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Role of cardiac FoxO1 in conditions of insulin resistance, nutrient excess, and diabetes Puthanveetil, Prasanth Nair 2012

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ROLE OF CARDIAC FOXO1 IN CONDITIONS OF INSULIN RESISTANCE, NUTRIENT EXCESS, AND DIABETES by PRASANTH NAIR PUTHANVEETIL  B.Pharm, Dr. MGR Medical University, India 2002 M.Pharm, MAHE, Manipal, India 2005 MSc, The University of British Columbia, 2008  A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in The Faculty of Graduate Studies (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  February, 2012 Prasanth Nair Puthanveetil, 2012  Abstract Glucocorticoids increase PDK4 mRNA and protein expression, which phosphorylates PDH, thereby preventing the formed pyruvate from undergoing mitochondrial oxidation. This increase in PDK4 expression is mediated by the mandatory presence of FoxOs in the nucleus. In the current study, we examined the importance of the non-genomic effects of dexamethasone (Dx) in determining the compartmentalization of FoxO, and hence its transcriptional activity. Rat cardiomyocytes exposed to Dx produced a robust decrease in glucose oxidation. Measurement of FoxO compartmentalization demonstrated increase in nuclear, but resultant decrease in cytosolic content of FoxO1 with no change in the total content. The increase in nuclear content of FoxO1 correlated to an increase in nuclear phospho p38 MAPK together with a robust association between this transcription factor and kinase. Dx also promoted nuclear retention of FoxO1 through a decrease in phosphorylation of Akt, an effect mediated by heat shock proteins binding to Akt. Measurement of the nuclear and total expression of Sirt1 protein showed no change following Dx.  Instead, Dx increased the association of Sirt1 with FoxO1, thereby  causing a decrease in FoxO acetylation. Our data suggests that FoxO1 has a major PDK4 regulating function.  In addition, given the recent suggestions that altering glucose  utilization can set the stage for heart failure, manipulating FoxO could assist in devising new therapeutic strategies to optimize cardiac metabolism and prevent PDK4 induced cardiac complications.  Obesity due to nutrient excess leads to chronic pathologies  including Type 2 diabetes (T2D) and cardiovascular disease. Related to nutrient excess, FoxO1 has a role in regulating fatty acid (FA) uptake and oxidation, and triglyceride storage by mechanisms that are largely unresolved. We examined the mechanism behind  ii  palmitate (PA) induced TG accumulation in cardiomyocytes. To mimic lipid excess, rat ventricular myocytes were incubated with albumin bound PA (1 mM) or rats were administered Intralipid (20%). PA treated cardiomyocytes showed substantial increase in TG accumulation, accompanied by amplification in nuclear migration of phospho-p38 and FoxO1, iNOS induction and translocation of CD36 to the plasma membrane. PA also increased Cdc42 protein and its tyrosine nitration, there by re-arranging the cytoskeleton and facilitating CD36 translocation.  Impeding this FoxO-iNOS-CD36  pathway could decrease cardiac lipid accumulation and oxidative/nitrosative stress, and help ameliorate the cardiovascular complications associated with obesity and diabetes. Cardiomyocyte cell death is a major contributing factor for diabetic cardiomyopathy, and multiple mechanisms have been proposed for its initiation. We hypothesized that the diabetes induced nuclear presence of FoxO1 and generation of nitrosative stress induces cardiac cell death, a process mediated by GAPDH.  Diabetes increased the nuclear  content of FoxO1 as a result of attenuated survival signalling. Increased nuclear FoxO1 augmented iNOS induction in the diabetic myocardium. The iNOS induced nitrosative stress increased the nitrosylation of GAPDH accompanied by its binding to Siah1 and translocation to the nucleus with an increased nuclear nitrosative stress. iNOS also nitrosylated caspase-3 there by hindering its ability to cleave PARP, a direct downstream target of Caspase-3.  The resultant effect is activation of PARP with an nuclear  compartmentalization of Apoptosis Inducing Factor (AIF) and resultant cell death. Overall, by regulating the FoxO1-iNOS pathway, we could dampen the cardiac damage observed during diabetes.  iii  Preface Chapter 2, 3 and 4 is based on work that I have published during my Ph.D. study. For each publication, I have indicated the contributions of my co-authors. 1  Puthanveetil P, Wang Y, Wang F, Kim MS, Abrahani A, and Rodrigues B. The  increase in cardiac pyruvate dehydrogenase kinase-4 after short-term dexamethasone is controlled by an Akt-p38-forkhead box other factor-1 signaling axis. Endocrinology, May 2010, 151(5). (doi: 10.1210/en.2009-1072) I was the first author in this manuscript. Myself and my supervisor, Dr Rodrigues were the principal sources of the idea, experimental design and writing of the manuscript. I was mainly responsible for acquiring and analyzing the data, and also designing and conducting all of the experiments. Ying Wang and Minsuk Kim helped with Western blot. Fang Wang helped with the immunohistochemistry. Ashraf Abrahani assisted in isolating rat cardiomyocytes and Dr Brian Rodrigues is the corresponding author. 2  Puthanveetil P, Wang Y, Zhang D, Wang F, Kim MS, Innis S, Pulinilkunnil T,  Abrahani A, and Rodrigues B. Cardiac triglyceride accumulation following acute lipid excess occurs through activation of a FoxO1-iNOS-CD36 pathway.  Free Radical  Biology & Medicine 51 (2011) 352–363. (doi:10.1016/j.freeradbiomed.2011.04.009). I did the experimental design, acquired most part of the data, analyzed and interpreted the results, performed statistical analysis and also wrote the manuscript. Ying Wang was involved in acquiring some of the Western blot data. Minsuk Kim and Fang Wang helped with the Immunofluorescence, Sheila Innis measured triglyceride content. Thomas Pulinilkunnil helped with membrane fraction isolation of CD36.  Ashraf  iv  Abrahani helped with the isolation of actin cytoskeleton. Dr Brian Rodrigues is the corresponding author. The investigation conforms to the guide for the care and use of laboratory animals published by the US National Institutes of Health and the University of British Columbia, and was approved by the Animal Care Committee in the University of British Columbia (Certificate No. A08-0627).  v  Table of contents Abstract .............................................................................................................................. ii Preface ............................................................................................................................... iv Table of Contents .............................................................................................................. vi List of Figures .................................................................................................................... xi List of Abbreviations ........................................................................................................xiv List of Symbols .................................................................................................................. xv Acknowledgements ...........................................................................................................xvi Dedication ....................................................................................................................... xvii Chapter 1: Introduction ....................................................................................................1 1.1 Diabetes.........................................................................................................................1 1.1.1 Type 1 diabetes .........................................................................................................1 1.1.2Type 2 diabetes .........................................................................................................3 1.1.2.1 High fat and obesity ............................................................................................4 1.1.2.2 Glucocorticoids and insulin resistance .................................................................4 1.2 Diabetic cardiomyopathy..............................................................................................7 1.3 Mechanisms behind cardiac injury ...............................................................................9 1.3.1  Pyruvate dehydrogenase kinase 4 (PDK4) and glucose metabolism .....................9  1.3.2  Nitrosative stress ............................................................................................... 11  1.3.2.1 Nitrosative signalling ........................................................................................ 13 1.3.3  CD36 induced lipotoxicity................................................................................. 15  1.3.4  Cell death .......................................................................................................... 17  1.4  FoxO1 ..................................................................................................................... 21  vi  1.4.1  Structure ........................................................................................................... 21  1.4.2  Transcriptional activity and post translational modifications .............................. 21  1.4.3  Physiological role .............................................................................................. 22  1.4.4  FoxO1 activation ............................................................................................... 23  1.4.3  Consequences of FoxO1 overactivation ............................................................. 24  1.4.3  Other roles ........................................................................................................ 25  1.5 Hypothesis and specific aims ...................................................................................... 26 Chapter 2: Methods.......................................................................................................... 30 2.1 Experimental animals ................................................................................................. 30 2.2 Isolation of cardiomyocytes ........................................................................................ 30 2.3 Plasma measurements................................................................................................. 31 2.4 Glucose oxidation in isolated cardiomyocytes ............................................................ 31 2.5 Pyruvate dehydrogenase activity assay ....................................................................... 32 2.6 Isolation of mitochondrial fraction ............................................................................. 32 2.7 Isolation of cytosolic and nuclear fractions ................................................................. 32 2.8 Plasma membrane isolation and determination of CD36 ............................................ 33 2.9 Filamentous and globular actin ................................................................................... 34 2.10  Immunoprecipitation .............................................................................................. 34  2.11  Western blotting .................................................................................................... 35  2.12  Immunoflourescence .............................................................................................. 35  2.13  Uptake of BODIPY................................................................................................ 36  2.14  Real time-PCR ...................................................................................................... 36  2.15  Nitrite and nitrate determination ............................................................................ 36  vii  2.16  PARP activity assay ............................................................................................... 37  2.17  Measurement of cardiac triglycerides ..................................................................... 37  2.18  Treatments ............................................................................................................. 37  2.19  Materials................................................................................................................ 38  2.20  Statistical analysis ................................................................................................. 39  Chapter 3: Results ............................................................................................................ 40 3.1 The increase in cardiac PDK4 following short term dexamethasone is controlled by an akt-p38-FoxO1 signalling axis ........................................................................................ 40 3.1.1  Reduction in cardiac glucose oxidation following Dx is mediated by PDK4 ...... 40  3.1.2  Dx promotes nuclear shuttling of FoxO1 but not FoxO3a .................................. 40  3.1.3  Nuclear transport of FoxO1 is mediated by Dx induced phosphorylation  of p38 MAPK ................................................................................................................. 41 3.1.4  Phosphorylated p38 exhibits an intense association with FoxO1 ........................ 41  3.1.5  Reduction in phosphorylation of FoxO1 is influenced by heat  shock dampening of Akt signalling ................................................................................. 42 3.1.6  Insulin dose dependently reverses the effect of Dx to promote  nuclear compartmentalization of FoxO ........................................................................... 42 3.1.7  Dx increases the association of Sirt1 with FoxO leading to its de-acetylation .... 43  3.1.8  Manipulation of FoxO1 can alter the increased expression of  PDK4 observed with Dx ................................................................................................. 43  viii  3.2  Cardiac triglyceride accumulation following acute lipid excess is through  activation of a FoxO1-iNOS-CD36 pathway........................................................................ 44 3.2.1  Cardiac TG accumulation following lipid excess is mediated by an increase  in membrane CD36 ......................................................................................................... 44 3.2.2  Palmitate induced increase in nuclear content of FoxO1 is  regulated by phospho-p38 and 14-3-3 ............................................................................. 44 3.2.3  iNOS and Cdc42 plays an important role in actin cytoskeleton  re-arrangement following lipid excess ............................................................................. 45 3.2.4  LPS and TNF α confirm the iNOS mediated increase in membrane CD36 ......... 46  3.2.5  Palmitate increases the nuclear content of p65 subunit of NF-k B  along with FoxO1 .......................................................................................................... 47 3.2.6  Nuclear expulsion of PGC-1 α following palmitate is linked to its  increased acetylation ...................................................................................................... 47 3.2.7 Intralipid infusion increases cardiac triglyceride with an associated increase in nuclear FoxO1 and iNOS expression ........................................................................... 48 3.3  Cardiac FoxO1 participates in hyperglycemia inducd cell death through  the mediation of iNOS and GAPDH............................................................................... 48 3.3.1  Diabetes increases nuclear FoxO1 as a consequence of attenuated  survival kinase signalling in the heart ............................................................................. 48 3.3.2  Increased nitrosative stress during diabetes is correlated to an  increase in iNOS protein ................................................................................................. 49 3.3.3  STZ diabetes promotes GAPDH bnding to Siah1 facilitating their  nuclear import................................................................................................................. 50  ix  3.3.4  Hyperglycemia increases cardiac PARP1 activation, nuclear migration  of AIF and externalization of phosphatidyl serine ........................................................... 50 3.3.5  Time dependent normalization of DZ-induced hyperglycemia can  reverse nitrosative stress mediated cardiac effects ........................................................... 51 3.3.6  Cardiomyocytes treated with high glucose mirror the effects of animal models  of hyperglycemia induced nitrosative stress ......................................................................... 52 Chapter 4: Discussion..................................................................................................... 106 4.1 The increase in cardiac PDK4 following short term dexamethasone is controlled by an akt-p38-FoxO1 signalling axis ...................................................................................... 106 4.2 Cardiac triglyceride accumulation following acute lipid excess is through activation of a FoxO1-iNOS-CD36 pathway ...................................................................................... 111 4.3 Cardiac FoxO1 participates in hyperglycemia inducd cell death through the ediation of iNOS and GAPDH ........................................................................................................ 116 Chapter 5: Summary and conclusions .......................................................................... 121 References ........................................................................................................................ 125  x  List of figures Figure 1  Glucose utilization in the cardiomyocyte ............................................................ 27  Figure 2  Regulation of FoxO ............................................................................................ 28  Figure 3  Structure of FoxO1 protein ................................................................................. 29  Figure 4 Dexamethasone induced inhibition of glucose oxidation is mediated by PDK4 ............................................................................................................. 53 Figure 5  Cytosolic to nuclear shuttling of FoxO1 is augmented by dexamethasone.......... 55  Figure 6  The increase in the nuclear content of FoxO1 after dexamethasone is  correlated to an increase in phosphorylation of p38 MAPK ................................................. 57 Figure 7  Augmenting p38 phpsphorylation with dexamethasone substantially increases  its attachment to FoxO1 and increases their joint nuclear import.......................................... 59 Figure 8  Dexamethasone attenuates Akt phosphorylation by means  of Heat shock proteins ......................................................................................................... 61 Figure 9  Insulin abolishes dexamethasone induced nuclear shuttling of FoxO1 ............... 63  Figure 10 Reduced acetylation of FoxO1 following dexamethasone is determined by the binding of Sirt1 to FoxO1 .................................................................................................... 65 Figure 11 FoxO1 inhibition lowers PDK4 expression seen with dexamethasone ............... 67 Figure 12 Cardiomyocyte glucose oxidation in the presence of insulin, SB202190, and nicotinamide ................................................................................................................. 69 Figure 13 Palmitate increases cardiomyocyte TG accumulation through participation of sarcolemmal CD36 .............................................................................................................. 70 Figure 14 Increase in nuclear content of FoxO1 following exposure to palmitate is mediated by p38 MAPK and 14-3-3 .................................................................................... 72  xi  Figure 15 Palmitate causes cytoskeletal re-arrangement through an iNOS-Cdc42 pathway ..................................................................................................... 74 Figure 16 iNOS is important for membrane translocation of CD36.................................... 76 Figure 17 Palmitate increases the nuclear presence and association between p65 subunit of NF kappa B and FoxO1 ................................................................................................... 78 Figure 18 Time dependent decrease in nuclear PGC-1 α following palmitate is accompanied by an increase in PGC-1 α acetylation ............................................................ 80 Figure 19 Intralipid infusion increases cardiac triglyceride with an increase in nuclear FoxO1 and iNOS expresssion .................................................................................. 82 Figure 20 Palmitate increases the association between p38 MAPK and FoxO1 in the nucleus .......................................................................................................... 84 Figure 21 Palmitate exposure reduces mitochondrial OXPHOS protein ............................. 86 Figure 22 PP2A induction following in vitro and in vivo lipid excess ................................ 87 Figure 23 Models of T1D and T2D also demonstrate an increase in nuclear FoxO1 and membrane CD36 ........................................................................................................... 88 Figure 24 STZ induced diabetes attenuates cardiac insulin signalling and increases the nuclear content of FoxO1 .................................................................................................... 89 Figure 25 Nitrosative stress following STZ-diabetes is evident in plasma and myocardial tissue ................................................................................................................. 91 Figure 26 Siah1 assists nuclear translocation of GAPDH following diabetes-induced nitrosative stress in cardiac tissue ........................................................................................ 93 Figure 27 Diabetes induced nitrosative stress increases PARP1 activation, nuclear migration of AIF and apoptosis ........................................................................................... 95  xii  Figure 28 Nitrosative stress and nuclear migration of apoptotic proteins fluctuates with the induction and reversal of hyperglycemia following diazoxide ........................................ 