclin synthase[ 194,195] . Based on these findings, we have performed studies to examine the effect of ONOO- on Kv channel function, particularly the Kv1 family. The

Diabetes Mellitus Research Advances, Nova Science Publishers, Incorporated, 2008. ProQuest Ebook Central,

Reactive Oxygen Species and K+ Channel Function in Diabetes and Insulin Resistance 75 contractile response to 4-AP was greatly attenuated by authentic ONOO-, but not decomposed ONOO- [ DC-ONOO-] . Further study identifying the involvement of Kv channel subtype family was performed in freshly isolated rat coronary smooth muscle cells using patch clamp whole-cell recording. Correolide, a specific blocker of Kv1 family[ 7,115] , reduced wholecell K+ current when cells were exposed to DC-ONOO-. NG

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Figure 4. Whole cell K+ currents in coronary VSMCs from arteries incubated with normal glucose [ NG] , L-glucose [ LG] and high glucose [ HG] . Outward currents in cells from arteries exposed to HG were reduced compared to cells from arteries incubated in NG or LG. 4-aminopyridine [ 4-AP, 3 mmol/L] blocked a large component of the outward current in cells from NG and LG arteries, but caused less inhibition in cells from HG arteries. B: I-V relationships of K+ current [ Ik] densities in cells from arteries exposed to NG, LG or HG. Density was significantly reduced in cells from HG arteries compared to cells from NG and LG arteries [ *P