97 Figure 29 High glucose mediated nuclear migration of GAPDH and AIF in cardiomyocytes is reversed by the iNOS inhibitor 1400W ................................................... 99 Figure 30 LPS dose dependently increases iNOS induction ............................................. 101 Figure 31 iNOS induction can increase the nuclear content of GAPDH ........................... 103 Figure 32 A summary diagram describing the mechanism of how cardiac FoxO1 regulates cell death through the mediation of iNOS and GAPDH....................................... 105 Figure 33 Diagram describing the consequences of FoxO1 induction under conditions of nutrient excess, insulin resistance and diabetes .............................................................. 123 Figure 34 Summary of the mechanisms by which cardiac FoxO1 can bring about cell death following its activation. ............................................................................................ 124  xiii  List of abbreviations and acronyms AMPK AIF DAPI DZ Dx ERK FA FoxO GC HDAC HAT Hsp LDH LPL MAPK NEFA PA PARP PDH PDK PPARα TG TNFα T1D T2D STZ  AMP-activated protein kinase Apoptosis inducing factor 4',6-Diamidino-2-phenylindole Diazoxide Dexamethasone Extracellular regulated kinase Fatty acid Forkhead box Other transcription factor Glucocorticoids Histone de-acetylase Histone acetyltransferase Heat shock protein Lactate dehydrogenase Lipoprotein lipase Mitogen-activated protein kinase Non-esterified fatty acid Palmitic acid Poly-ADP-ribose polymerase Private dehydrogenase Private dehydrogenase kinase Peroxisome proliferator-activated receptor α Triglycerides Tumor necrosis factor α Type 1 diabetes Type 2 diabetes Streptozotocin  xiv  List of symbols  °C  Degree Celsius  g  Gram  h  Hour  i.p  Intraperitoneal  i.v  Intravenous  Kg  Kilogram  mg  Milligram  ml  Milliliter  mM  Millimolar  nM  Nanomolar  g  Microgram  g  Microgram  l  Microliter  xv  Acknowledgements  To begin with I would like to express my intense gratitude to my mentor and guide, Dr. Brian Rodrigues, Department of Pharmacology and Toxicology, University of British Columbia. I am extremely thankful to Almighty for providing such a wonderful human being as my teacher and guide. I am extremely thankful to Dr. Rodrigues for being with me during my times of crisis, and also supporting and moulding me to become what I am. I completely owe this degree to him with full heart. I am grateful to each and every member of my committee starting from Dr. Granville D. Laher, and Dr. Marzban for their valuable suggestions and encouragement and also the faculty for providing me such a wonderful opportunity to come here and experience this academic and research setting. I am thankful to Dr. Burt for being extremely supportive and Rachel, Suzanna and Barb for providing right information at the right time. I thank the faculty, technical and administrative staff for the help, support and encouragement during this program I would like to express my sincere thanks to Mr.Abrahani for training me with technical skills and all my lab members for providing a wonderful support and friendly lab atmostphere. I sincerely acknowledge the support and encouragement provided to me by Dr. Pulinnilkunnil, Dr Fang Wang, Dr Dake Qi and also to lab mates Ying and Dahai and also to my dear friends Anton, Jayakumar&family and Hesham. I express my deep love and gratitude for Manuettan, and my dear ones for being with me all time and for their sincere prayers. I would also like to express my gratitude for the support from Achemmas, mamas and ammayi and also to my grandparents souls who has showered their blessings on me. I would definitely like to take this time to express my gratitude and offer prayers to all those souls of the rats who sacrificed their lives for my study. I would like to express my thanks to CDA and Heart and Stroke Foundation of BC and Yukon for their financial support during my graduate program. My special thanks to all my friends and well-wishers within and outside UBC for making my stay comfortable. Last but not the least; I express my love and affection for my loving Amma, Ammu and Achan. Without my Amma’s and Ammu’s prayers, wishes and support this would not have been possible.  xvi  TO LORD THE ALMIGHTY  xvii  Chapter 1: Introduction 1.1 Diabetes Diabetes, a growing global epidemic, is estimated to affect approximately 350 million people. The World Health Organization projects that this number will almost double by 2030, and recommends a standard diagnostic measure for diabetes to include fasting blood glucose levels equal to or more than 7 mmol/l or 11 mmol/l post-prandially together with the recently introduced HbA1c ≥6.5% (http://www.who.int/diabetes/en/).  A key feature associated with  chronic diabetes includes micro and macrovascular complications, with the latter being a prominent reason for the development of coronary artery disease, atherosclerosis and myocardial infarction1, 2. Diabetes, on the basis of origin, is classified into 2 major types; Type1 and Type2 diabetes. 1.1.1 Type 1 diabetes (T1D) T1D is mostly a chronic autoimmune disorder, resulting in the destruction of pancreatic βcells. The autoimmune attack involves helper T-cells and their co-mediators (CD8+, CD4+), and other immune cells (B cells, natural killer cells, natural killer T cells and dendritic cells) that can cause β-cell damage3, 4. T1D is polygenic in origin with environmental factors also playing a role in its onset. Genetic factors include genes that are primarily located at the HLA/IDDM1 region and these comprise of approximately 35-40% of the genes responsible for T1D5, 6. The HLA class 2 alleles (HLA-DQA1, DQB1 and DRB1) form the most common factor in humans and animals developing T1D7. Apart from class 2 alleles, a class 4 and class 1 allele have also been reported to cause T1D. Some of the genes that belong to the latter classes include tumour necrosis factor and lymphotoxin8, 9. Apart from HLA/IDDM1, other prominent locus which has  1  been proposed to induce T1D includes IDDM2 and IDDM3-18 regions. Major gene mutations arising from these regions are FADD, FasL, SEL1L, and CTLA4 genes5, 8, 9. Environmental factors include attack by viral pathogens like B3 and B4 coxsackie virus, cytomegalovirus, encephalomyocarditis virus, parvo virus, mumps virus, and rubella virus 10, 11. The auto-immune attack due to CD8+ and CD4+ involves the endogenous peptides which act as antigens and are captured either by MHC class 1 or 2 molecules12, 13. Following this, they bring about apoptosis by activation of FAS ligand and programmed cell death ligand-113. The immune cells like macrophages, natural killer cells and dendritic cells can initiate an immune response by their ability to secrete cytokines like interleukin-1 β (IL-1 β), IL-12, interferon-γ (IFNγ) and tumour necrosis factor α (TNFα)14, which then bring about β-cell apoptosis through their respective receptor actions. Microbial secretions can also induce immune response attack on βcell through their stimulatory effect on Toll like receptors 3 and 4 15, 16. Regarding virus induced T1D, they can attack by themselves or through the initiation of an immune response in the host cells. The viral particles can themselves turn on the inflammatory pathways or through the mediation of macrophages that release inflammatory molecules, turn on inflammatory pathways in the β-cells resulting in cell death10, 17, 18. Many animal models have also been developed to study and understand the exact mechanisms responsible for induction of T1D. Some of the genetic animal models include the biobreeding rats and non-obese diabetic mice19-21. Pharmacological agents have also been widely used to destroy pancreatic β-cells in animals and these include streptozotocin (STZ), alloxan and cyclophosphamide21, 22.  2  1.1.2 Type 2 diabetes (T2D) Compared to T1D, T2D is the major form of diabetes and 90% of patients have this form of diabetes. The major causes for T2D include a high fat diet and obesity, lack of exercise, increased and prolonged cytokine attack, glucotoxicity, glucocorticoids (both endogenous or exogenous as drugs), and stress23, 24. An emerging and highly promising role for genetic factors and chemicals in T2D has also been proposed25. The genetic factors include single nucleotide polymorphism of genes like HNF4 alpha, HNF 1 alpha, glucokinase, Pdx1, Kir6.2, Sur1, and PPARG25-31.  The chemicals that are known to accumulate in the pancreatic β cells causing their  destruction include pesticides like DDT, industrial wastes like polychlorinated biphenyls, dioxins and bisphenol-A32. Whatever may be the source of origin, the disease starts with a decrease in insulin sensitivity or increase in insulin resistance. Excess lipids or cytokines are known to increase the serine phosphorylation of the insulin receptor through their action on receptors like TNF-α and TLR4 or intracellular mediators like DAG and ceramide33, 34. As a result of this serine phosphorylation on  insulin receptor substrate, there is an attenuation in tyrosine phosphorylation of IRS proteins with an accompanied decrease in downstream kinases like Akt that can result in decreased efficiency of the survival kinase pathway and reduced glucose uptake in muscle and adipose 33, 35, 36. As a result of this decreased glucose uptake due to loss of insulin sensitivity in these tissues, the body tries to compensate by increasing more insulin secretion from β cells.  Due to prolonged  activation, β cells get exhausted, start declining in function, and eventually die. Over time, there is onset of hypoinsulinemia followed by hyperglycemia and T2D. The Westernized diet with inclusion of high fat has been one of the major cause for the development of obesity and obesity induced T2D34-38. Chronic use of anti-inflammatory drugs like glucocorticoids have also been associated with development of insulin resistance and T2D39, 40. 3  1.1.2.1 High fat and obesity. Obesity is a rapidly growing epidemic, with an estimated 1 billion overweight adults worldwide, of which 300 million are obese (http://www.who.int/en/).  Obesity can lead to  chronic pathologies including cancer, stroke, T2D, hypertension and cardiovascular disease, which in turn can increase morbidity and mortality41-45. Major reasons for obesity include a genetic pre-disposition (key genes implicated in obesity include adiponectin, leptin and its receptor, PPAR-ã and UCP 1, 2 and 3), and an imbalance between food intake (nutrient excessboth carbohydrate and saturated lipids) and energy expenditure (lack of exercise-sedentary lifestyle)46-48. Related to nutrient excess, in addition to their contribution towards weight gain, lipids are considered a major initiator of inflammatory pathways through increased expression of cytokines, chemokines and stimulation of their respective receptors, culminating in activation of the insulin resistance with resultant T2D and associated complications 49, 50. Some of the animal models which are used currently to study the effect of obesity include monogenic mouse strains with genetically altered leptin signaling like ob/ob and db/db, and polygenic strains like New Zealand Obese mouse, M16 mouse, Kuo Kondo mouse and rats like Zucker fatty rats and Otsuka Long Evans Tokushima Fatty rats. The non-genetic strains include high fat fed C57BL/J6 mice, sand mice, spiny mice and Sprague Dawley and Long Evans rats which can develop obesity and obesity induced T2D51-55. 1.1.2.2 Glucocorticoids and insulin resistance. Excess glucocorticoids (GC), whether endogenous (stress induced cortisol) or exogenous (as anti-inflammatory drugs), have adverse effects on multiple organs with resultant induction of osteoporosis, growth retardation in children, muscle atrophy, cardiovascular disorders and insulin resistance associated with diabetes24, 56-61. The physiological role of GC includes: a) 4  protective function to combat stress by induction of anti-oxidant genes,62-64 b) providing blood cells and neurons with enough glucose to protect them from starvation induced damage by activating  glycogenolysis  and  gluconeogenesis  through  increased  expression  of  phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression in the liver65-67, c) reducing glucose usage by muscle tissue by causing muscle-specific insulin resistance and induction of pyruvate dehydrogenase kinase-4 (PDK4) expression68, 69, and d) protecting the body from inflammatory signals turned on by endogenous cytokines or pathogens or pathogenic products through their inhibitory effects on NF kappa B activity70-72. GCs act by means of their receptor which are localized intracellularly; the glucocorticoid receptor, GR (a nuclear receptor family member). GCs on binding to its receptor in the cytosol releases the chaperones Hsp90 and FKBP which acts as a checking system for the GR by preventing their auto activation and nuclear migration24.  This nuclear migration action is also brought by a homodimerization  between two GRá subunits. The ligand bound receptor complex moves into the nucleus, which through the aid of glucocorticoid response element GRE, brings about either activation or repression of targets73. Apart from this conventional receptor pathway, they are also known to act by means of a rapid non-genomic action by means of G-protein coupled receptors, MAP kinase pathways, and the heat shock protein axis74-80. Despite these actions they have been reported to induce whole body insulin resistance when in excess68. First reported by Caro et al, continuous administration of 1 mg/kg of dexamethasone for 1-4 week caused insulin resistance in liver, with resultant depletion in insulin receptors and there by insulin action81. This was followed by Kajita’s et al study using adipocytes where GCs were able to induce insulin resistance with a reduced glucose uptake82. In humans having Cushing’s syndrome or who are administered GCs for a prolonged period, insulin resistance developed with symptoms similar to 5  the metabolic syndrome83, 84. Results from our group showed that a single dose of GC produced whole body insulin resistance followed by an alteration in cardiac metabolism68. In follow up studies, we reported that the whole body insulin resistance was due to the effect of glucocorticoids on skeletal but not cardiac muscle. In skeletal muscle, there was an attenuation of IRS-1 and Akt phosphorylation following insulin, an effect which was absent in cardiac muscle69. In the heart, they caused accumulation of both triglycerides and glycogen due to their stimulatory effects on LPL and GLUT4 translocation respectively24,  69  .  With regards to  oxidation, even though fatty acid oxidation increased, glucose oxidation was reduced in the heart24, 68. Endogenous GC excess have also been reported to have complications similar to metabolic syndrome. One of the major mediators which enhance the activity of endogenously secreted GCs are the 11 beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)85. They convert the inactive endogenous cortisone to cortisol which can then bring about their GC actions in specific tissue, with cardiac tissue being one of the tissues showing prominent expression of 11beta-HSD186. 11beta-HSD1 activity enhancement has been shown to increase fat depots by their action on adipose tissue, visceral obesity, insulin resistance, dyslipidemia, leptin resistance, and hypertension24,  85, 87  .  Recently, it has been shown that the enhanced  adipogenesis following GC treatment is due to their ability to enhance insulin sensitivity but only in adipose tissue; this occurred even in the presence of whole body insulin resistance and hyperinsulinemia88. Paterson et al have shown that an increased expression of 11beta-HSD1 in liver can exhibit features similar to metabolic syndrome even without obesity89. Cushing’s syndrome, a natural condition which has the characteristics of metabolic syndrome is known to have excess GC in the circulation followed by an enhanced activity or expression of 11betaHSD1. Thus, with the GC associated adverse events like insulin resistance and cardiovascular 6  complications, the role of 11beta-HSD1 is inevitable90. Indeed, due to these actions of 11betaHSD1 to initiate metabolic disturbances accompanied by fat deposition in conditions of cortisol excess, it has become a pharmacological target for Cushing’s and metabolic syndrome 91, 92. Apart from 11beta-HSD1, some of the target genes for GR that play an important role in metabolism like AMPK, PDK4, GLUT4, LPL, ATGL and HSL could also be playing a role in GC mediated metabolic complications accompanied by cardiovascular events. Overall, GCs when present in excess in the body can lead to central obesity, hypertension, muscle atrophy, and insulin resistance followed by T2D. 1.2 Diabetic cardiomyopathy  Independent of vascular complications, T1D and T2D can provoke a direct injury to cardiomyocytes, eventually leading to cardiac death termed diabetic cardiomyopathy. Diabetic cardiomyopathy is a multifaceted term used to describe cardiac muscle damage-induced heart failure, and multiple structural and biochemical reasons have been suggested to induce this disorder. These include an imbalance in calcium homeostasis (due to decreased expression of sarco-endoplasmic calcium ATPase and Na+-Ca2+ exchanger activity), defects in contractility (as a consequence of decreased expression of contractile proteins like myofibrillar Ca 2+-Mg2+ ATPase, myosin Ca2+-ATPase and increased Troponin-I phosphorylation), fibrosis (caused by collagen deposition due to increased TGF-β or angiotensin-II and their respective receptor activities), metabolic changes (resulting in increased lactate and changes in pH, accumulation of toxic metabolites like ceramides, and hyperglycemia induced insulin resistance) and cell death (either apoptosis, necrosis, or both)2, 93-96. Even though the above mentioned factors have been proposed for its origin, an alteration in metabolism holds the key for inducing myopathy by itself and also by the factors mentioned above2. Glucolipotoxicity due to insulin resistance of the heart 7  can bring about multiple biochemical, structural and functional changes in the heart 97. An increased serine phosphorylation of IRS-1 can bring about dampening of the growth/survival signal pathway, there by resulting in a deficit in function of glucose processing by cells, accompanied by unregulated lipid utilization and storage98, 99. The excess glucose and lipids in the system can induce ROS in cardiac cells bringing about a change in calcium homeostasis100102  . Apart from this, ROS accompanied by hyperglycemia and hyperlipidemia can increase the  formation of glycated end products and also stimulate the hexosamine biosynthetic pathways 103106  . Glycation of collagen, SERCA2a and RyR2 can increase the damage to cardiac muscle.  Hexosamine biosynthetic pathways can form excessive O-linked N-acetyl glucosamine products and one of the major targets of O-linked N-acetyl glucosamine modification is SERCA2a, leading to decrease in its function106-109.  Lipotoxicity induced ceramide and triglyceride  accumulation in the heart can cause cell death through mediation of PP2A110, 111. Glucolipotoxic events can lead to an increase in fatty acid oxidation and decrease in glucose oxidation by induction of PDK4 thereby increasing lactate formation and decreasing pH of myocardial tissue69. This altered pH can bring about changes in cardiac ionic balance (by altering H + and Ca2+ ions) which can then result in structural and functional changes. Cardiomyocyte cell death is another major contributing factor for cardiomyopathy, and multiple mechanisms have been proposed for its initiation. Extrinsic factors that include attack from neighbours like endothelial cells, vascular smooth muscles and macrophages, play a significant role in cell death. These cells release cytokines and toxic metabolites during diabetes which turn on cell death signalling cascades inside the myocardium, either through their effects on peripheral receptors like TNF-α and TRAIL, or their downstream protein targets112. Apart from this, intrinsic causes of cell death have also been reported. However, the most prominent 8  feature of the diabetic myocardium is attenuated insulin signalling that reduces survival signalling kinases (Akt), which could switch on potential protein targets like FoxOs, an initiator for cell death.  Additionally, increased stress signalling (MAPK) kinases, reactive nitrogen  species generation (peroxynitrite), reduced anti-oxidant capacity, altered mitochondrial efficiency, and insulin resistance have been implicated in this process 113-118. Some of the clinical features of diabetic cardiomyopathy include systolic and diastolic dysfunctions. The systolic impairment is due to left ventricular hypertrophy (LVH), an after effect of increased LV mass. Due to LVH there is a decrease in ejection fraction of left ventricle. Diastolic dysfunction also holds equal importance as systolic impairment in diabetic cardiomyopathy119-121. This is characterised by a perturbations in filling and relaxation during diastole which is accompanied by distension and stiffness due to fibrosis 122,  123  . Data from  Framingham study has revealed that both males and females who are diabetic are more susceptible to heart failure than the non-diabetic controls. Another interesting observation was that the risk of heart failure was also independent of other confounding factors like age, lipid profile and coronary artery disease124-126. The outcome of this study was that the cardiac reserves in diabetic hearts was depleted, as a consequence of cell death pathways getting turned on. 1.3 Mechanisms behind cardiac injury 1.3.1  Pyruvate dehydrogenase kinase-4 (PDK4) and glucose metabolism  The metabolism of glucose comprises of a number of processes which include cellular uptake and glycolysis, with the formed pyruvate undergoing oxidation in the mitochondria to yield ATP2. Insulin is a mandatory mediator for all of these glucose catabolic pathways. For example, in the heart, although basal glucose uptake is mediated by GLUT1, the major amount of glucose transported is through GLUT4. Insulin signaling plays an important role in enhancing glycolysis 9  through its influence on phosphofructokinase-1, the rate limiting enzyme for glycolysis2. Following glycolysis, glucose undergoes oxidation through the catalytic activity of pyruvate dehydrogenase complex (PDC)69.  PDC is also controlled by insulin through its ability to  suppress the expression and activity of pyruvate dehydrogenase kinase-4 (PDK4). PDK4 is known to phosphorylate the E1 moiety of pyruvate dehydrogenase of the PDC, thereby preventing the formed pyruvate from undergoing mitochondrial oxidation69. In this situation, pyruvate is converted to lactate rather than getting oxidized to acetyl CoA. Formed lactate can itself be utilized by the heart following its conversion to pyruvate (Figure 1). T1D, with its associated reduction in circulating insulin, increases PDK4 gene expression.  Evidence of  increased PDK4 expression is also documented in high fat and obesity models of insulin resistance, and is linked to both inefficient insulin action and fatty acid (FA) stimulation of PPARs and their co-activators127, 128. Like PPAR-α, liver X receptor and retinoid X receptor also play an important role in the induction of PDK4. Physiological conditions of reduced insulin like that observed during fasting is also accompanied by augmentation of PDK4 128, 129. Other triggers that are known to increase PDK4 mRNA and protein expression, both in vivo and in vitro, are stress hormones like glucocorticoids127. Our lab had shown that in vivo, a single dose of 1 mg/kg of dexamethasone showed an increase in PDK4 expression, with an altered metabolism (decreased glucose oxidation and increased fatty acid oxidation)68, 130. In conditions of nuclear receptor mediated PDK4 induction, transcription factors like FoxO1 and FoxO3 have been shown to play a crucial role in effective transcription of PDK4 in muscle tissue 131, 132. In hepatic tissue, HNF4, another transcription factor, has been shown to regulate PDK4 transcription133. Apart from hormone receptors, metabolites like pyruvate also regulate PDK4 acutely, but in a negative manner, by suppressing its activity134, 135. Increased pyruvate is a 10  consequence of an increased glucose uptake or glycogenolysis, which can then form pyruvate, and redirect the metabolism towards glucose oxidation134. By inhibiting glucose oxidation, they facilitate fatty acid oxidation, with lactate being the fate of entered glucose 2. An increased lactate could bring about a decrease in pH, which can initiate certain cell death pathways or could affect the membrane stability and structural and functional integrity through their effects on H+ ions. Increased fatty acid oxidation also contributes towards this condition along with resultant ROS induction and nitrosative stress2. Inhibition of PDK4 has been shown to be a promising target for the treatment of diabetes and metabolic syndrome, with radicicol and M77976, known PDK4 inhibitors having beneficial effects136. 1.3.2  Nitrosative stress  Nitric oxide (NO) was discovered as a wonder molecule known to have an impact on the cardiovascular system. This discovery by Robert F. Furchgott, Louis J. Ignarro and Ferid Murad obtained the Nobel prize in physiology and medicine for these scientists. NO is known to be synthesised from nitric oxide synthases. NOS family members are classified as NOS1, NOS2 and NOS3137. NOS1, known as the neuronal NOS is a 161 kDa protein, NOS2 or inducible NOS is a 131 kDa protein and third member NOS3 also known as endothelial NOS is a 133 kDa protein. All the three have similarity in their genetic constitution with their existing bidomain structure138, 139. The N-terminal has the oxygenase domain in which the haem group and BH4 group is attached. The c-terminal is a reductase domain, which has binding regions for FAD, FMN and NADPH140. The L-arginine which is a part of the oxygenase domain is the connecting link between the 2 domains through its CaM binding motif137, 139. The structural difference is mostly exhibited in the NH2 terminal, with nNOS having a PDZ-binding site and eNOS having myristoylation and palmitoylation sites, which are unique to only these NOS forms 141, 142. nNOS 11  and eNOS have the autoinhibitory loops between 2 FMN moieties, which are absent in iNOS 138, 143  . Overall, the synthesis of NO is an oxido-reductase reaction, with electrons being transferred  from NADPH in the reductase domain through the FAD-FMN path towards Fe and BH4 in the N-terminal; this catalyses the reaction between L-arginine and oxygen to produce NO141. NOS dimerization is required for its activation137, 139,  140  . Regarding nNOS and eNOS, they have  dimeric interactions between oxygenase and reductase domains of the complimentary monomers, whereas for iNOS the dimer formation is facilitated only by the oxygenase domain138, 143. iNOS has the highest potential to produce reactive nitrogen species among its family members 137, 140. The reactive nitrogen species include nitroxyl moieties, peroxynitrite and nitrosothiols. The conversion of NO to peroxynitrite happens in the presence of superoxides, the free radicals present in the system.  Systemic scavengers like SOD prevent the conversion of NO to  peroxynitrite thereby protecting the cells from increasing reactive nitrogen species attack. These increased reactive nitrogen species forms the major mediator in initiating problems during conditions like ischemia, stroke, diabetes, cancer, sepsis and inflammation. Preventing excess NO or superoxide generation could limit the complications during these conditions.  NOS  inhibitors are known to competitively but non-covalently bind to its substrate with L-arginine, biopterin and heam being the major binding sites142, 144. There are partially-selective (L-NIL and L-NIO) and highly selective (1400W, GW273629 and GW274150) iNOS inhibitors 142, 144. For example, the inhibiting potential of the selective iNOS inhibitor, 1400W on iNOS is 32 and 4000 fold more compared to nNOS and eNOS respectively. NO is physiologically well known for its vasodilation action on vascular tissue, but is also known to have anti-hypertrophic and anti-proliferative actions. Because of these properties, NO is considered a wonder molecule in conditions like hypertension, ischemia and aortic stenosis 145, 12  146  . The half life of NO in vivo or inside a living system is as low as 5 sec, but in vitro they have  been shown to have a half-life of a few minutes. In the presence of an aqueous environment and in the presence of oxygen, NO is known to get converted to nitrite which is a much more stable form147-149. 1.3.2.1 Nitrosative signalling. In the cardiovascular system, eNOS is known to increase NO with resultant ONOO-, with resultant PKCå activation.  This activated PKCå increases activation of proteins having  transcription functions like Erk, HIF-1, and NF-kappa B150-152.  These proteins induce  transcription of iNOS with resultant nitrosative stress. This NO induced NO pathways gets turned on in cardiovascular system following ischemia/hypoxic conditions. In neuronal tissues, following ischemia, nNOS mediated NO production is known to offer a protective function through the mediation of Ras-Raf-Mek-Erk pathway, with resultant induction of anti-apoptotic proteins150, 151. Apart from signalling, they are also known to bring about actions by their ability to modify proteins, and includes nitration and nitrosylation.  NO through the mediation of  intermediates like ONOO- and NO2 form 3-nitrotyrosine153.  Nitric oxide also reacts with  superoxide to form peroxynitrite which is highly unstable compared to NO.  This formed  nitrotyrosine is one of the prominent markers for nitrosative stress especially in conditions like diabetes, metabolic syndrome, hypoxia, inflammation and sepsis. Inside the body, a stable concentration of NO is maintained by superoxide dismutase, which prevents the frequent conversion of NO to peroxynitrite153. Thus, peroxynitrite and superoxide dismutase share an inverse relationship. For an effective 3-nitrotyrosine synthesis, along with NO availability, it also requires the presence of either carbonate ion, or an oxo-metal complex or hydroxyl ion. There are only few target proteins which are known to undergo nitration modification with either 13  loss or gain of function of the target154. Because of this nitration modification, they are also known to affect other types of modifications like phosphorylation. IRS-1 and MnSOD tyrosine nitration are the types of protein modification with loss of function, whereas cytochrome c and PKC€ are known to have a gain of function following protein nitration155. Nitration has also been shown to have an effect on differential compartmentalization of certain proteins; for example, cytosolic to mitochondrial distribution of aldolase-A and GAPDH156-158. Apart from having an effect on proteins, nitration also has an impact on organelles like mitochondria. By nitration of some of the major mitochondrial proteins like cytochrome c, MnSOD, aconitase, voltage gated anion channel, succinate dehydrogenase, ATP synthase, ATPase and voltage-gated K+ channels, they regulate mitochondrial function.  Apart from  mitochondria, they also have effects on protein pumps like Na+-K+-ATPase, and SERCA2A, and proteins that make up the cytoskeletal framework like actinin, synuclein, desmin, myosin heavy chain, profilin, and tubulin159-161. Lipoproteins like VLDL, LDL and HDL and proteins involved in the clotting process like fibrinogen and plasminogen are also affected by nitration 162,  163  .  Some prominent effects of nitration are also evident in cells like red blood cells. Increased secretion of reactive nitrogen species from vascular system can intrude into blood cells if their concentrations are elevated.  Uric acid is one of the known inhibitors of this nitration  modification both in vitro and in vivo. Low molecular weight porphyrin complex with Mn and Fe have also been shown to have SOD mimetic effects with reactive nitrogen species scavenging activity164, 165. Studies using genetic models have shown that MnSOD or ZnSOD overexpression has an inhibitory effect on reactive nitrogen species generation, where as their respective knock outs have proved to aggravate the nitration process associated with damage in various tissues. Increased myeloperoxidase and eosinophil peroxidase levels increase nitration of proteins and 14  increasing iron regulatory proteins hinder the overproduction of reactive nitrogen species165-170. S-nitrosylation is another major nitrosative stress mediated protein modification where a nitrosonium group has been attached to thiolate in cysteine 171,  172  .  The process of trans-  nitrosylation being another sub-process within S-nitrosylation which involves interchange of NO+ groups between a nitrosothiol and thiolate group171-173. There are more than 100 substrates that can be S-nitrosylation modified, and it includes caspase (1-8), haemoglobin, myoglobin, GAPDH, NMDA receptor complex, aquaporin-1, glutathione, ferritin, homocysteine, serum albumin, aldehyde dehydrogenase and hexokinase-1173. S-nitrosylation of proteins has been shown to regulate synaptic function, redox response, cell death, protein quality control, mitochondrial function and transcription function173-179. Irrespective of nitration or nitrosylation, nitrosative stress induced complications have a significant role in sepsis, neurodegeneration, vascular complications, myocardial infaction, cardiac ischemic disease, insulin resistance, diabetes, and diabetic cardiomyopathy180-183. Tyrosine nitration or S-nitrosylation modifications brought about by nitrosative stress can change the activity of target proteins which can then cause the above mentioned complications, with iNOS being the major nitrosative agent among the members of NOS family in these conditions. 1.3.3 CD36 induced lipotoxicity On a molar basis, as ATP derived from FA is several fold higher than that produced by glucose, cardiac tissue prefers FA over glucose2. Exogenous FA are delivered to the heart by LPL-mediated lipolysis of TG-rich lipoproteins and albumin bound FA2. After traversing the interstitial space, FA are taken up by the cardiomyocyte using a transporter system which includes FABPpm, FATP and FAT/CD36184-186. Among these transporters CD36, plays an important role as lipid provider in the heart. CD36 or clustered domain 36 is also known as the 15  collagen type 1 or thrombospondin receptor 187-189. It has multiple ligands inside the body starting from free fatty acids, collagen, thrombospondin, anionic phospholipids and oxidized LDL190-192. It also has multiple functions like fatty acid transport, internalization of oxidized lipids, phagocytosis, cell migration, cell adhesion, cholesterol efflux and anti-angiogenesis187-189. CD36 contains 472 amino acids; with a molecular weight of 53 kDa in unglycosylated state and 85-88 kDa in the glycosylated state193-195. They exhibit N-glycosylation and O-glycosylation with N-glycosylation showing a greater abundance than the O-glycosylation form.  Post  translation modifications that determine their activity are glycosylation, palmitoylation and ubiquitination.  While glycosylation and palmitoylation increase its activity, ubiquitination  decreases CD36 activity191-196. To show their function, it is mandatory that they reach the plasma membrane surface and there are various mechanisms to determine this 191, 192, 195, 196. The most prominent stimulants include insulin, exercise and oligomycin. It has been shown that PI3 kinase, Akt and AMPK can regulate CD36 membrane translocation acutely, and this has been confirmed with the use of stimulants and inhibitors of these proteins192.  Cytoskeletal and  vesicular proteins also play a major role in effective translocation of this protein to the membrane surface192. Apart from this, some studies have shown that dimerization of CD36 is also mandatory for effective membrane translocation197, 198. An increased expression of CD36 at the membrane surface has shown to be associated with triglyceride accumulation as droplets192, 198  . Increased fatty acid flux inside the cell can lead to an uncoupling between fatty acid uptake  and oxidation leading to the generation of other fatty acid metabolites like DAG and ceramides which are toxic to the cell199, 200. Increased intracellular triglyceride can generate more free fatty acid moieties within the cell, whereas increased ceramide can lead to increased expression of PP2A, a causative factor for induction of apoptosis through dephosphorylation of BAD and 16  survival kinases200-202. Increased ceramide has also shown to promote caspase-independent cell death and even necrosis203. With increasing accumulation of triglyceride and ceramide inside the cardiac muscle tissue, contractile force and frequency is hampered due to actions on structural and contractile proteins203, 204. Increased fatty acid oxidation due to increased fatty acid flux can also lead to increased ROS generation2, 205. Increased ROS can in turn lead to stress signal and cell death signal activation there by causing total catastrophic events like necrosis and apoptosis. ROS has shown to decrease the anti-oxidant capacity of the cells directly and also through their ability to form reactive nitrogen species2,  205  . ROS is also known to regulate metabolism,  structural integrity and biochemistry of the cell. Thus, regulating CD36 could help in controlling all of the damage associated with excess lipid induced cell damage and cell death. CD36 induced lipotoxicity has been shown to play a major role in cardiovascular pathology and even induction of insulin resistance and diabetes206-208.  With the given evidence that selective  overexpression of CD36 in the heart is sufficient to cause high fat induced cardiomyopathy, and CD36 KO has shown alleviation in lipotoxic events208, regulation of this major fatty acid transporter could offer a cure during metabolic disturbances like diabetes. 1.3.4 Cell death Cell death can basically be classified into programmed and non-programmed cell death. From the name, it suggests that programmed cell death is under total control and will of the cell. Programmed cell death is structured to either save the tissue/organ system from loss of ATP or to prevent the cell from other malicious forms of death where there is a disposal of cellular contents to the neighbourhood there by bringing about or turning on cell death of neighbouring cells 209-212. Apoptosis, autophagy and apoptosis like programmed cell death are some examples of a programmed death process, whereas necrosis is an example of non-programmed cell death212-216. 17  In apoptosis and apoptosis like programmed cell death, there is chromatin condensation whereas autophagy involves a sequential process whereby there is sequestration of cell organelles into autophagic vesicles followed by their degradation by lysosomal particles214, 216, 217. Necrosis is characterized by organelle swelling, especially the cytosol, followed by plasma membrane rupture213, 217, 218. Apoptosis is initiated by either intrinsic or extrinsic factors219. The intrinsic cascade starts with a destabilized mitochondrial membrane pore opening and subsequent release of cytochrome c to the cytosol220-222. This released cytochrome c in the cytosol forms a complex with Apoptosis Activating Factor-1 (Apaf-1), and procaspse-9, to create an active complex which is called apoptosome220, 222-226. Apaf-1 has a head domain called CARD, which is kept inert by placing it in-between 2 β sheets.  Cytosolic cytochrome c displaces CARD from the clutches of the  inhibitory β sheet like conformation, allowing them to form a complex with caspase-9 and cytochrome c222,  225, 226  .  This active apoptosome complex then cleaves and activates the  executional caspases like caspase-3 and 7221, 224. These caspases then activate caspase activated DNAse or inactivate PARP1 and bring about DNA damage219. The extrinsic pathway starts with the activation by Fas ligand or TNF α on their respective receptors. This results in activation of procaspase-8 into an active dimerized caspase-8 through the mediation of death induced signaling cascade, which involves adapter proteins like FADD and TRADD 227, 228. The activated caspase-8 then progress towards an apoptotic pathway through the cleavage of caspase-3 and 7, with resultant cell death as described above226. The initiation of apoptotic cell death pathways are under the control of Bcl-2 family members which includes both pro-apoptotic and antiapoptotic family members229, 230. The Bcl-2 members are homologues to CED-9 as seen in C. elegans231, 232. The 2 groups under the proapoptotic proteins include the ones containing BH-1, 2 18  and 3 domains but lacking BH-4 domain (Bax, Bak), and the ones which contain only the BH-3 domain (Bid, Bim, Bad, Noxa and Puma) 230, 233-237. Anti-apoptotic Bcl-2 members contain all the BH-(1-4) domains, which includes Bcl-2, Bcl-xL and Mcl-1235, 236, 238. Among the proapoptotic family members, each and every protein has different sites of localization and ways of activation229, 233. For Bax to get activated, it needs conversion from an inactive monomer to an active dimer, along with its cytosolic to mitochondrial translocation239. Both Bak and Bax require oligomerization for their activation. Bax is normally localized along with dynamin like protein-1 and BAD is coupled with glucokinase240. On truncation mediated activation, Bax and Bid moves to the mitochondrial outer membrane and stimulate the leak of the transition pore 239, 241  .  Bcl-2 and Bcl-xL normally inhibit the Bax and Bid mediated mitochondrial leak by  preventing their dimerization on the mitochondrial membrane surface.  In conditions like  diabetes, metabolic syndrome and sepsis, when there is BAD activation, the activated BAD inhibit the anti-apoptotic property of Bcl-2 and Bcl-xL234, 240, 242. Apart from cytochrome c mediated apoptosis, there are other major mediators of cell death like endonuclease-G and apoptotic inducing factor (AIF)243. Under conditions where there is activation of pro-apoptotic Bcl-2 members, there is promotion of endonuclease-G and AIF mitochondrial to nuclear translocation with induction of apoptotic cell death that is caspaseindependent243, 244. Necrosis belong to the class of non-programmed cell death pathway. It ultimately leads to depletion of ATP and release of cellular contents into the neighbouring regions promoting further cell death245,  246  Conditions like calcium increase, and formation of excess reactive  oxygen/nitrogen species forms the initiator for necrosis pathway. Receptor interacting protein-1 (RIP-1) is one of the major upstream regulators of necrotic cell death. RIP-1 is always inhibited 19  by caspases and protected by the chaperones Hsp-90245-247. Nitric oxide has always shown to inhibit capases by its nitrosative modification and promote a necrotic pathway. Calpains are also known to bring about apoptosis through the mediation of calcium increase, calcium mediated cathepsin activation and lysosomal membrane leak. This in turn can lead to cPLA2 cleavage and activation. Activated cPLA2 in the presence of excess fatty acids and lipoxygenase cause lipid hydroperoxide formation and plasma and mitochondrial membrane rupture, leaking the cell contents to the surrounding environment 248. Apart from this pathway, necrosis can also result from incidents or agents that cause DNA damage249. Increased DNA damage can result in polyADP ribose polymerase (PARP-1) activation, with resultant ATP and NAD+ depletion. An increased ATP depletion can result in RIP-1 activation with resultant necrotic death. Autophagy is another form of cell death which comes under the programmed category. Beclin-1 is the major protein involved with autophagocytic cell death250, 251. Some of the typical features include increased lysosomal formation, increased lysosomal hydrolase like cathepsin-D activation, acidic pH induction, and lysosomal leakage with formation of autophagosomes 251-253. Apart from becoming a mediator in necrotic cell death, PARP-1 can initiate a novel cell death pathway which is independent of apoptosis and necrosis, called parthanatos 254. If activated, PARP-1 can result in the formation of increased poly-ADP ribose formation which when formed in excess, leads to mitochondrial membrane leak 255. This results in mitochondrial to nuclear translocation of proteins like apoptosis inducing factor and endonuclease-G, which can lyse DNA and cause a caspase-independent cell death254, 255. Under conditions of diabetes, the different cell death pathways gets turned on at different stages, based on the severity of nitrosative/oxidative stress, levels and duration of  20  hyperglycemia, hypoinsulinemia and dyslipidemia. A proper understanding about all these cell death pathways could provide multiple targets to minimize cell loss due to death during diabetes. 1.4  FoxO1  FoxO1, one among the 100 family members of the forkhead family has been known to play an important role in cell survival, oxidative stress resistance, energy metabolism, cell cycle arrest and cell death. The Fox-gene was first discovered in 1989 in drosophila melanogaster and till date, around 15 Fox (A-L) and subfamilies have been discovered under the Fox superfamily. Human FoxO is analogues to drosophila dFoxO and C. elegans abnormal dauer formation-16 (DAF-16)256-258. 1.4.1 Structure. FoxOs have a DNA binding Fork head /winged helix (DBD) which is highly conserved for all the FoxO members, nuclear localization sequence, nuclear export sequence and a C-terminal containing a transactivation domain259,  260  .  The Forkhead/winged helix domain is highly  evolutionary conserved, and contains 110-amino acids, which is quite common for most Fox family members. It comprises of 3 α, 3 β and 2 winged helices. The configuration is in such a way that the α, β, and winged helices are aligned in an antiparallel manner, and interconnected to form a 3D structure260. The DNA binding of this transcription factor is facilitated by DBD, specific regions in N-terminal domains and C terminal domains257, 259, 260 (Figure 3). 1.4.2 Transcriptional activity and post translational modifications. Many post translational modifications are known to mediate the transcriptional activity of FoxO. Some of the major post-translational modifications include phosphorylation, acetylation, ubiquitination, arginine methylation and O-glycosylation260-263. FoxO family members (FoxOs1, 3, and 4) are expressed in almost every tissue (with FoxO1 being the dominant member in the 21  adult heart), are highly regulated by insulin, and bind to an identical DNA target sequence, thus regulating similar target genes262, 264-266. Depending upon the site and upstream target, these modifications can either increase or decrease transcriptional activity.  By binding to DNA  through their forkhead domain, these proteins either inhibit or activate target protein gene expression.  Stress and growth signals determine the nuclear compartmentalization, DNA  binding, and cytosolic degradation of FoxO260, 265. Akt/growth signal and serum glucocorticoid kinase are known to phosphorylate and down regulate the activity of FoxOs (FoxO1, 3 and 4), where as FoxO6 is not insulin regulated261. Stress signals including AMPK, p38 MAPK, Erk, JNK and MST-1 are known to phosphorylate and increase nuclear import.  The latter  phosphorylation is known to disrupt the binding of 14-3-3 to FoxO, thereby promoting its nuclear retention264. FoxO1 has been shown to be regulated by insulin and insulin like growth factors through phosphorylation at the Ser256 moiety making this transcription factor prone to phosphorylation at Ser319 and Thr24 by other kinases261, 265. This is followed by its nuclear export, trafficking that is mediated by 14-3-3s which can bind to FoxO only in its phosphorylated state261, 264 (Figure 3). Binding of 14-3-3 to phosphorylated FoxO weakens its DNA binding ability and facilitates nuclear export and subsequent degradation by ubiquitination by E3 ubiquitin ligase264. The DNA binding ability of FoxO is also determined by de-acetylases like Sirtuins which increase the nuclear retention and transcription activity of FoxO (Figure 2). Where as CBP/p300 binds and acetylates FoxO1 attenuating its DNA binding and transcriptional function260, 262, 263. 1.4.3 Physiological role. FoxO1, a prominent member of the forkhead box family and subfamily O of transcription factors, and produced from FKHR gene (forkhead domain in rhabdomyosarcoma), is involved in 22  regulating metabolism, cell proliferation, oxidative stress response, immune homeostasis, pluripotency in embryonic stem cells and cell death258, 266, 267 FoxO1 has important roles in systemic homeostasis. In mice, loss of FoxO1 is embryonically lethal, whereas FoxO3 deletion results in normal birth but these mice are prone to cardiac hypertrophy and eventually failure. FoxO1 has an important role in controlling oxidative stress resistance, cell cycle arrest, apoptosis, and metabolism268-271. In oxidative stress, they are known to increase the expression of antioxidant genes (superoxide dismutase, catalase) and GADD45, thereby promoting reactive oxygen species scavenging activity and preventing DNA damage, thus safeguarding cells from further damage267, 272. In cancer, their ability to regulate genes involved in cell cycle progression (cyclin B, PlK), together with their inhibition of cell proliferation (p27 KIP1) makes them a major therapeutic target. FoxOs are also involved in inducing apoptosis and atrophy by upregulating genes like FasL, Bim and MURF-1269, 273, 274. Recently, a role for FoxO1 in regulating cardiac glucose and fatty acid (FA) metabolism has also been suggested 275.  FoxOs upregulate  gluconeogenesis by increasing hepatic glucose-6-phosphatase and phosphoenolpyruvate carboxykinase mRNA and protein276-278. In skeletal muscle and HepG2 cells, they are known to increase pyruvate dehydrogenase kinase (PDK4) protein expression there by regulating glucose oxidation279 1.4.4 FoxO1 activation. FoxO1 has shown to be activated in conditions like fasting, nutrient excess, insulin resistance, diabetes, inflammation, sepsis, and ischemia. Under conditions of fasting, when there is an attenuation of Akt signalling followed by activation of stress kinases like AMPK, JNK and p38 MAPK, an increase in nuclear FoxO1 is evident together with its increased transcriptional function. It has also been shown that increased expression or activity of Sirt1 could play a role 23  in the nuclear entrapment and enhancement in activity of FoxO1280-282. In conditions of nutrient excess, whether its high fat or high glucose, studies have shown that excess ROS and reactive nitrogen species generation results in concomitant activation of MAP kinase pathways and nuclear ingress of FoxO1283.  Obesity is one of the conditions of nutrient excess with the  activation of above mentioned pathways. In conditions of chronic hyperglycemia, advanced glycated end products and their receptor activation can bring about O-linked beta-Nacetylglucosamine modification of FoxO1 at the threonine moiety which has been shown to enhance transcriptional function independent of its phosphorylation state 284. In conditions like insulin resistance and diabetes, there is a decreased check on FoxO1 phosphorylation/activity by the insulin signalling pathway, which does not allow the 14-3-3 chaperone to come and bind to FoxO1, making them less prone to ubiquitination mediated degradation132. In addition, during diabetes, stress signals are also known to get activated along with the presence of increased ROS and nitrosative stress signalling in the body282. In conditions of inflammation and sepsis, along with nitrosative stress signalling, toll like receptor signalling and transcription factors like NFkappa B have shown to either enhance the nuclear presence or transcriptional activity of FoxO1285, 286. Under conditions of hypoxia/ischemia, transcription factors like hypoxia inducible factor (HIF-1α) are shown to interact with FoxOs and enhance their transcriptional function to combat these situations287. 1.4.5 Consequences of FoxO1 overactivation. An increase in FoxO1 activation can cause various effects depending on the type of tissue. In skeletal muscle starvation induced activation of FoxO1 causes upregulation of atrogin-1 (MAFbx) and MuRF-1 (muscle ring finger-1) genes which are involved with muscle loss288. In adipose tissue, FoxO1 overexpression leads to suppression of p21 and PPARγ which are mostly 24  involved in adipocyte differentiation289. In liver, following FoxO1 activation, vital enzymes involved with gluconeogenesis like glucose-6-phosphatase (G6Pase), fructose-1,6-biphosphatase and phosphoenolpyruvate carboxykinase (PEPCK) are increased 276. G6Pase, whose expression pattern is unique to the liver, releases enormous amounts of glucose into the system, and could be a contributor for hyperglycemia induced complications including insulin resistance and diabetes. Regarding lipids, in the liver FoxO1 is known to enhance lipogenesis and lipid induced steatosis276. In beta cells, pancreatic and duodenal homeobox factor 1 (Pdx1) is involved with the survival, metabolism and regulation of beta cell function through its ability to regulate genes like insulin, Glut 2 and glucokinase. An activated FoxO1 in the beta cells has been shown to inhibit Pdx 1 with resultant deterioration of beta cell function and ultimately beta cell death. This can ultimately result in the development of T2D 290. In the heart, following increased FoxO1 activation, they cause autophagy through the induction of genes like Gabarapl1 and Atg12291. 1.4.6 Other roles. In cancer, they are shown to play important roles like tumor suppression and tumor cell death. Due to their ability to promote cell cycle arrest by activation of p27kip and c-myc, they have a tumor suppressive role292. Through the activation of pro-apoptotic proteins like Bim and caspases, they are known to increase tumor cell death. Another important role of FoxO1 in inflammation and sepsis has been explored through their ability to co-ordinate with TLR4 and NF-kappa B293. FoxO1 has also been shown to play an important role in longevity through the collaborative effect of FoxO1 with Sirt1 resulting in turning on of anti-oxidant genes like MnSOD and catalase294. A very recent role of FoxO1 has emerged in stem cell research due to  25  its ability to maintain pluripotency of embryonic stem cells and is suggested to be due to FoxO’s ability to regulate OCT4 and SOX2 genes by a direct effect on their respective promoters 295. 1.5  Hypothesis and specific aims We hypothesize that in response to nutrient excess, insulin resistance and diabetes, changes  in glucose and FA utilization by the heart requires the participation of FoxO transcription factors. Understanding these distinctive functions of FoxOs, especially FoxO1 would be useful in restoring metabolic equilibrium and limiting cardiac damage due to cell death. Following insulin resistance/nutrient excess/diabetes, our specific goals are to: i) Substantiate the importance of FoxO in regulating cardiac glucose metabolism. ii) Elucidate the role of FoxO in promoting unregulated FA uptake and oxidation. iii) Explore the role of FoxO1 in causing cardiac cell death.  26  Figure 1.  Glucose utilization in the cardiomyocyte. Glucose uptake into cardiomyocyte  occurs through GLUT1 and GLUT4 transporters. Once inside, glucose is broken down through glycolysis, with the resultant formation of pyruvate. The pyruvate generated is transported into mitochondria and decarboxylated to acetyl-CoA through pyruvate dehydrogenase (PDH). Acetyl-CoA then enters tricarboxylic acid cycle and with the generation of ATP. PDH is phosphorylated and inactivated by pyruvate dehydrogenase kinase 4 (PDK4), which decreases glucose utilization and promotes fatty acid utilization  27  Foxo  Degradation  MAPK  Cytosol  Akt Foxo  14-3-3  Sirt  Nucleus  Transcription  Figure 2. Regulation of FoxO. Cellular location, DNA binding, and degradation of FoxO is determined by its nuclear expulsion following its phosphorylation by Akt and binding to 14-3-3, or its nuclear intrusion by stress signals like MAPK. Nuclear retention and DNA binding is also under the control of Sirt mediated de-acetylation.  28  14-3-3 binding  Domains  P  P  N  P  FHDBD  NLS  NES  TAD C  Figure 3. Structure of FoxO1 protein. FoxOs have a DNA binding Fork head /winged helix domain (FHDBD), nuclear localization sequence (NLS), nuclear export sequence (NES) and a Cterminal containing a transactivation domain (TAD).  The Akt phosphorylation and 14-3-3  binding sites are dispersed throughout the DNA binding domain and NLS of FoxO1.  29  Chapter 2: Methods 2.1  Experimental animals  The investigation conforms to the guide for the care and use of laboratory animals published by the US NIH and the University of British Columbia. Adult male Wistar rats (260-300 g) were injected with Streptozotocin (STZ, 100 mg/kg i.v. through the tail vein), a β-cell toxin, to induce hyperglycemia within 24 h. Animals remained hyperglycemic up to 4 days and plasma insulin, glucose, and NEFA levels and proteins from heart tissues analyzed from day 0 to 4. To induce acute reversible hyperglycemia, we used diazoxide (DZ), a selective K+-ATP channel opener that hyperpolarizes the β-cell, decrease insulin secretion and causes maximum hyperglycemia (within 2 h). DZ (100 mg/kg) was administered i.p, and blood glucose was monitored from time of injection till reversal of hyperglycemia was achieved (12 h). Animals were euthanized at 0, 4 and 12 h after DZ injection, and heart samples used for Western blot analysis. 2.2  Isolation of cardiomyocytes This investigation conformed to the Guide for the Care and Use of Laboratory Animals  (National Institutes of Health) and the University of British Columbia and was approved by the Animal Care and Use Committee (Protocol No. A08-0627). Adult male Wistar rats (260–300 g) were obtained from the University of British Columbia Animal Care Unit. Ventricular myocytes were prepared by a previously described procedure296. Hearts were removed from anesthetized rats (60 mg/kg pentobarbital sodium, i.p.) and digested by perfusing collagenase retrogradely. Myocytes were made calcium-tolerant by successive exposure to increasing concentrations of calcium.  Our method of isolation yields a highly enriched population of calcium-tolerant  myocardial cells that are rod-shaped in the presence of 1 mM Ca2+ with clear cross striations. Intolerant cells are intact but hypercontract into vesiculated spheres. Yield of myocytes (cell 30  number) was determined microscopically using an improved Neubauer hemocytometer. Myocyte viability (over 80%) was assessed as percentage of elongated cells with clear cross striations that excluded 0.2% Trypan blue. Cardiomyocytes were plated on laminin-coated, 100 mm culture plates to a density of 1,000,000 cells/plate. Cells were maintained using Media-199, and incubated at 37°C under an atmosphere of 95% O2-5% CO2. Where indicated, various agents were added to the culture media for different intervals (0-12 h). Where indicated, Dx (1 M), Insulin (1-100 nM), SB202190 (20 μM, a relatively specific inhibitor of p38 MAPK at this concentration) and Nicotinamide (20 mM, a potent and specific Sirt inhibitor which binds to the site where the co-factor NAD+ attaches to Sirt, there by decreasing its activity), high glucose (25 mM), D-mannitol (25 mM), Palmitate (1 mM), LPS (0.1-10 μg/ml) and 1400W (10 µM) were used for the time periods indicated were added to the culture medium. 2.3  Plasma measurements  Blood samples collected were used to measure glucose using a glucometer and glucose test strips (Accu-Chek Advantage, Roche). Non-esterified fatty acid and insulin were measured using assay kits from Wako and Cayman respectively. 2.4  Glucose oxidation in isolated cardiomyocytes  Glucose oxidation was performed as described briefly.  Following attachment of  cardiomyocytes to laminin-coated, 60-mm center-well organ culture dishes, cells were incubated in M199 and 0.15 μCi/mL D-[U-14C]glucose. Incubation was carried out for 6 h at 37°C with 95% O2-5% CO2 gassing. 5% KOH was then injected into the center wells, and oxidation stopped by injecting 1 M H2SO4 into the incubation buffer. The dishes were sealed, stored at  31  4°C overnight, and KOH assessed for 14CO2 by scintillation counting. Oxidation was calculated as a % change of 14CO2 released by the control. 2.5  Pyruvate dehydrogenase (PDH) activity assay  A Mitoprofile rapid microplate assay kit (which follows the reduction of NAD+ to NADH) was used to measure PDH activity in cardiomyocytes (MSP18, Mitosciences, Oregon, USA). Mitochondrial fractions from control and Dx treated myocytes were made to similar protein concentrations with the provided diluent, reagent mix was added and absorbance measured at 450 nM at room temperature for a period of 15 min. 2.6  Isolation of mitochondrial fraction 1 x 106 cardiomyoctes were scraped and suspended in a homogenization buffer (10 mM Tris-  HCl pH 7.5, 10 mM KCl, 0.15 mM MgCl2, 0.5 mM EDTA, 1 mM PMSF, 1 mM DTT, and a 1% mixture of protease and phosphatase inhibitors), centrifuged at 4000 x g for 15 min, and subjected to further lysis and sucrose gradient (10 mM Tris-HCl pH 7.5, 0.15 mM MgCl2, 250 mM sucrose, 0.5 mM EDTA, 1 mM PMSF, and 1 mM DTT) separation at high speed (20000 x g) for 15 min. The isolated pellet was re-suspended using a lysis buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, 0.2 mM PMSF, 1% Triton-X 100, and a 1% mixture of protease and phosphatase inhibitors) as described previously. Prohibitin antibodymitochondrial marker (Abcam) was used to confirm the purity of mitochondrial fractions. 2.7  Isolation of cytosolic and nuclear fraction  Hearts from control, STZ and DZ treated rats were cleared of blood by washing thoroughly in tyrode buffer and aortic and atrial sections removed from the ventricles. Ventricular tissue was freeze-clamped in liquid nitrogen, and tissue stored until fractionated.  Following Dx and 32  palmitate treatment, cardiomyocytes were scraped and washed twice with 0.5 ml PBS. Ventricular tissue and cardiomyocytes were lysed in ice-cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 0.5 % NP40) for 15 min. Following centrifugation at 13,000 rpm for 5 min, at 4°C, the supernatant was separated (as the cytosolic fraction). The separated pellet was vigorously mixed and vortexed with buffer B (20 mM HEPES pH 7.9, 0.42 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10% glycerol, 1 mM PMSF), and kept on ice for 15 min. Samples were then centrifuged at 13,000 rpm for 10 min at 4°C, and the resulting supernatant collected as the nuclear fraction. Proteins were quantified using a Bradford protein assay, and used for Western blot to determine nuclear and cytosolic contents of FoxO1, phospho-p38 MAPK, and PGC-1α., GAPDH, Siah1 and AIF. Histone H3 was used as a nuclear marker and β-actin and GAPDH were used as cytosolic markers. 2.8  Plasma membrane isolation and determination of CD36  Following treatment, cardiomyocytes were scraped, homogenized, and subjected to centrifugation at 1200 x g for 10 min. The separated supernatant was then centrifuged at 16,000 x g for 30 min. From this, the separated pellet was resuspended and layered on top of a 40% sucrose cushion and then centrifuged at 100,000 x g for 1 h at 4°C, as described previously. The suspended top layer was further subjected to centrifugation at 30,000 x g for 30 min at 4°C. The pellet obtained was re-suspended, and Western blot performed to obtain membrane CD36. Cardiomyocyte homogenates were also used to separate triton-soluble and insoluble fractions, using a previously described method. In brief, homogenates were centrifuged at low speed to separate nuclear fractions. The resulting supernatant was centrifuged at 100,000 x g for 20 min to separate cytosol and pellet. The pellet was resuspended in a buffer containing 1% Triton X33  100 and kept on ice for 30 min. This sample was then subjected to centrifugation at 100,000 x g for 30 min, a process which separates supernatant (Triton-soluble membrane fraction) from the pellet (Triton-insoluble fraction). 2.9  Filamentous and globular actin  The ratio of globular to filamentous actin (G-actin/F-actin ratio) in the cardiomyocytes was determined using an assay kit. Myocytes were isolated and lysates centrifuged at 2,000 rpm for 5 min. Total actin content of the supernatant was centrifuged at 100,000g for 1 h at 37°C to isolate F-actin (pellet) and G-actin (supernatant). The pellets were resuspended to the same volume as the G-actin fraction using ice-cold Milli-Q water plus 10 μmol/l cytochalasin D and left on ice for 1 h to dissociate F-actin. The ratio of G-actin to F-actin was determined using Western blot and densitometry. 2.10  Immunoprecipitation  Proteins isolated from control and treated (STZ and DZ) heart tissues and cardiomyocytes were lysed in buffer (20 mM Tris-HCl, 2 mM EDTA, 5 mM EGTA, 50 mM 2-mercaptoethanol, 25 μg leupeptin, 4 μg aprotinin, pH 7.5), and immunoprecipitated using phospho-p38, FoxO1, 14-3-3, Sirt1, PGC-1α, GAPDH, caspase-3, and cleaved caspase-3 antibodies overnight at 4 C. The immunocomplex was pulled down with protein A/G-sepharose (1 h), and heated for 5 min at 95°C. The immunocomplex was subjected to Western blot for determining protein interactions. SB202190 (20 μM), a selective and potent inhibitor of p38 MAP kinase was used to prevent interaction between phospho-p38 MAPK and FoxO1. Myocytes were pre-incubated for 1h with SB202190, followed by incubation with PA.  After 6h, nuclear fractions were isolated for  immunoprecipitation of FoxO1 followed by Western blot for phospho-p38 and FoxO1.  34  2.11  Western blotting  Western blot was done by a well described method.  In brief, plated myocytes were  homogenized in ice-cold lysis buffer. Samples were quantified, boiled with loading dye, and 40 g used in gel electrophoresis. After blotting, membranes were incubated with goat anti-PDK1 and rabbit PDK2 and 4, rabbit anti-PDH, rabbit anti-phospho PDHE1alpha (Ser 293), rabbit antip38 MAPK, anti-phospho p38 MAPK (Thr180/Tyr182), anti-phospho-Akt (Ser-473), anti-Akt, anti-FKHR (FoxO1), anti-FKHRL (FoxO3), anti phospho-FoxO1 (Ser-256), anti-Ac FKHR (D19), anti-Sirt1, anti-CD36, anti-14-3-3δ, anti-NOS1 (nNOS), anti-NOS2 (iNOS), anti-NOS3 (eNOS), anti-Cdc42, anti-phospho-AMPK (Thr172), anti-Ac-lysine, anti-PGC-1, anti-NF-κB p65 and anti-PP2A-Aα/β, and goat anti-14-3-3, anti-VAMP (VAMP1/2/3), and mouse antiOXPHOS antibodies, and subsequently treated with secondary goat anti-rabbit and donkey-anti goat or anti-mouse HRP-conjugated antibodies. Bands were visualized using an ECL® detection kit, and quantified by densitometry. 2.12  Immunoflourescence  Isolated hearts from control and STZ treated rats were fixed by storing in 10% formalin for 24 h. Cardiac tissue was sectioned (3-μm), and placed on glass slides which were paraffin embedded and silane coated. These slides were subjected to immunostaining. Cardiomyocyte were fixed for 10 min with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS for 3 min, blocking with goat serum serum for 20 min, and finally rinsed with PBS. Cells were incubated with rabbit polyclonal phospho-p38 MAPK, goat-polyclonal FKHR antibodies anti-CD36 antibody, rabbit anti-nitrotyrosine or anti-annexin V followed by incubation with secondary antibodies (Alexa Flour 488 goat anti-rabbit IgG, Alexa flour 594 donkey anti-goat 35  IgG) and DAPI, respectively. Slides were visualized and photographed using Leica fluorescent and Zeiss Pascal confocal microscopes. 2.13  Uptake of BODIPY  For BODIPY uptake, cardiomyocytes were treated with control media or media containing 1 mM palmitate, and then exposed to 10 µM concentration of fluorescent dye, BODIPY FL C16 for 15 min. Slides were visualized and photographed using a Zeiss Pascal Confocal microscope. 2.14  Real time PCR  PDK4 mRNA expression was analyzed by real-time quantitative PCR using a light cycler and SYBR Green PCR Mix (Roche). Total RNA from cardiomyocytes was extracted using Trizol (Invitrogen). After spectrophotometric quantification, reverse transcription was carried out using an oligo-(dT) primer and superscript II RT (Invitrogen). The real time PCR mixture with cDNA was amplified using PDK4 primers. PDK4: 5′-CCTTTGGCTGGTTTTGGTTA-3′ (left) and 5′CACCAGTCATCAGCCTCAGA-3′ (right). The amplification parameters were set at 95°C for 1 s and 60°C for 6 s and 72°C for 10 s (40 cycles total). The fluorescence signals were analyzed using light cycler relative quantification (Roche).  Relative expression was evaluated by  normalizing to  actin. 2.15  Nitrite and nitrate determination  The nitrite and nitrate content in the plasma and tissue protein was determined using a kit from R&D systems. Equal volumes of plasma or similar concentrations of tissue proteins were used for this determination. Nitrite is determined based on the conversion of nitrate to nitrite by nitrite reductase. This is followed by colorimetric determination of nitrite content as an azo dye formed by means of a Greiss reaction. Nitrate is determined by subtracting endogenous nitrite  36  (not formed from nitrate conversion) from the total nitrite (endogenous + nitrate converted) concentration. OD values are measured using a microplate reader at wavelengths 540/690 nm. 2.16  PARP activity assay  PARP activity was assayed using a HT universal colorimetric PARP assay kit from Trevigen. Equal protein concentrations from control and STZ treated rat hearts were used for this activity determination. This assay is a 96 well format and makes use of the incorporation of biotinylated poly (ADP-ribose) onto histone proteins. Following the incubation of samples and reagents in contact with the well surface, absorbance was measured at 450 nm. 2.17 Measurement of triglycerides  Total cardiac lipids were extracted and solubilized in chloroform:methanol:acetone:hexane (4:6:1:1 v/v/v/v). Separation and quantification of TG was achieved using HPLC (Waters 2690 Alliance HPLC, Milford, MA), as described previously. 2.18  Treatments  To study the role of glucocorticoid induced insulin resistance, we used 1 μM Dx (0-12 h) and to study the early events associated with FA induced lipotoxicity, albumin bound Palmitate (1 mM), Oleate (1 mM), and Br-Palmitate (1 mM) (molar ratio 1:1), LY 294002 (20 μM), LPS (0.1-10 μg/ml), 1400W (10 µM) high glucose (25 mM), D-mannitol (25 mM), and TNF- (5 ng/mL) were added to the culture medium for 0-12 h. PA was chosen as it a common saturated fat that represents 10-20% of the normal human dietary fat intake. Regarding its concentration, plasma NEFA usually increases to 1-1.5 mM during diabetes. PA typically accounts for no more than ~30% of total FA. Hence, a predicted value for PA in this NEFA fraction during diabetes would be approximately 0.5 mM. However, FA derived from the albumin bound fraction does not account for all of the FA provided to the heart. Thus, other relevant sources like hydrolysis 37  of lipoproteins by cardiac LPL also play a role in the provision of PA. Given that the molar concentrations of FA in lipoproteins are approximately 10-fold higher than that of FA bound to albumin and that circulating plasma TG concentrations increase following diabetes, a more realistic concentration of PA supplied to the heart following obesity and diabetes would likely be around 1 mM. We chose the treatment time upto 12 h to evaluate signaling pathways leading to triglyceride accumulation, prior to the initiation of cell death pathways. For invivo studies, STZ (100 mg/Kg) for 1-4 days, and DZ(100 mg/Kg) for 0-12 h were used. 2.19  Materials  [U-14C] glucose was purchased from American Radiolabeled Chemicals Inc (MO, USA). The ECL® detection kit was obtained from Amersham Canada. Rabbit anti-p38 MAPK, antiphospho p38 MAPK, anti phospho-FoxO1, anti-phospho-AMPK, anti-NF kappa B p65, antiphospho FKHR (p-FoxO1, Ser-256), anti-phospho AKT (Ser-473), anti-Histone-H3, anti-NOS1 (nNOS), anti-GAPDH, anti-cleaved caspase-3, anti-caspase-3, anti-PARP1, anti-cleaved PARP1, anti-AIF and anti-Sirt1 were from Cell Signaling. Rabbit anti PDK2 from Abcam, anti-PDK1 PDH, FoxO1, FoxO3, Ac FKHR, goat anti-14-3-3, PGC-1, Ac lysine, anti-14-3-3δ, antiVAMP-1/2/3, Anti-phospho IRS (Tyr-989), anti-NOS2 (iNOS), anti-NOS3 (eNOS), anticytochrome C, goat anti-Siah1, mouse anti-β-actin, goat anti-rabbit, donkey-anti goat and goat anti-mouse HRP-conjugated antibodies obtained from Santa Cruz Biotechnology Inc. BODIPY FL C16 was obtained from Invitrogen molecular probes. PDK4 antibody was purchased from Abnova Corporation whereas phospho-PDHE1- was from Novus Biological. PDH activity kit was acquired from Mitosciences.  Annexin-V and nitrosylated cysteine was obtained from  38  Abcam.  Alexa Fluor 594 goat anti-rabbit IgG was obtained from Invitrogen.  All other  chemicals were from Sigma Chemicals. 2.20  Statistical analysis  Values are means  SE. Wherever appropriate, one-way ANOVA followed by the Tukey or Bonferroni tests were used. To evaluate differences between group mean values, we used unpaired Student’s t-test. Statistical significance level was set at value; P < 0.05.  39  Chapter 3: Results 3.1  The increase in cardiac PDK4 following short term dexamethasone is controlled by  an Akt-p38-FoxO1 signaling axis 3.1.1 Reduction in cardiac glucose oxidation following Dx is mediated by PDK4 Exposing adult rat cardiomyocytes to 1 μM Dx for 6 h produced a robust decrease in glucose oxidation (Figure. 4A). This was associated with a significant increase in mitochondrial PDH phosphorylation (Figure 4C) together with a drop in its activity (Figure. 4B). We did not observe any change in total PDH protein expression with Dx (Figure. 4C)). An upstream kinase which regulates PDH phosphorylation in heart is PDK4.  Measurement of PDK4 revealed an  augmentation in both protein (Figure. 4D, upper panel) and gene (Figure. 1D, lower panel) expression when cardiomyocytes are treated with Dx. Unlike PDK4, Dx did not influence the protein expression of PDK1 and 2 (Figure. 1D). 3.1.2 Dx promotes nuclear shuttling of FoxO1 but not FoxO3a Stress stimuli are known to facilitate cytoplasmic to nuclear shuttling of Forkhead transcription factors (specifically FoxO1 & FoxO3a) to induce genes that control glucose metabolism297.  Determination of the effects of Dx on FoxO1 compartmentalization  demonstrated an increase in the nuclear (Figure 5A, upper panel), but a resultant decrease in the cytosolic (Figure. 5A, lower panel) content of this transcription factor. This change occurred in the absence of any alterations in the total content of FoxO1 protein (Figure. 5A blots). The increase  in  the  nuclear  content  of  FoxO1  following  Dx  was  confirmed  using  immunofluorescence microscopy (Figure. 5B). Interestingly, with this duration of Dx treatment, FoxO3a demonstrated no cytoplasmic to nuclear shuttling (Figure. 5C). 40  3.1.3 Nuclear transport of FoxO1 is mediated by Dx induced phosphorylation of p38 MAPK Incubation of cardiomyocytes with Dx increased cytosolic phosphorylation of p38 MAPK, an effect that was evident within 4 h, and remained high up to 6 h following Dx (Figure. 6A). An increase in the nuclear content of phosphorylated p38 MAPK was also evident within 4 h, but further increased after 6 h of Dx treatment (Figure. 6B). Interestingly, the increase in nuclear content of FoxO1 correlated to an increase in nuclear phospho p38 MAPK (Figure. 6B blots). We used a serine protease thrombin to phosphorylate p38 MAPK. As predicted, and comparable to Dx, nuclear p38 MAPK phosphorylation following thrombin corresponded to an enhanced nuclear FoxO1 (Figure. 6B blots). As pre-incubation of myocytes for 1 h with SB202190 prevented the effects of Dx to increase nuclear phospho p38 and FoxO1 (Figure. 6C), our data suggests that p38 MAPK is a likely mediator responsible for the nuclear shuttling of FoxO1. 3.1.4 Phosphorylated p38 exhibits an intense association with FoxO1 Using immunofluorescence, we confirmed the property of Dx to phosphorylate p38 (Figure. 7 upper panel). This increase in p38 phosphorylation paralleled a robust attachment of phosphop38 to FoxO1 (Figure 7 merge-upper panel). More importantly, binding of p38 to FoxO1 promoted a consequent migration of the bound complex to the nuclear compartment, as measured by immunofluorescence (Figure 7 merge-upper panel) and Western blot (Figure 7 lower panel).  41  3.1.5 Reduction in phosphorylation of FoxO1 by Dx is influenced by heat shock protein dampening of Akt signaling Phosphorylation of FoxO in the nucleus by insulin/insulin like growth factors (growth signals that increase Akt/PKB) cause their nuclear exclusion and segregation in the cytoplasm in 293T cells261. Using LY 294002 to inhibit Akt phosphorylation, we validated this mechanism in cardiomyocytes and observed a decrease in FoxO1 phosphorylation, and increase in its nuclear content (Figure 8 right box). Incubation of cardiomyocytes with Dx also produced a decrease in phosphorylation of Akt and FoxO1 in the total fraction, effects that were reversed by simultaneous incubation with insulin (Figure 8 A and B). Binding of Dx to its receptor releases heat shock proteins (Hsps) from the receptor complex, which are known to bind with kinases like Akt. Immunoprecipitation of total Akt followed by Western blot showed a robust association between Hsps (Hsp 25 & Hsp 90) and Akt after Dx treatment (Fig. 5 C). This effect of Dx was prevented by insulin (Figure 8 C). Dx had no influence in increasing the expression of total Hsp25 and Hsp90 protein.  Unlike insulin, inhibition of p38 MAPK phosphorylation using  SB202190 had no effect on phosphorylation of Akt and FoxO1 in the total cell fraction (Figure 8 D), suggesting that Dx causes both nuclear import (by p38 phosphorylation) and nuclear retention (by reducing Akt phosphorylation). 3.1.6 Insulin  dose  dependently  reverses  the  effect  of  Dx  to  promote  nuclear  compartmentalization of FoxO In the presence of Dx, the cytosolic content of FoxO1 decreased whereas its nuclear compartmentalization increased (Figure 9 A & B).  Insulin, when incubated with Dx, was  capable of reversing this effect, but only at a 100 nM concentration (Figure 9 A & B). 14-3-3s 42  are binding proteins known to interact with FoxO to facilitate their nuclear export following phosphorylation by Akt. Interestingly, in the presence of Dx, the association between 14-3-3 and FoxO1 decreased, an effect that was negated by 100 nM insulin (Figure 9 C & blot). 3.1.7 Dx increases the association of Sirt1 with FoxO leading to its de-acetylation Cardiomyocytes incubated with Dx demonstrated a robust decrease in FoxO acetylation (Figure 10 A blots). De-acetylases like sirtuins increase the nuclear retention and DNA binding ability of FoxO. Measurement of the nuclear and total expression of Sirt1 protein showed no change following Dx (Figure 10 A blots). Instead, Dx increased the association of Sirt1 with FoxO1 (Figure 10 B Lower panel). Using the Sirt inhibitor nicotinamide (NM), the effect of Dx on the acetylation of FoxO1 was reversed (Figure 10 B Upper panel). NM had no impact in decreasing nuclear FoxO1 content (Figure 10 B Upper panel) even though it was able to disrupt the association of Sirt1 with FoxO1 (Figure 10 B Lower panel).  Similar to NM, insulin also  reversed the acetylation of FoxO1 observed with Dx (Figure 10 C Upper blot), an effect that was absent when SB202190 was used (Figure 10 C Lower blot). 3.1.8 Manipulation of FoxO1 can alter the increased expression of PDK4 observed with Dx Making use of different agents that alter FoxO compartmentalization or transcriptional activity, we tested their influence on PDK4 protein expression.  Insulin (which promotes cytosolic  shuttling and acetylation of FoxO1) and NM (a specific Sirt inhibitor) were effective in reducing Dx induced increase in PDK4 protein expression and phosphorylation of PDH (Figure 11 A). SB202190, which only prevents the nuclear entry of FoxO, was not as effective as insulin or NM in reducing Dx induced increase in PDK4 protein expression and phosphorylation of PDH (Figure 11 A).  43  3.2 Cardiac triglyceride accumulation following acute lipid excess is through activation of FoxO1-iNOS-CD36 pathway 3.2.1 Cardiac TG accumulation following lipid excess is mediated by an increase in membrane CD36 Exposure of adult rat cardiomyocytes to 1 mM palmitic acid acutely for 12 h caused a robust increase in TG accumulation (Figure 12 A). This increase in myocellular lipid was accompanied by an increase in membrane CD36. The latter effect was time dependent, with increases in membrane CD36 observed as early as 4 h after incubation with palmitate, remaining elevated at 6 h (Figure 12 B). No significant change in total protein content of this transporter was observed (Figure 12 B, blot).  The augmented membrane presence of this FA transporter following  palmitate was confirmed using immunofluorescence (Figure 12 C) and Western blot of the triton soluble and insoluble fractions (Figure 12 C, blot). In addition to the membrane presence of CD36, the FA uptake capacity was also determined using the FA fluorescent analogue BODIPY FL.  Cardiomyocytes incubated with 1 mM palmitate for 6 h showed a brighter green  fluorescence compared to control, likely as a consequence of increased uptake of the fluorescent dye (Figure 12 D). 3.2.2 Palmitate induced increase in nuclear content of FoxO1 is regulated by phospho-p38 and 14-3-3 Previously, it has been reported that an increase in membrane CD36 is linked to an amplified presence of nuclear FoxO1 in C2C12 cells275. In our study, the increase in membrane CD36 was also associated with a time dependent increase in nuclear FoxO1 (Figure 13 A), which was accompanied by a decrease in the cytosolic content of this transcription factor (Figure 13 B). No change in the total content of FoxO1 protein was observed.  We have reported that 44  phosphorylation of p38 MAPK promotes its nuclear translocation, in addition to facilitating the nuclear import of FoxO1.  Interestingly, the palmitate-induced increase in nuclear FoxO1  matched the increase in the nuclear/cytosolic ratio of phospho-p38 (Figure 13 C and D). No change in the total content of p38 MAPK protein was observed. Validation of the p38 MAPKFoxO1 association was confirmed using immunoprecipitation; palmitate induced a strong association between FoxO1 and phosphorylated p38 MAPK in the nucleus (Figure 20 A). As SB 202190 prevented the effects of PA to increase the nuclear association between phospho p38 and FoxO1, our data suggests that p38 MAPK is a likely mediator that shuttles FoxO1 into the nucleus (Figure 20 B). The effects of palmitate to increase nuclear FoxO1 was not evident with oleate (unsaturated FA) or bromo-palmitate (non-hydrolysable analogue) (Figure 20 C). In addition to import, export mechanisms can also determine the nuclear content of FoxO1; 14-3-3 proteins are major chaperones involved with export of FoxO1 297. We chose 14-3-3δ as this chaperone is a target for both MAPK signaling and nuclear export of FoxO. MAPK has been shown to interfere with dimerization and activation of 14-3-3δ, whereas down regulation of 14-33δ increases FoxO’s transcriptional activity298. Palmitic acid caused a time dependent decrease in the association between FoxO1 and 14-3-3δ as shown by immunoprecipitation (Figure 13 E). There was no effect of palmitate on the total protein content of 14-3-3δ. 3.2.3 iNOS and Cdc42 play an important role in actin cytoskeleton re-arrangement following lipid excess iNOS protein is reported to be a major target of FoxO1 299. In the presence of palmitate, an induction of iNOS protein was observed, as early as 4 h after treatment. The other sources of nitric oxide, eNOS and nNOS, remained unchanged under similar conditions (Figure 14 A). The relationship between FoxO1 and iNOS protein was confirmed using LY compound. This Akt 45  inhibitor increased iNOS protein concomitantly with nuclear FoxO1 (Fig. 3A, insets). Cdc42, a Rho family small GTPase, has a prominent role in regulating cytoskeletal re-arrangement and vesicular exocytosis300. Tyrosine nitration of Cdc42 has been shown to increase its activity301. Immunoprecipitation of palmitate treated samples with Cdc42 demonstrated an increase in Cdc42 tyrosine nitration and its association with the vesicular protein, VAMP (Figure 14 B). Along with the augmented tyrosine nitration of Cdc42, we also observed an increase in the total Cdc42 following palmitate exposure (Figure 14 B, inset).  Adult cardiomyocytes have a  predominance of F-actin, and upon stimulation, further conversion of G to F-actin occurs with Cdc42 being a major facilitator of this process. Interestingly, a time-dependent increase in G to F conversion was observed with palmitate (Figure 14 C). 3.2.4 LPS and TNF- confirm the iNOS mediated increase in membrane CD36 The relationship between iNOS and membrane CD36 was confirmed using the iNOS inhibitor, 1400W. This specific iNOS inhibitor was capable of reversing both iNOS induction (Figure 15 A) and CD36 translocation (Figure 15 B) following palmitate. To corroborate the role of iNOS in regulating membrane CD36, cardiomyocytes were exposed to a potent iNOS inducer, LPS. LPS produced a substantial increase in iNOS expression, which correlated well with an increase in membrane CD36. These increases were abolished by 1400W (Figure 15 C and D). In addition to high concentrations of plasma FA, obesity is also accompanied by increased levels of inflammatory cytokines like TNF-. Interestingly, similar to palmitate, cardiomyocytes treated with TNF- also displayed an increase in nuclear FoxO1 (Figure 15 E), iNOS protein expression, and plasma membrane CD36. These effects of the inflammatory cytokine were prevented by 1400W (Figure 15 F).  46  3.2.5 Palmitate increases the nuclear content of p65 subunit of NF-κB along with FoxO1 The transcription function of FoxO1 is enhanced when its protein-protein interaction with other partners increase293. Another major player involved with the inflammatory process and iNOS induction is NF-κB. In addition to FoxO1, we observed a time dependent increase in the nuclear migration of p65 subunit of NF-κB with palmitate and TNF- (Figure 16 A and inset). Like FoxO1, p38 MAPK has also been shown to play an important role in the nuclear entry of p65 subunit of NF-κB.  Interestingly, immunoprecipitation of nuclear FoxO1 followed by  Western blot showed an association of p65 subunit of NF-κB and phospho-p38 MAPK (Figure 16 B and C), with FoxO1. 3.2.6 Nuclear expulsion of PGC-1α following palmitate is linked to its increased acetylation Storage of FA as TG is an outcome of either increased FA uptake or reduced FA utilization. The latter process is under the control of AMPK (a metabolic switch which regulates β-oxidation through acetyl CoA carboxylase) and PGC-1α (a transcription factor that is a major determinant of mitochondrial oxidative phosphorylation through its regulation of OXPHOS proteins) 302. No change in phosphorylation of AMPK was observed with palmitate (Figure 17 A, blot). For PGC1α to have an effective transcription function, they require retention within the nucleus303. In cardiomyocytes treated with palmitate, a time dependent decrease in nuclear content of PGC-1α was observed, accompanied by an increase in cytosolic PGC-1α (Figure 17 A).  This  compartmentalization occurred without any changes in the total content of PGC-1α. One likely mechanism facilitating shuttling of PGC-1α involves its acetylation, which decreases its nuclear presence and transcription function303. Interestingly, palmitate increased the lysine acetylation of PGC-1α, an effect which was accompanied by a decreased association of PGC-1α with Sirt1 (a major de-acetylase) (Figure 17 B), with no change in total Sirt1 at 6 h. The net outcome of this 47  decreased nuclear presence of PGC-1α was a decreased expression of OXPHOS proteins (Figure 21). 3.2.7 Intralipid infusion increases cardiac triglyceride with an associated increase in nuclear FoxO1 and iNOS expression To determine the in vivo effects of lipid excess, a 20% Intralipid or saline infusion was administered into the left jugular vein of rats for 3 h. Following Intralipid administration, there was a robust increase in the cardiac TG content (Figure 18 A).  Similar to our in vitro  observations using palmitate, this increase in TG was associated with a significant augmentation in nuclear FoxO1 and iNOS expression (Figure 18 B). In our model, which focused on the early signaling events associated with lipid excess, we were unable to detect any significant increase in caspase activity or cell death upto 12 h. It is possible that extending the duration of palmitate treatment will induce lipotoxic cell death. Nevertheless, we did evaluate the content of PP2A, a ceramide target, in both in vitro (PA-12 h) and in vivo (IL-infusion) models of lipid excess. In both cardiomyocytes treated with PA and hearts from IL infused animals, there was a robust increase in PP2A expression suggesting that pathways which can induce cell death have indeed been activated (Figure 22). 3.3 Cardiac FoxO1 participates in hyperglycemia induced cell death through the mediation of iNOS and GAPDH 3.3.1 Diabetes increases nuclear FoxO1 as a consequence of attenuated survival kinase signaling in the heart. In male Wistar rats, 100 mg/kg of STZ induced severe hypoinsulinemia. The reduction in insulin was evident from day 1 post-STZ, and continued to drop such that at day 4, a 2.5 fold decrease was noted (Figure 24 A-left panel). This hypoinsulinemia closely matched the increase 48  in plasma glucose. Plasma NEFA levels only increased by day 3 following STZ (Figure 24 Aright panel). To confirm whether this decrease in insulin influenced downstream signaling of the insulin receptor, we measured phosphorylation of IRS (tyrosine-989) and Akt (serine-473), and observed a decline in cardiac IRS and Akt phosphorylation starting from day 1, and almost abolished by day 4 (Figure 24 B-blots). Akt, a prominent survival kinase, is known to increase phosphorylation of FoxO1 at serine-256, promoting FoxO1’s nuclear exit and decreasing its transcriptional function. Interestingly, phosphorylation of FoxO1 decreased from day 1 postSTZ (Figure 24 C-right panel). As anticipated, this decrease in FoxO1 phosphorylation was coupled to an increase in nuclear (Figure 24 C-left panel) and a decrease in cytosolic (Figure 24 C-centre panel) FoxO1. 3.3.2 Increased nitrosative stress during diabetes is correlated to an increase in iNOS protein. In human aortic endothelial cells, overexpression of constitutively active FoxO1 increases iNOS mRNA and protein299. We determined whether the increase in nuclear FoxO1 in cardiac tissue after STZ administration could increase iNOS protein. Western blot analysis of the three NOS isoforms revealed that following 4 days post-STZ, iNOS protein increased. There were no changes in the expression of eNOS and nNOS protein (Figure 25 A and blots). Increased iNOS expression is accompanied by formation of peroxynitrite, which increases tyrosine nitration. Microscopic evaluation of ventricular sections of STZ-diabetic hearts revealed an increase in nitrotyrosine staining compared to control (Figure 25 B) Additionally, measurement of nitrite and nitrate levels, indices of nitrosative stress, were augmented in both plasma (Fig. 2 C upper panel) and cardiac tissue (Figure 25 C lower panel) of STZ-diabetic animals.  49  3.3.3 STZ-diabetes promotes GAPDH binding to Siah1 facilitating their nuclear import. In HEK293 cells, nitrosative stress increases S-nitrosylation of GAPDH, promoting its binding to Siah1, a E3 ubiquitin ligase, thus facilitating their combined nuclear entry304. Determination of nuclear and cytosolic GAPDH using Western blot revealed a profound increase in the nuclear content of GAPDH in diabetic hearts, with no significant change in the cytosol (Figure 26 A). Diabetes also increased nuclear Siah1 (Figure 26 B), which was accompanied by a reduction in its cytosolic content (Figure 26 B-inset). To corroborate the relation between GAPDH, Siah1 and nitrosylation, we immunoprecipitated GAPDH and immunoblotted for Siah1 and nitrosylated cysteine of GAPDH. Our results indicated an increased binding of Siah1 to GAPDH along with augmented cysteine S-nitrosylation of GAPDH following diabetes (Figure 26 C). 3.3.4 Hyperglycemia increases cardiac PARP1 activation, nuclear migration of AIF and externalization of phospatidylserine. Following STZ-diabetes, cytosolic cytochrome-c and caspase-3 cleavage were found to be increased (Figure 27 A). Cleaved caspase-3 can induce cell death by two mechanisms. One is by cleaving PARP1 (a nuclear protein which acts as a stabilizer for nicked DNA there by maintaining structure and function) there by increasing DNA breaks, and the other through their activation of caspase-activated DNAses which disintegrate DNA218. In addition to GAPDH, nitrosylation targets many proteins leading to their inactivation, and caspase-3 is one such target protein305.  Interestingly, under conditions of STZ-diabetes, a substantial increase in S-  nitrosylation of both (non-cleaved and cleaved) forms of caspase-3 were observed (Figure 27 B). As we were unable to detect any increase in PARP1 cleavage (Figure 27 C-blot) in STZ-diabetic hearts, our data suggests that following nitrosylation, cleaved caspase-3 is unable to cleave 50  PARP1. Nevertheless, even though there was no change in cleavage of PARP1, its activity following diabetes was increased compared to control (Figure 27 C-left panel).  Activated  PARP1 increases PAR synthesis bringing about mitochondrial permeability transition pore opening and translocation of apoptosis inducing factor (AIF) from mitochondria to cytosol and nucleus. In the nucleus, AIF causes DNA damage 255. In the cytosol, they increase the activity of phospholipid translocase scramblase, an enzyme responsible for flipping phosphatidylserine to the outer membrane compartment, an indication of early stage apoptosis. In this study, following PARP activation, we observed an increased nuclear compartmentalization of AIF (Figure 27 Cright panel), followed by increased phosphatidyl serine externalization as observed using Annexin-V staining (Figure 27 D). 3.3.5 Time dependent normalization of DZ-induced hyperglycemia can reverse nitrosative stress mediated cardiac effects. Given that STZ induced diabetes is irreversible, we used diazoxide, a K+ channel opener. We have previously reported that this agent can induce hyperglycemia without β-cell death. Injection of DZ increased blood glucose within 2 h, with a maximum effect observed between 2 to 4 h. This increase in blood glucose was reversed completely 12 h post DZ (Figure 28 A). Similar to STZ-hyperglycemia, hearts from DZ treated animals demonstrated a decrease in Akt phosphorylation, increased nuclear presence of FoxO1 and total iNOS expression, and augmented nuclear translocation of GAPDH, Siah1 and AIF at 4 h. All of these effects were completely revoked at 12 h, once these animals achieved normal blood glucose level (Figure 28 B and C).  51  3.3.6 Cardiomyocytes treated with high glucose mirror the effects of animal models of hyperglycemia induced nitrosative stress. High glucose is a potent inducer of iNOS in endothelial cells and cardiomyocytes 299. In our current study, incubation of cardiomyocytes with high glucose for 12 h increased iNOS protein expression, an effect which was preceded by an increase in nuclear FoxO1 at 4h. The osmotic control mannitol was unable to influence nuclear FoxO1. The iNOS effects were also seen with LPS, another known potent inducer of iNOS (Figure 29 A). This effect of high glucose on iNOS was reversed by the iNOS inhibitor 1400W (Figure 29 A).  In these in vitro conditions,  nitrosative stress was also able to promote a nuclear increase in GAPDH and Siah1 as seen with in vivo models of hyperglycemia, effects that were reversed when 1400W was used (Figure 29 B). Like high glucose, palmitate also had similar effects on iNOS and GAPDH (Figure 31). The increase in nuclear GAPDH with high glucose and LPS were correlated to an increase in nuclear AIF, an effect that was reversed with 1400W (Figure 29 C), a clear demonstration that hyperglycemia induced FoxO1-mediated nitrosative stress can switch on cardiac cell death signalling both in vivo and in vitro.  52  53  Figure 4.  Dexamethasone (Dx) induced inhibition of cardiac glucose oxidation is  mediated by PDK4. A) Following incubation of cardiomyocytes with 1 M Dx for 6 h, glucose oxidation was assessed by measuring 14CO2 released from (U-14C)glucose. Results are the average value of 4 separate experiments, and are expressed as % change from control (Con). B) Mitochondrial fractions from Con and Dx treated myocytes were isolated and assayed for PDH activity. The activity assay follows the reduction of NAD+ to NADH with an increase in the absorbance at 450 nM. The activity was determined over a period of 15 min. Results are the average value for 3 separate experiments. C) Mitochondrial fractions from Con and Dx treated myocytes were used for Western blot to determine total and phospho-PDH. Prohibitin (PHB) was used as a mitochondrial marker. Bands were visualized using an ECL® detection kit and quantified by densitometry. Results are meansSE for 3 independent experiments. D) The mitochondrial fractions were also used to determine PDK1, 2 and 4 protein using Western blot, and anti-PDK1 goat and anti-PDK2 and 4 polyclonal rabbit antibodies (upper panel). The lower panel describes PDK4 gene expression measured using quantitative real-time PCR. Results are the meansSE for 3 separate experiments for each group. AU, arbitrary units. *Significantly different from control, P<0.05.  54  55  Figure 5. Cytosolic to nuclear shuttling of FoxO1 is augmented by Dx. Ventricular cardiomyocytes were treated with Dx (1 M) for 6 h. Following incubation, cells were lysed and centrifuged to isolate the nuclear (Nu) and cytosolic (Cyt) fractions. A) Nu and Cyt fractions were used for Western blot of FoxO1, using anti-FoxO1 antibody. Tot, total cell fraction.  Results are meansSE for n=3 experiments in each group.  B) A single  representative image of 3 separate experiments showing immunoflourescent nuclear localization of FoxO1 in 4,6-diamidino-2-phenylindole (DAPI) stained cardiomyocytes. C) Nu and Cyt fractions were used for Western blot of FoxO3a using anti-FoxO3a antibody. Results are meansSE for n=3 experiments in each group. *Significantly different from control, P<0.05.  56  57  Figure 6. The increase in nuclear content of FoxO1 after Dx is correlated to an increase in phosphorylation of p38 MAPK. A) Following treatment of cardiomyocytes with Dx (2-6 h), total and phospho-p38 MAPK contents in the cytosolic fraction was determined by Western blot using rabbit polyclonal total and phospho-p38 MAPK antibodies. GAPDH was used as a cytosolic marker. Results are the meansSE for 3 different experiments. B) In cardiomyocytes time dependently treated with Dx (2-6 h), nuclear fractions were also isolated to measure phospho-p38 MAPK and FoxO1 content using Western blot with rabbit polyclonal phospho-p38 MAPK and FoxO1 as the primary antibodies. Thrombin (Tb) was used as a positive control to phosphorylate p38 MAPK and enhance the nuclear content of FoxO1. Histone-3 was used as a nuclear marker.  Results are the meansSE for 3 different  experiments. C) Cardiomyocytes were pre-incubated with SB202190 for 1 h prior to 6 h incubation with Dx. Western blot of nuclear fractions was used to measure phospho-p38 MAPK and FoxO1 proteins.  Results are the meansSE for 3 different experiments.  *Significantly different from control; +significantly different from 2 h Dx; @significantly different from all other groups, P<0.05.  58  59  Figure 7.  Augmenting p38 phosphorylation with Dx substantially increases its  attachment to FoxO1 and increases their joint nuclear import. A) Control and Dx treated (6 h) cells were fixed, permeabilized and incubated with primary antibodies (rabbit polyclonal phospho-p38 MAPK and polyclonal goat against FoxO1) followed by incubation with secondary antibodies [goat anti-rabbit IgG-Alexa Fluor (green) and donkey anti-goat IgGAlexa Fluor (red)] and visualized with a confocal microscope. DAPI was used for nuclear staining 5. The merged image of FoxO1 and phospho-p38 MAPK is described in the fourth panel. Bar = 20 m. Data is from a representative experiment done thrice. B) In a separate experiment, nuclear fractions were also isolated from control myocytes and myocytes time dependently treated with Dx (2-6 h). To examine the association between phospho-p38 and FoxO1, phospho-p38 was immunoprecipitated (IP) using a phospho-p38 antibody and immunoblotted (WB) for anti-FoxO1 and anti-phospho-p38. Results are the meansSE for 35 experiments for each group. *Significantly different from control; +significantly different from 2 h Dx; @significantly different from all other groups, P<0.05.  60  61  Figure 8. Dx attenuates Akt phosphorylation by means of heat shock proteins. A) Cardiac cells were incubated with Dx (1 M) or Dx+I (Insulin, 100 nM) for 6 h, and total cell fractions were used to determine phospho-Akt using an anti-phospho Akt (Ser 473) antibody. Results are the meansSE for 3 different experiments. B) Total cell fractions were also used for Western blot to determine phospho-FoxO1 using an anti-phospho FoxO1 (Ser 256) antibody. Results are the meansSE for 3 different experiments. The inset (right panel) illustrates the influence of LY 294002 (20 M, 6 h) on cardiomyocyte total phospho-Akt and phospho-FoxO1, and nuclear FoxO1 content determined by Western blot. Results are the meansSE of 3 experiments. C) Cells incubated with Dx and I were used to examine the association between Akt and heat shock proteins (Hsp25 & Hsp90).  Total-Akt was  immunoprecipitated from cytosolic fractions using an anti-Akt antibody, and immunoblotted with anti-Hsp90, anti-Hsp25, and anti-Akt antibodies.  Total cell fractions were used to  determine total Hsp90 and Hsp25 protein expressions.  Results are the meansSE for 3  experiments in each group. D) Cardiomyocytes were pre-incubated with SB202190 for 1 h prior to a 6 h incubation with Dx. Western blot of total fractions was used to measure phospho-Akt proteins. Results are the meansSE of 3 experiments. E) Cardiomyocytes were pre-incubated with SB 202190 for 1 h prior to 6 h incubation with Dx. Western blot of total fractions was used to measure phospho-FoxO1 proteins. Results are the means  SE of 3 experiments. T, total protein. *Significantly different from control; @significantly different from all other groups P<0.05.  62  63  Figure 9. Insulin abolishes Dx-induced nuclear shuttling of FoxO1. A) Cardiomyocytes were treated with Dx in the presence or absence of increasing concentrations of insulin (1-100 nM) for 6 h. Cytosolic fractions were isolated and Western blot performed to determine FoxO1 using an anti-FoxO1 antibody. GAPDH was used as a cytosolic fraction marker. Results are the meansSE for 3 different experiments. B) Nuclear fractions from the above groups were also subjected to Western blot for FoxO1. Histone-3 was used as a nuclear marker and the results expressed as meansSE for 3 separate experiments. C) The association between FoxO1 and 14-3-3 was determined by immunoprecipitating 14-3-3 using an anti 143-3 antibody and the immunocomplex was immunoblotted for 14-3-3 and FoxO1 proteins. Results are the meansSE for 3-5 experiments for each group. *Significantly different from control; +significantly different from all other Dx treated groups; @significantly different from all other groups P<0.05.  64  65  Figure 10. Reduced acetylation of FoxO1 following Dx is determined by the binding of Sirt1 to FoxO1.  A) Ventricular cardiomyocytes were treated with Dx (1 M) for 6 h.  Following incubation, cells were lysed, centrifuged to isolate the Nu fractions, and used to determine acetyl (Ac) FoxO1 and Sirt1 proteins by Western blot using anti-acetyl FoxO1 and anti-Sirt1 antibodies.  Results are the meansSE for 3 experiments for each group. B)  Nicotinamide (NM) was used to inhibit Sirt1, and acetylation of FoxO1 subsequently determined (upper panel). In these groups, the nuclear content of FoxO1 was determined and H3 used as a nuclear marker. The lower panel describes the association between Sirt1 and FoxO1 following Dx or Dx+NM, and was determined by immunoprecipitation of nuclear Sirt1 using anti-Sirt1 antibody and the complex then immunoblotted for anti Sirt1 and anti FoxO1. Results are the meansSE for 3 experiments for each group. C) Effects of Insulin (I) and SB202190 on the acetylation of FoxO1 induced by Dx was determined by Western blot using an anti-Acetyl FoxO1 antibody. Results are the meansSE for 3 experiments for each group. *Significantly different from control; @significantly different from all other groups P<0.05.  66  67  Figure 11. FoxO1 inhibition lowers PDK4 expression seen with Dx. A) Ventricular cardiomyocytes were simultaneously treated with Dx along with insulin, SB202190 and nicotinamide for 6 h. Following incubation, cells were lysed, centrifuged to isolate the mitochondrial fractions, and used to determine PDK4 protein and phosphorylation of PDH by Western blot using anti-PDK4 and anti phospho-PDH antibodies. Results are the meansSE for 3 experiments for each group.  *Significantly different from control.  +significantly  different from Dx treated group. @significantly different from all other groups, P<0.05. B) Summary of the mechanism by which Dx induces PDK4 and reduces glucose oxidation. Incubation of cardiomyocytes with Dx stimulates p38 phosphorylation permitting nuclear entry of FoxO1.  In addition, Dx stimulates the heat shock protein axis, inhibits Akt  phosphorylation, induces Sirt1 association with FoxO1, and aids in its nuclear retention. These effects, coupled to the nuclear entry of glucocorticoid receptor following Dx, increases the expression of PDK4, decreases PDH activity and glucose oxidation is inhibited.  68  120  Production of 14CO2 (% change from basal)  100  80  60  40  20  0 Con  Dx  Dx+I  Dx+SB  Dx+NM  Figure 12. Cardiomyocyte glucose oxidation in the presence of Insulin, SB202190 and nicotinamide. Ventricular cardiomyocytes were simultaneously treated with Dx along with insulin, SB202190 and nicotinamide for 6 h. Following this incubation, glucose oxidation was assessed by measuring 14CO2 released from (U-14C)glucose. Results are the average value of 4 separate experiments, and are expressed as % change from control (Con).  69  70  Figure 13. Palmitate increases cardiomyocyte TG accumulation through participation of sarcolemmal CD36. Adult cardiomyocytes were treated with albumin bound palmitate (molar ratio 1:1) (PA, 1 mM) for 12 h, and intracellular TG content measured using HPLC (A). The influence of PA at different time intervals (2-6 h) on plasma membrane CD36 was determined by extracting membrane proteins using a sucrose cushion. Isolated proteins were subjected to Western blot using rabbit anti-CD36 and goat anti-rabbit HRP as primary and secondary antibodies respectively (B). For Confocal microscopy, cardiomyocytes were fixed, incubated with primary antibody (anti-CD36) and Alexafluor-594 (Red) was used as the secondary antibody. DAPI was used to mark the nucleus (C). Verification of membrane localization of CD36 was done using Triton-soluble and insoluble fractions following palmitate exposure for 6 h and Western blot performed for CD36 protein (C, blot). The ability of cardiomyocytes to uptake FA following exposure to palmitate for 6 h was measured using the fatty acid fluorescent analogue BODIPY FL C16 dye (10 μM) for 10 min (D). Results are the means  SE of 3-5 +  experiments in each group. *Significantly different from control; Significantly different from 2 h palmitate treatment, @Significantly different from all other groups; P<0.05.  71  72  Figure 14.  Increase in nuclear content of FoxO1 following exposure to palmitate is  mediated by p38 MAPK and 14-3-3.  Cardiomyocytes were treated with albumin bound  palmitate (PA, 1 mM) for different time intervals (2-6 h) and the nuclear (A), cytosolic (B), and total (blots) proteins isolated were subjected to Western blot for FoxO1 proteins using antiFoxO1 antibody. The same samples were used for detecting total (blots) and phospho-p38 MAPK using respective antibodies (C, D). GAPDH and Histone H3 were used as cytosolic and nuclear markers respectively. Proteins isolated from the total cardiomyocyte lysates following PA treatment were also subjected to immunoprecipitation using FoxO1 antibody, and immunoblotted for 14-3-3ς (E). Results are the means  SE of 3-5 experiments in each group. +  *Significantly different from control, Significantly different from 2 h palmitate treatment, @  Significantly different from all other groups; P<0.05.  73  74  Figure 15. Palmitate causes cytoskeletal re-arrangement through an iNOS-Cdc42 pathway. Cardiomyocytes were exposed to albumin bound palmitate (PA, 1 mM) for 2-6 h. Total protein was isolated and subjected to Western blot using anti-iNOS, eNOS, nNOS (A) and Cdc42 (B, inset) antibodies. GAPDH was used as a loading control. The same fractions were also used for immunoprecipitation using an anti-Cdc42 antibody and immunoblotted for VAMP (B, lower panel) and nitration of tyrosine residues of Cdc42 protein using an anti-nitrotyrosine antibody (B, upper panel). Actin re-arrangement was measured using a G/F actin assay kit, which separates F-actin (pellet) from G-actin (supernatant). An increase in the conversion of G to F actin represents the cytoskeletal re-arrangement.  This ratio was determined using Western blot  followed by densitometry (D). LY 294002 was used as a positive control to inhibit growth signaling and increase nuclear FoxO1 (A, inset). Results are the means  SE of 3-5 experiments +  in each group. *Significantly different from control, Significantly different from 2 h palmitate treatment, @Significantly different from all other groups; P<0.05.  75  76  Figure 16. iNOS is important for membrane translocation of CD36. Cardiomyocytes were exposed to albumin bound palmitate (PA, 1 mM, 6 h) in the presence or absence of 1400 W (10 µM), an iNOS specific inhibitor, total and membrane fractions isolated, and Western blot performed for iNOS and CD36 using anti-iNOS (A) and anti-CD36 (B) antibodies.  LPS  (1μg/ml, 12 h) was used as a positive control for iNOS induction (C) and CD36 membrane localization (D). Cardiomyocytes treated with the inflammatory cytokine, TNF- (5 ng/mL, 12 h), were used to measure nuclear FoxO1 (E), iNOS induction (F, upper panel) and membrane CD36 translocation (F, lower panel). GAPDH, Histone H3, and Na +-K+ ATPase were used as cytosolic, nuclear and plasma membrane markers respectively. Results are the means  SE of 35 experiments in each group. *Significantly different from control; @Significantly different from all other groups; P<0.05.  77  78  Figure 17. Palmitate increases the nuclear presence and association between p65 subunit of NF-κB and FoxO1. Cardiomyocytes were treated with albumin bound palmitate (PA, 1 mM, 26 h) and nuclear fractions isolated to measure p65 NF-κB (A). TNF- (5 ng/mL, 12 h) was used as a positive control (A, inset). The same fractions were used for immunoprecipitation using an anti-FoxO1 antibody and immunoblotted for p65 NF-κB (B) and p38 MAPK (C). Results are the means  SE of 3-5 experiments in each group.  *Significantly different from control;  +  @  Significantly different from 2 h palmitate treatment,  Significantly different from all other  groups; P<0.05.  79  80  Figure 18.  Time-dependent decrease in nuclear PGC-1α following palmitate is  accompanied by an increased PGC-1α acetylation.  Cardiomyocytes were treated with  albumin bound palmitate (PA, 1 mM, 2-6 h) and the nuclear (A, upper panel), cytosolic (A, lower panel) and total proteins (A, blot) isolated were subjected to Western blot for PGC-1α using anti- PGC-1α antibody.  The total protein samples were also subjected to  immunoprecipitation using PGC-1α antibody, and immunoblotted for acetyl-lysine (B, upper panel) and Sirt1 (B, lower panel). Results are the means  SE of 3-5 experiments in each group. +  *Significantly different from control; Significantly different from 2 h palmitate treatment, @  Significantly different from all other groups; P<0.05.  81  82  Figure 19. Intralipid infusion increases cardiac triglyceride with an increase in nuclear FoxO1 and iNOS expression. Animals were anaesthetized and the left jugular vein cannulated. Intralipid (IL, 20%; 5 ml/kg/h) was infused over a period of 3 h. At the third hour, hearts were removed and cardiac samples were used for HPLC analysis of TG (A) and Western blot of nuclear FoxO1 and iNOS protein expression (B).  GAPDH and Histone H3 were used as  cytosolic and nuclear markers respectively. Data are mean±S.E. for 3 rats in each group, except for the TG measurement which represents an average of 2 hearts in each group. *Significantly different from control (CON), P<0.05.  The right panel describes a mechanism by which  palmitate increases cardiomyocyte TG content. Incubation of cardiomyocytes with palmitate increases nuclear entry of FoxO1.  Nuclear FoxO1 causes an increase in the total protein  expression of iNOS and Cdc42. The formed iNOS also nitrates the Cdc42 protein. Increased Cdc42 protein with tyrosine nitration increases the membrane translocation of CD36 by rearranging the actin-cytoskeleton thereby permitting further entry of fatty acids. This results in the increased influx of fatty acids which decrease the association between PGC-1α and Sirt1 thereby decreasing PGC-1α activity, culminating in channelizing of fatty acids towards storage as triglyceride inside the cell.  83  FoxO1 following i.p of Nuclear Phospho-p38 (AU) Phospho-p38 following i.p of Nuclear FoxO1 (AU)  2.5  A  Con  *  2.0  PA I.P Phospho-p38 WB FoxO1 I.P FoxO1 WB Phospho-p38  1.5 1.0 0.5 0.0 2.5  *  2.0 1.5 1.0 0.5 0.0 Con  PA  B Con  PA  SB+PA I.P FoxO1 WB Phospho-p38 I.P FoxO1 WB FoxO1  2.5  Con  C  @  OA  Br-PA FoxO1  2.0  Nuclear FoxO1 (AU)  PA  Histone-H3  1.5  1.0  0.5  0.0  Con  PA  OA  Br-PA  84  Figure 20. Palmitate increases the association between phospho-p38 MAPK and FoxO1 in the nucleus. Adult cardiomyocytes were treated with albumin bound palmitate (PA, 1 mM) for 6 h, and nuclear proteins isolated. Proteins isolated from the nuclear fractions were subjected to immunoprecipitation using phospho-p38 MAPK antibody and immunoblotted for FoxO1 (A, upper panel). The same nuclear fractions were immunoprecipitated using FoxO1 antibody and immunoblotted for phospho-p38 MAPK (A, lower panel). Myocytes were pre-incubated for 1h with SB202190, followed by incubation with PA. After 6h, nuclear fractions were isolated for immunoprecipitation of FoxO1 followed by Western blot for phospho-p38 and FoxO1 (B). Cardiomyocytes were also treated for 6 h with oleate (unsaturated FA, 1 mM) or bromopalmitate (non-hydrolysable analogue, 1 mM), and nuclear FoxO1 determined. Histone H3 was used as a nuclear marker. Results are the means  SE of 3-5 experiments in each group. *Significantly different from control, @Significantly different from all other groups; P<0.05.  85  PA  OXPHOS Complex  Con  1 11 111 1V V  OXPHOS Complex (AU)  5 4 3  *  2  *  1 0  1  11  * 111  * 1V  * V  Figure 21. Palmitate exposure reduces mitochondrial OXPHOS protein. Cardiomyocytes were treated with albumin bound palmitate (1 mM) for 12 h and mitochondrial fractions isolated to measure the OXPHOS (І, ІІ, ІІІ, ІV & V) protein complex. Results are the means  SE of 3-5 experiments in each group. *Significantly different from control; P<0.05.  86  Con  PA-12 h PP2A  Con  IL PP2A  Figure 22. PP2A induction following in vitro and in vivo lipid excess. Adult cardiomyocytes were treated with albumin bound palmitate (PA, 1 mM) for 12 h or a 20% Intralipid or saline infusion was administered into the left jugular vein of rats for 3 h, and total proteins isolated to measure PP2A by Western blot. The results are representative of 2 different experiments.  87  Con  Dx  STZ Nuclear FoxO1 PM-CD36  Figure 23. Models of T1D and T2D also demonstrate an increase in nuclear FoxO1 and membrane CD36. To induce insulin resistance and T2D, we used dexamethasone (Dx, 1 mg/kg for 7-days).  We have previously reported that with this dose of Dx, whole body insulin  resistance develops early (a decrease in glucose infusion rate occurs within 4h), and extending the treatment for 7 days maintains insulin resistance. Chronically, there was an increase in blood glucose and plasma NEFA, whereas plasma insulin decreased. For T1D, we have successively and repeatedly used streptozotocin (STZ, a selective -cell toxin). After i.v. injection of 100 mg/kg STZ, we have demonstrated significant hypoinsulinemia, hyperglycemia, and hyperlipidemia. In hearts from Dx treated or STZ diabetic rats, we demonstrate amplification in the nuclear content of FoxO1 along with an increase in plasma membrane CD36.  88  A 30  *  Blood Glucose (mmol/dl)  Plasma Insulin (ng/ml)  2.5  2.0  *  *  *  20  10  1.5  * *  1.0  *  Plasma NEFA (mmol/L)  0 1.5  *  0.5  *  *  1 2 3 Days STZ  4  1.0  0.5  0.0  0.0  1  0  B  0  3 2 Days STZ  1  2  4  0  3  4  Days STZ Phospho-Akt (Ser-473) Phospho-IRS (Tyr-989) Beta actin  C 1  2  Days STZ  4  3  0  1  2  4  3  *  *  *  0.6 0.4 0.2 0.0  0  1  2  3  4 Days STZ  2  3  4  Days STZ Ph-FoxO1 Tot-FoxO1  2.0  0.20 Mean OD of Cytosolic FoxO1/ Total FoxO1 (AU)  Mean OD of Nuclear FoxO1/ Total FoxO1 (AU)  *  1  Beta actin  Histone-H3  0.8  0  Days STZ Cyt-FoxO1  Nu-FoxO1  Mean OD of PhosphoFoxO1/ Total FoxO1 (AU)  0  0.15  * *  0.10  *  0.05 0.00  0  1  2  3  * 4 Days STZ  1.5  *  1.0 0.5 0.0  0  1  *  *  *  2  3  4 Days STZ  89  Figure 24. STZ-induced diabetes attenuates cardiac insulin signaling and increases the nuclear content of FoxO1.  Male Wistar rats (270-290 g) were treated with 100 mg/kg  streptozotocin to induce diabetes, and the animals killed after 1-4 days. Using diagnostic kits, plasma insulin and NEFA were measured, and a glucometer was used to determine blood glucose (A).  Frozen heart tissue collected at termination were homogenized and proteins  extracted were used for measuring phosphorylation of IRS at tyrosine-989 and phosphorylation of Akt at Serine-473 using Western blot. Beta actin was used as a loading control (B). The frozen heart tissue from the same groups were also subjected to fractionation, and nuclear and cytosolic fractions isolated. The nuclear fraction was used to measure the content of FoxO1, which was normalized to total FoxO1. Histone (H3) was used as a nuclear marker (C, left panel). The cytosolic fraction was also used to determine the content of FoxO1 which was again normalized to the total FoxO1 content. Beta actin was used as a loading control for the cytosolic fraction (C, middle panel). Total homogenates from the heart tissue was used to measure the phosphorylation of FoxO1 at Serine 256, and was compared to control (day 0). This was then normalized to total FoxO1 content (C, right panel).  Results are the means±SE of 3-5  experiments in each group. *Significantly different from control (day 0); P<0.05.  90  C  Protein Nitrite Plasma Nitrite (Fold increase to control) (Fold increase to control) 3.0  6 5 4 3 2 1 0  2.0  1.5  1.0  0.5  0.0  *  *  Plasma Nitrate Protein Nitrate (Fold increase to control) (Fold increase to control)  iNOS protein expression (AU)  A Con  * STZ  2.5  2.0 Con  Con  6 5 4 3 2 1 0  2.0  STZ  1.5 iNOS  1.0 nNOS  0.5 eNOS  0.0  actin  B  STZ  Nitro-Tyrosine  *  *  1.5  1.0  0.5  0.0  91  Figure 25. Nitrosative stress following STZ-diabetes is evident in plasma and myocardial tissue. Male Wistar rats were treated with 100 mg/kg streptozotocin to induce diabetes, and the animals killed after 4 days. Total iNOS, nNOS and eNOS protein contents were measured in heart homogenates and compared to control. Beta actin was used as a loading control (A). Ventricular cross sections of control and STZ treated rats were fixed and then immunolabeled using a nitrotyrosine specific antibody. Tissue sections were visualized and photographed using a confocal microscope (B). The plasma and tissue homogenates from control and STZ treated animals were used to measure both nitrite (C, left panels) and nitrate (C, right panels) levels. Results are the means±SE of 3-5 experiments in each group. *Significantly different from control; P<0.05.  92  15  * CON  Nuclear GAPDH Nuclear Histone H3 Cytosolic GAPDH  5  Cytosolic Actin  0  Con  STZ  2.0  Nuclear Siah1 (AU)  B  STZ  10  *  1.5  Cytosolic Siah1 (AU)  Nuclear GAPDH (AU)  A  2 1  *  0  Con STZ CON  STZ  1.0  Nuclear Siah1 Nuclear Histone H3  0.5  Cytosolic Siah1 Cytosolic Actin  0.0  IP GAPDH WB: Siah1 (AU)  1.2  STZ  *  0.6  CON  STZ I.P GAPDH  WB Siah1  0.0  IP GAPDH WB: Nitrosylated Cys (AU)  C  Con  WB Nitrosyl-Cys  *  2  WB GAPDH  1  0  Con  STZ  93  Figure 26.  Siah1 assists nuclear translocation of GAPDH following diabetes-induced  nitrosative stress in cardiac tissue. STZ-diabetes induced male Wistar rats were euthanized after 4 days and hearts isolated and frozen.  The frozen heart tissues were subjected to  fractionation, to obtain nuclear and cytosolic fractions.  Using Western blot, GAPDH was  determined in these fractions (A). Histone H3 and Beta actin were used as loading controls for nuclear and cytosolic fractions respectively. The same fractions were used to measure Siah1 in nucleus (B) and cytosolic compartments (B, inset). Histone H3 and Beta actin were used as loading controls for nuclear and cytosolic fractions respectively. The homogenates from control and STZ treated animals were used for immunoprecipitation using anti-GAPDH antibody. The pulled down precipitate was used to measure Siah1 (C, upper panel), cysteine nitrosylation (C, lower panel) and GAPDH respectively (C). Results are the means±SE of 3-5 experiments in each group. *Significantly different from control; P<0.05.  94  PARP activity (Fold increase to control)  D 1.0  0.5  0.0 Cytosolic AIF (AU)  0.15  B 15  *  10  5  0  2.5  2.0  *  1.5  Con i.p Cleaved Caspase-3 Nitrsoylated Cys (AU)  Cytosolic Cyt C (AU)  *  0.10  0.05  0.00  Cleaved Capase 3 (AU)  0.20  Nuclear AIF (AU)  i.p Caspase-3 Nitrsoylated Cys (AU)  Con STZ  A 2.0  25  1.0  0.0 0.6  *  1.5  1.0  0.5  0.0  FL-Caspase-3  Nitro-Cys  20  *  15  10 5  0  C Cleaved PARP Nu-AIF  Cyt-AIF  0.5  *  0.3  *  0.0  STZ  Annexin - V  95  Figure 27.  Diabetes induced nitrosative stress increases PARP1 activation, nuclear  migration of AIF and apoptosis . STZ-induced diabetic rats were euthanized after 4 days, and cytosolic fractions isolated from heart tissue was used for measuring cytosolic cytochrome C (A, left panel) and cleaved caspase-3 (A, right panel). The homogenates from control and STZ treated animals were used for immunoprecipitation using anti-caspase-3 (full length, FL) and anti-cleaved caspase-3 antibodies, and the immunoprecipitates used to measure cysteine nitrosylation of full length caspase-3 and full length caspase-3 (B, left panel blots) and nitrosylated cleaved caspase-3 (B, right panel). From the heart homogenates, Western blot was performed to measure PARP cleavage (C, left panel, blot) and PARP activity using an assay kit (C, left panel). From the same heart samples, nuclear and cytosolic fractions were isolated to measure AIF using Western blot (C, right panel). Cross sections from myocardial ventricles of control and STZ treated rats were fixed and then immunolabeled using Annexin-5 specific antibody for the detection of phosphatidylserine translocation as a marker of apoptosis. Tissue sections were visualized and photographed using a confocal microscope (D). Results are the means±SE of 3-5 experiments in each group. *Significantly different from control; P<0.05.  96  35  Blood glucose (mmol/L)  A  *  *  30 25  *  *  20  *  15 10 5 0 0  1  2  4  6  8  10  h  12  Time after DZ treatment  B Con  3  *  DZ- 12h  3  # iNOS protein (AU)  Phospho-Akt (Ser 473) (AU)  6  DZ- 4h  *  DZ  2 Con 1  4h  12h Phospho-Akt  #  iNOS Beta actin  0  0  * #  0.3 0.0  0.3  *  0.2 0.1 0.0  #  Nuclear GAPDH (AU)  0.6  Nuclear AIF (AU)  Nuclear Siah1 (AU)  Nuclear FoxO1 (AU)  C 15  * DZ  10 5  #  0  3 2 1  Con  4h  12h Nuclear FoxO1 Nuclear Siah1  *  Nuclear GAPDH  #  Nuclear AIF Histone-H3  0  97  Figure 28. Nitrosative stress and nuclear migration of apoptotic proteins fluctuates with the induction and reversal of hyperglycemia following diazoxide Male Wistar rats were treated with diazoxide (100 mg/Kg) and the animals followed for blood glucose levels from 1-12 h (A). The whole heart homogenates from control, 4 h and 12 h diazoxide treated hearts were used to measure the phosphorylation of Akt at Serine-473 (B, left panel) and iNOS induction (B, right panel). Beta actin was used as a loading control. Samples from the above mentioned groups were also used to isolate nuclear fractions. In these nuclear fractions, expression of FoxO1 (B, left upper panel), Siah1 (B, left lower panel), GAPDH (B, right upper panel) and AIF (B, right lower panel) were determined using Western blot. Histone (H3) was used as a nuclear loading control. Results are the means±SE of 3-5 experiments in each group. *Significantly different from control; # Significantly different from 4 h diazoxide treated group; P<0.05.  98  Con  HG  LPS  HG + 1400W  H G  S LP  on C  iNOS Protein (AU)  H G  *  1.0  +  14 00 W  A  *  iNOS  0.5  Beta-Actin  @  14 00 W  0.0  Nuclear GAPDH (AU)  + G H  S LP  H G  C  *  4 3  on  B  Nu-GAPDH  *  Nu-Siah1  2  Hisone- H3 1  @  0  + G H  S  Nu-AIF  0.6 @ 0.4  LP  on  * H G  0.8  *  C  Nuclear AIF (AU)  1.0  14 00 W  C  Histone-H3  0.2 0.0  99  Figure 29.  High glucose mediated nuclear migration of GAPDH and AIF in  cardiomyocytes is reversed by the iNOS inhibitor 1400W. Isolated rat adult cardiomyocytes were incubated with normal glucose (5 mM), high glucose (25 mM), LPS (10 µg/ml) and high glucose+1400W (10 μM). Total cell lysates were used for measuring iNOS protein expression using Western blot (A). Beta actin was used as a loading control. Cells from the same treatment groups were used to isolate nuclear fractions, which were then used to measure GAPDH (B) and Siah1 (B, blots). The nuclear fractions isolated from the treatment groups were also used to measure nuclear compartmentalization of AIF (C). Histone (H3) was used as a nuclear loading control. Results are the means±SE of 3-5 experiments in each group. *Significantly different from control; @ Significantly different from high glucose treated group; P<0.05.  100  A M  6h  4h  2h  C on  HG  A  Nu-FoxO1 Histone-H3  Nuclear FoxO1 (AU)  0.15  *  *  0.12  0.09  0.06  0.03  0.00  Con  2h  4h  MA  6h  HG  10  0 1.  1 0.  C on  LPS-12 h  B  iNOS Beta-Actin  *  iNOS protein expresion (AU)  1.2  *  1.0 0.8 0.6 0.4 0.2 0.0  Con  0.1  1.0  10  g/ml  LPS-12 h  101  Figure 30 LPS dose dependently increases iNOS induction. Isolated cardiomyocytes were incubated with high glucose (25 mM, 0-6 h) and mannitol (25 mM, 6 h, as osmotic control). Nuclear proteins separated were used to measure FoxO1 by Western blot (A). Cardiomyocytes were also treated with increasing concentrations of LPS (0.1-10 µg/ml, 12 h). Proteins isolated from whole cell fractions were used to measure iNOS induction by Western blot (B). Histone and Beta actin were used as loading controls. Results are the means±SE of 3-5 experiments in each group. *Significantly different from control, P<0.05.  102  LP S  HG  PA  Co n  A iNOS protein expression (AU)  0.5 iNOS  *  0.4  Beta-Actin  0.3 0.2  *  *  PA  HG  0.1 0.0 Con  LPS  Nuclear GAPDH expression (AU)  S LP  HG  PA  Co n  B 2.1  *  1.8 1.5  *  *  PA  HG  GAPDH Histone- H3  1.2 0.9 0.6 0.3 0.0 Con  LPS  103  Figure 31  iNOS induction can increase the nuclear content of GAPDH.  Isolated rat  cardiomyocytes were treated with control media, albumin-bound palmitate (1 mM), high glucose (25 mM), and LPS (10 µg/ml) for 12 h. From the treated groups, cell lysates were used to measure iNOS protein using Western blot (A), and nuclear fractions separated were used to measure GAPDH protein (B).  Histone (H3) was used as a nuclear loading control.  *Significantly different from control; P<0.05.  104  Figure 32 A summary diagram describing the mechanism of how cardiac FoxO1 regulates cell death through the mediation of iNOS and GAPDH  105  Chapter 4: Discussion 4.1  The increase in cardiac PDK4 following short term dexamethasone is controlled by  an Akt-p38-FoxO1 signaling axis In a healthy heart, glucose oxidation accounts for approximately 30% of energy provided to the cardiac muscle2. PDH is the rate limiting enzyme responsible for glucose oxidation in the mitochondria.  A notable regulator of PDH activity in the heart is PDK4 2, which by  phosphorylating the 1 subunit (at Serine 293) of the E1 moiety of PDH, prevents its catalytic conversion of pyruvate to acetyl CoA, and eventually glucose oxidation306. During insulin deficiency, PDK4 expression is augmented, PDH is inhibited and glucose oxidation is reduced. Previously, we have reported that in vivo administration of Dx induces whole body insulin resistance and increased PDK4 expression in the heart 68.  In the current study, our data  demonstrate that isolated cardiomyocytes, when incubated with Dx, also exhibit reduced glucose oxidation and PDH activity. As Dx increased a) phospho-PDH (surrogate marker for PDH activity) and b) PDK4 protein and gene expressions, our data suggests that the effects of Dx to inhibit cardiac glucose oxidation likely involves mediation at the juncture of the PDK4 gene. In conditions of attenuated insulin signaling like fasting, diabetes and high fat feeding, PDK4 is upregulated307. Traditionally, nuclear receptors like PPAR, PPAR, and glucocorticoid and retinoid receptors have been suggested to be involved in the upregulation of this kinase127. With glucocorticoids, PDK4 gene expression is mediated through a GRE that has been identified in the PDK4 promoter279. However, for glucocorticoids to induce receptor mediated transcription, a requirement of p300/CBP complex to be bound to this transcriptional machinery is necessary, and this is mediated by nuclear FoxO proteins279. Thus, in HepG2 cells, upregulation of FoxO 106  further increased the expression of PDK4 normally seen with Dx279. In the current study, the increase in cardiac PDK4 protein seen with Dx correlated well with the observed increase in the nuclear content of FoxO1, but not FoxO3. As this effect of Dx occurred in the absence of any change in total FoxO1 protein, but a decrease in cytoplasmic FoxO content, our data suggests that nuclear shuttling of FoxO1 is likely responsible for this PDK4 induction seen with short term Dx treatment. In cardiomyocytes, glucocorticoids are known to increase phosphorylation of p38 MAPK and their nuclear translocation308. Incubation of cardiomyocytes with Dx increased cytosolic p38 phosphorylation after 4 h. This increase was observed at 4 h, remained high up to 6 h, and was apparent in the absence of any change in total protein. Nuclear p38 phosphorylation lagged behind the cytosolic increase, suggesting potential shuttling of p38 from cytosol to nucleus. FoxO is known to undergo nuclear ingress following p38 activation. In the present study, p38 activation with thrombin or Dx increased nuclear FoxO content. The effect of Dx was reversed by simultaneous incubation with SB202190. The interaction between phospho p38 and FoxO1 was confirmed using immunoprecipitation and immunofluorescent imaging.  Thus, in the  presence of Dx, phosphorylated p38 time dependently associates with FoxO1, increasing their nuclear appearance. Overall, our data suggests that phosphorylated p38 is a key regulator for the nuclear translocation of FoxO proteins. In addition to nuclear import, retention of FoxO within the nucleus is also important for increasing transcriptional activity. Akt is known to phosphorylate FoxO and promote its nuclear exit297.  Recently, in beta cells exposed to Dx, downregulation of Akt signaling has been  reported. In the present study, cardiomyocytes incubated with Dx also showed a decrease in Akt phosphorylation. As expected, the decrease in Akt phosphorylation was accompanied by a 107  reduction in phosphorylation of FoxO1 (Ser-256). Both these effects were reversed with a pharmacological concentration of insulin. The importance of an intact Akt signaling and its relation to phosphorylation and nuclear retention of FoxO was verified using LY compound. On Dx attachment to the glucocorticoid receptor, heat shock proteins (Hsps) are released from the receptor complex. More recently using HepG2 cells, a function of Dx to increase the expression of Hsps has also been described309. Both these properties of Dx to release Hsps and increase their expression act to prevent protein unfolding and hence degradation. Indeed, in human neuroblastoma (SH-SY5Y) cells, Hsps have been shown to bind to Akt, resulting in decreased phosphorylation and degradation310. In our study, using immunoprecipitation, addition of Dx to isolated myocytes elicited binding of Akt with Hsp90 and Hsp25. As this increased interaction between Hsp and Akt occurred in the absence of any increase in total protein expressions of Hsps, our data suggests that this interaction between Hsps and Akt is a consequence of a receptor mediated event. Interestingly, addition of insulin was able to reverse this Hsp-Akt interaction. It is possible that, as Hsps have been suggested to act as chaperones for insulin fragments internalized following their receptor activation, addition of excess insulin competitively binds to Hsp reducing its interaction with Akt. Other signals that interfere with Akt phosphorylation is p38. Using SB compound to inhibit p38 we were unable to reverse the attenuation of Akt following Dx. These data suggest that inhibition of Akt phosphorylation is independent of the p38 MAPK pathway. On Dx administration, activation of p38 MAPK (which promotes nuclear entry of FoxO) and attenuation of Akt signaling by Hsps (which allows nuclear retention) play prominent roles in increasing the nuclear content of FoxO. Thus, to augment nuclear exclusion of FoxO, a stimulus would be required to overcome both these effects induced by Dx. Interestingly, although insulin 108  up to a concentration of 10 nM marginally increased nuclear exit and cytoplasmic compartmentalization of FoxO, these changes were not significant. The insulin concentration had to be increased to 100 nM before normalization of cytoplasmic and nuclear FoxO were achieved, suggesting that only high concentrations of insulin are able to overcome the effects of Dx in cardiomyocytes. Insulin initiates its cascade of effects on FoxO1 by Akt dependent phosphorylation. Once Akt phosphorylates FoxO, the trafficking chaperones 14-3-3 bind to FoxO, facilitating their nuclear exit and preventing their nuclear re-entry. On the other hand, MAPK signaling has been shown to phosphorylate 14-3-3, and disrupt its binding to FoxO297. Thus, in the presence of Dx, the reduced 14-3-3 binding to FoxO could be a result of reduced Akt or increased MAPK signaling, or both. As high concentration of insulin was able to reverse the binding of 14-3-3 to FoxO, resulting in its nuclear exit, our data suggests that simply manipulating the Akt pathway is able to normalize nuclear content of FoxO. In addition to nuclear shuttling and retention, an enhancement in the DNA binding ability of FoxO is also required for its transcriptional activity. For effective binding, nuclear FoxO needs to de-acetylated at the lysine residue by a class III histone de-acetylase protein like sirtuins, which also enhances their nuclear retention. When glucose entry and glycolysis are diminished, the ratio of NAD+/NADH increases, and Sirt activity is augmented. In Sv40 hepatocytes, Sirt1 is known to de-acetylate FoxO and increase its transcriptional activity for gluconeogenic enzymes311. In our study, we also report that following Dx, a decrease in the acetylation status of FoxO is evident, without any change in the nuclear and total protein content of Sirt1. Moreover, using a Sirt specific inhibitor like NM, we were able to reverse the acetylation status of FoxO1 without changing the nuclear FoxO1 protein content. This implies that even in the presence of an abundance of protein in the nucleus, simply manipulating the acetylation status of FoxO could 109  likely determine their transcriptional activity. Using immunoprecipitation, we demonstrated a close association between Sirt1 and FoxO1 following Dx. This effect was reversed by NM, and suggests that the de-acetylation of FoxO1 following Dx is likely a consequence of an increased association to Sirt1 and not due to an increase in nuclear Sirt1. Similar to NM, insulin reversed the acetylation status induced by Dx, whereas SB202190 was ineffective. As insulin normalizes the Akt pathway (and hence glucose metabolism) whereas the p38 signaling pathway could not, our data suggests that the increase in Sirt1 activity which we observe with Dx is a consequence of attenuated Akt signaling. PDK4 is a major rate limiting enzyme involved in glucose oxidation. As FoxO has a major role in PDK4 regulation by glucocorticoids, we used different strategies to regulate FoxO and hence PDK4 expression.  Manipulating FoxO’s nuclear entry (using SB202190), nuclear  retention (with insulin) or acetylation status (by means of nicotinamide) was able to alter the expression of its target protein PDK4. These data suggests that in the heart, FoxO1 have a major PDK4 regulating function. One limitation of this study is that all of our data were obtained using isolated cardiomyocytes. In addition, in an in vivo setting, the heart is exposed to multiple substrates including fatty acids and triglycerides, which were absent in our experimental conditions. PDK4 and PDH activities can be modulated by pyruvate levels and ratios of NADH/NAD + and AcetylCoA/CoA. In the absence of fatty acids in the incubation buffer, coupled to an altered insulin signaling following Dx, the control of PDK4 and PDH activities by metabolites is likely minimal in our in vitro setting, and may not completely reflect the situation in an intact myocardium.  110  4.2  Cardiac triglyceride accumulation following acute lipid excess is through activation  of a FoxO1-iNOS-CD36 pathway Obesity due to lipid excess is a major feature of the current Western diet, and is associated with a number of cardiovascular complications202.  Lipids, either through induction of  inflammatory cytokines, or accumulation of intracellular TG, have detrimental effects on the heart. FA can induce cardiomyocyte cell death by stimulating cytokines like TNF-α and IL-1β. In addition, FA, through formation of diacylglycerol and TG, can also induce ceramide formation and cell death202,  206  .  In the present study, we report that acute treatment of  cardiomyocytes with saturated FA increases TG accumulation by a process that requires activation of a FoxO1-iNOS-CD36 pathway. To mimic nutrient excess, cardiomyocytes were incubated with a saturated FA, palmitate. Within 12 h, a substantial increase in intracellular TG stores was observed. In humans and rodents, TG accumulation subsequent to FA uptake is an outcome of an increased plasma membrane presence of the FA transporter, CD36, whose genetic ablation dramatically increases plasma FA levels. Additionally, overexpression of CD36 in C2C12 cells is known to direct FA to TG312. CD36 is an 88 kDa membrane glycoprotein which acts as a receptor for multiple ligands including thrombospondins, low density lipoproteins, fibrillar β-amyloids, bacterial components, infected erythrocytes and FA187. Thus, in settings where FAs are increased like Type 1 (STZ-induced) and Type 2 (db/db mice) diabetes, increased membrane presence of CD36 has been implicated in delivering surplus lipids to the heart 313, 314. In this study, our data suggest that in cardiomyocytes, palmitate can augment membrane CD36 mediated lipid uptake and increase intracellular TG stores.  111  In vitro and in vivo studies have reported that an increase in the nuclear presence of FoxO1 is associated with an increased membrane presence of CD36. This increase in membrane CD36 occurred without augmentation in mRNA or total protein expression275.  In adult  cardiomyocytes, we observed that the augmentation in membrane CD36 was accompanied by an increase in nuclear and decrease in cytosolic compartmentalization of FoxO1. FoxO1’s nuclear presence is mostly determined by growth (Akt) and stress (MAPK) signals. Indeed, in models of diabetes and high fat feeding283, decreased growth or increased stress signals increased the nuclear presence and transcriptional activity of FoxO164, 315. Unlike these chronic models, in our study using acute palmitate incubation, stress (p38 MAPK) and not growth signals (data not shown) appeared to play a major role in the nuclear ingress of FoxO1. Similar effects were observed in MIN6 cells incubated with saturated FA316. This influence of p38 MAPK on cardiomyocyte FoxO1 nuclear compartmentalization appeared to be a consequence of a direct interaction of these proteins. Previously, we have reported a similar interaction between p38 MAPK and FoxO1, together with their nuclear import, in cardiomyocytes treated with glucocorticoids. Interestingly, in HEK293T cells, phosphorylation sites for p38 MAPK have been identified on FoxO1 which are different from sites phosphorylated by growth signals like Akt317. In addition, p38 MAPK binding to FoxO1 is known to upregulate the transcriptional activity of FoxO1317. Overall, our data suggests that early activation of p38 MAPK following acute lipid excess is central for FoxO1 to shuttle into the nucleus to exhibit its transcriptional function.  Once inside, FoxO1 can undergo nuclear exit, a process mediated by 14-3-3  chaperones297. Previously, we have reported that following chronic diabetes, fatty acids decrease the intracellular content of 14-3-3. In the current study, short term palmitate incubation had no effect on the total protein content of 14-3-3. Instead, palmitate reduced the binding between 112  FoxO1 and 14-3-3. As stress kinases are known to decrease the binding between FoxO1 and 143-3, our data suggests that p38 MAPK is important for both nuclear import and retention of FoxO1 following acute palmitate treatment. As these effects were only observed with saturated FA, and not with unsaturated FA or non-metabolizable analogues of palmitate, our data imply that it is these mechanisms which make palmitate a notorious instigator of lipotoxicity. In this study, although nuclear FoxO1 was accompanied by an increase in membrane CD36, no change in the total protein content of this transporter was observed. Recently, C2C12 cells also showed an increase in membrane CD36 independent of any changes in CD36 mRNA or protein following an increase in nuclear FoxO1275. These observations suggest that independent of direct transcriptional control of CD36 protein, a role for FoxO1 to activate the transport machinery that drives CD36 to the plasma membrane is possible. Nitric oxide synthase (NOS) is an enzyme involved in the production of nitric oxide.  In the heart, of the three isoforms  expressed, iNOS (NOS2), a calcium-calmodulin independent enzyme has the highest potential for producing peroxynitrite unlike its counterparts, nNOS (NOS1) and eNOS (NOS3) 318. Intriguingly, overexpression of FoxO1 in vascular endothelial cells has been shown to increase iNOS expression299. In the current study, the palmitate induced increase in nuclear FoxO1 was associated with an increase in iNOS protein expression and membrane CD36 presence, effects that were reversed by the specific iNOS inhibitor 1400W, or duplicated by LPS, an endotoxin which acts as a potent iNOS inducer. Apart from an inflammatory role, iNOS has also been implicated in protein trafficking through nitration of proteins like Cdc42 (cell division cycle 42)319. Cdc42, a small rho GTPase family member, is involved with cytoskeletal re-arrangement and vesicular exocytosis320. Like the GTP-bound form, nitration of Cdc42 causes it’s activation, allowing this protein to rearrange the cytoskeleton in addition to increasing its association with 113  VAMP proteins there by facilitating vesicular exocytosis320. The iNOS-Cdc42 interaction has been shown to have a significant impact in the migratory function of RAW264.7 cells and primary macrophages by regulating the cytoskeleton319. It should be noted that Cdc42 per se is also known to increase class A scavenger receptor-activity and lipid transport in mouse peritoneal macrophages and aged skin fibroblasts respectively321,  322  . Given the increase in  nitration and protein expression of Cdc42, its association with VAMP, and cytoskeletal rearrangement, our data suggests that on exposure of cardiomyocytes to palmitate, the increase in membrane CD36 is largely a consequence of a FoxO1-iNOS-Cdc42 axis. Whether iNOS could also disrupt the microtubular network, thereby preventing the re-cycling of CD36, allowing it to be permanently located at the membrane surface during obesity and Type2 diabetes is currently unknown and will be investigated. In conditions of obesity and Type 2 diabetes, there is increased production of TNF-α from adipocytes. TNF-α acting through its receptors, can bring about cardiomyocyte cell death, cardiac hypertrophy, contractile dysfunction and heart failure. Like FA, TNF-α is also known to increase the nuclear FoxO1 transcriptional activity in diabetes, and iNOS expression in cardiomyocytes323, 324. In our study, TNF-α increased nuclear FoxO1, iNOS protein expression, and membrane CD36, effects that were reversed by 1400W. Hence, in conditions of lipid excess, this pro-inflammatory cytokine could act synergistically with FA to bring about activation of the FoxO1-iNOS-CD36 pathway resulting in cardiac TG accumulation and cardiovascular complications. Following toll like receptor activation or insulin resistance, a protein-protein interaction between FoxO1 and NF-κB in the nucleus has been suggested to contribute towards the inflammatory process293. Additionally, like their effects on FoxO1, FA and TNF-α are able to 114  increase the nuclear content of active NF-κB, a process that involves nuclear migration of the p65 subunit of the NF-κB complex. In our study, following palmitate or TNF-α, we observed an increased nuclear presence of the p65 subunit of NF-κB. As p38 MAPK phosphorylation has been shown to increase the nuclear content of p65 in this and other studies 325, our data suggest that following palmitate, phoshpo-p38 in a trimeric complex with FoxO1 and p65 undergoes nuclear entry allowing their mutual transcriptional activation. Following entry into the cardiomyocyte, FA can either be oxidized to generate ATP or stored as TG. The oxidization machinery is under the control of AMPK and PGC-1α. AMPK increases β-oxidation by phosphorylation and inhibition of ACC2. PGC-1α is a transcription factor coactivator which regulates oxidative phosphorylation of substrates through its target, OXPHOS (oxidative phosphorylation proteins). OXPHOS is a complex of five proteins residing in the mitochondria which derive ATP by a chain of redox reactions302. PGC-1α’s nuclear presence is mandatory for its transcription co-activation function. In cardiomyocytes treated with palmitate, no change in AMPK was observed. However, the nuclear content of PGC-1α decreased with a concomitant increase in its cytosolic levels.  The metabolic sensors AMPK and Sirt1, by  phosphorylation and de-acetylation of PGC-1α respectively, increases its nuclear presence and DNA binding ability326, 327. In the absence of any alteration in AMPK phosphorylation, the decreased nuclear presence of PGC-1α following lipid excess was likely a consequence of increased lysine acetylation of PGC-1α due to reduced Sirt1 binding327. It should be noted that in addition to de-acetylation by Sirt1, acetylation of PGC-1α by acetyl transferase general control of amino acid synthesis (GCN5) can also determine its nuclear retention and DNA binding ability, and should be examined. As the decreased nuclear presence of PGC-1α coincided with a decrease in the protein content of the OXPHOS complex, our data implies that in addition to 115  augmented lipid uptake through CD36, excess lipids interfere with FA oxidation thereby promoting storage of FA as TG. One limitation of the present study is that in addition to CD36, other transporters like FABP and FATP have also been implicated in transport of FA.  FABP and CD36 work  synchronously to supply lipids to the heart, and an increased membrane presence of FABP has been demonstrated to cause lipotoxicity328. FATP (FATP1 and 6) is the other major cardiac fatty acid transporter, and is co-localized with CD36. Cell lines overexpressing FATP have been shown to increase lipotoxicity329. Whether the FoxO1-iNOS pathway can affect FABP and FATP to deliver excess lipid to the heart is currently unknown, and should be investigated. 4.3  Cardiac FoxO1 participates in hyperglycemia induced cell death through the  mediation of iNOS and GAPDH Diabetes is a cellular crisis, not only for energy generation but also for cell survival. Although, Type1 diabetes begins with β-cell death, its long term complications include damage to organs including the eye, kidney and heart 330-332. Diabetic cardiomyopathy is a condition accompanied by increased cardiomyocyte cell death, the prevention of which is crucial as cardiac cells lack an intrinsic regenerative capacity. Major mechanisms that have been proposed to bring about cell death during diabetes include altered metabolism, an imbalance in calcium homeostasis, mitochondrial inefficiency, increased free radical generation, high levels of cytokines, increased stress signal stimulation and decreased/impaired survival signaling 333-337. With diabetes, the survival kinase Akt, which is downstream of the insulin receptor, is blocked. As a consequence, Akt’s ability to regulate the transcriptional activity of FoxO1 is weakened, turning on FoxO1 function.  In the current study, using in vivo and in vitro models of  116  hyperglycemia, we report a novel role for FoxO1 to switch on cardiomyocyte cell death through an iNOS-GAPDH pathway. FoxO1, a prominent member of the Fox family and subfamily O, is known to regulate metabolism, stress response and cell death269,  297  .  For example, in the liver, it promotes  gluconeogenesis, whereas in skeletal muscle, it inhibits glucose oxidation through its ability to induce PDK4. In adipose, FoxO1 has a PPAR-γ suppressive action with inhibition of adipocyte differentiation, and in β-cells, it inhibits Pdx-1 thus decreasing insulin synthesis and secretion and promoting cell death269. Related to the heart, this transcription factor can increase autophagy under conditions of stress by inducing Gabarapl1 and Atg12 genes291. In insulin resistance and nutrient excess, we have reported a role for cardiac FoxO1 to regulate metabolism through its ability to induce PDK4 and iNOS thereby decreasing glucose oxidation and increasing triglyceride accumulation in the heart respectively. In the present study, we uncover a novel role of FoxO1 to switch on cardiac cell death pathways in the heart following hyperglycemia. FoxO1’s activity is determined by phosphorylation, acetylation and ubiquitination, with Akt mediated phosphorylation being a major regulator as it decreases the nuclear presence of FoxO1264. Using STZ, we were able to attenuate phosphorylation of Akt and FoxO1 at Ser-256, and observed an increase in nuclear migration of FoxO1. These effects were evident as soon as insulin levels dropped (day1), and persisted for 4 days following STZ. At present, an established role of FoxO1 in initiating cell death in cancer cell line is through its ability to induce death receptor ligand (FasL) or the pro-apoptotic gene, Bim338. In the heart, we report an additional mechanism by which hyperglycemia induced nuclear increase in FoxO1 can switch on cell death. This mechanism involves S-nitrosylation signalling due to iNOS induction.  117  iNOS, one among the 3 NOS members, has a higher potential than its NOS counterparts to produce abundant NO. Its induction is dependent on different conditions like insulin resistance, nutrient excess, sepsis and inflammation. iNOS induction can lead to an increase in reactive nitrogen species with resultant protein modifications, changes in cellular redox status, and induction of cell death339, 340. For example, in vascular endothelial cells, gain of function of FoxO1 increased iNOS mRNA and protein levels with resultant endothelial dysfunction 299. In this study, both in vivo and in vitro models of hyperglycemia showed an increase in the nuclear content of cardiac FoxO1 and nitrosative stress, the latter effect likely a consequence of increased iNOS and not other NOS isoforms. Dual mechanisms can explain target protein modifications by excess NO; tyrosine nitration and cysteine S-nitrosylation. Tyrosine nitration plays an important role in insulin resistance and lipotoxicity, conditions that could lead to cell death if present chronically. Additionally, tyrosine nitration can directly activate cytochrome c, inactivate MnSOD, and initiate mitochondrial mediated cell death pathways 170, 341, 342. Unlike tyrosine nitration, S-nitrosylation can directly induce cell death by their effects on DNA and nuclear proteins304. To do this, a mechanism is required to transfer reactive nitrogen species into the nucleus, a process that is likely facilitated by nuclear migration of S-nitrosylated proteins like GAPDH. GAPDH, primarily a glycolytic protein, also has DNA stabilizing and transcriptional activation functions in the nucleus. More recently, a role for GAPDH in apoptosis, cancer, and neuro degeneration has also been proposed, making it a major therapeutic target 343-345. In HEK293 cells, iNOS mediated S-nitrosylation of GAPDH augmented its nuclear translocation followed by cell death304. Following hyperglycemia, we also observed an iNOS mediated Snitrosylation of GAPDH and nuclear import. As GAPDH does not have a nuclear localization 118  sequence, this nuclear translocation was facilitated by formation of a complex between nitrosylated GAPDH and Siah1, allowing the complex to translocate to the nucleus. It should be noted in addition to hyperglycemia, fatty acids were also capable of increasing iNOS, with a resultant increase in nuclear GAPDH, suggesting that nitrosylated GAPDH is an impressive NOtransferring agent to the nucleus. As nitrosative stress in the nucleus is known to cause DNA damage by its effect on histone proteins, we measured PARP1 activity, a major enzyme involved in DNA repair. Our data suggests that following hyperglycemia-induced nitrosative stress, there is increased cytosolic to nuclear shuttling of GAPDH followed by PARP1 activation. Interestingly, over activation of PARP1 can increase poly-ADP ribose (PAR) synthesis. Increased PAR can induce mitochondrial membrane leakiness, allowing mitochondrial AIF to translocate to cytosolic and nuclear compartments254. The latter events were also observed in our in vivo and in vitro models of hyperglycemia. AIF, a 67 kDa FAD dependent mitochondrial protein is known to cause cell death termed parthanatos due its ability to directly or indirectly, through its collaborative action with endonucleases, fragment DNA in the nucleus346. In the cytosol, AIF is known to stimulate phospholipid translocase scramblase mediated phosphatidylserine flipping, an event that precedes cell phagocytosis. As externalization of phosphatidylserine was a prominent feature observed with persistent hyperglycemia in our STZdiabetes model, we suggest that PARP1-AIF signalling could be a mechanism to bring about this novel form of cell death in diabetic hearts. Caspase-3, a cysteine dependent aspartate specific protease orchestrates apoptosis, a programmed cell death pathway, following its cleavage/activation by cytochrome c in the cytosol347. Thus, in STZ-diabetic FVB mouse hearts and H9c2 myoblasts incubated with high glucose, the increase in cytosolic cytochrome c and caspase-3 cleavage is associated with 119  apoptosis348. We also report an increase in the cytosolic content of cytochrome c and caspase-3 cleavage following STZ-hyperglycemia. Caspase-3 mediates apoptosis through its ability to cleave and inactivate PARP1, and/or cleave and activate caspase activated DNAse 347. Unexpectedly, caspase-3 cleavage in our study was accompanied by an increase rather than a reduction in PARP1 activity. Interestingly, in cultured hepatocytes, nitric oxide has an inhibitory effect on caspase-3 through its modification by S-nitrosylation349. Additionally, in SNAP-treated hepatocytes, caspase-3 was nitrosylated and inactivated, with reduced PARP cleavage349. We also demonstrate an increase in S-nitrosylation of uncleaved and cleaved caspase-3 following STZ-hyperglycemia, with no change in PARP cleavage.  Overall, our data imply that S-  nitrosylation on GAPDH accompanied by PARP1 activation, together with caspase-3 Snitrosylation mediated inhibition of PARP1 cleavage act together to bring about cardiac cell death during diabetes (Figure 32). Limitations of the study: Conclusive evidence for the contribution of FoxO1 in cardiomyocyte cell death during diabetes is only possible with FoxO1 deletion. However, global knock out of FoxO1 has been shown to be embryonically lethal whereas cardiac specific deletion can induce heart failure per se. Another strategy for determining the importance of FoxO1 is the use of specific pharmacological inhibitors, but no such agents are currently available.  120  Chapter 5: Summary and conclusions In summary, Dx stimulates p38 MAPK phosphorylation permitting nuclear entry of FoxO1. In addition, with stimulation of the glucocorticoid receptor by Dx, heat shock proteins are released to bind and inhibit Akt phosphorylation as a means of protecting this kinase. Reduced Akt phosphorylation decreases FoxO phosphorylation and enables its nuclear retention. Moreover, the resulting decrease in glucose uptake and glycolysis due to attenuated Akt signaling induces Sirt1 association with FoxO1, increases its de-acetylation, and makes it transcriptionally active.  Together, these effects result in increasing the nuclear content of  transcriptionally active FoxO1. When coupled to nuclear entry of the glucocorticoid receptorligand complex following Dx, an increased expression of PDK4 is evident, which inhibits PDH activity and can lower glucose oxidation (Figure 11). These data are relevant given recent suggestions that altering glucose utilization can set the stage for heart failure. Regarding excess lipids, activation of stress kinase pathways increase the nuclear content of FoxO1. This nuclear presence of FoxO1 per se or accompanied by an increased protein interaction with other nuclear transcription factors like NF-kappa B could turn on inflammatory markers like iNOS and vesicular proteins like Cdc42 along with their tyrosine nitration (Figure 19). Collectively, these proteins increase CD36 at the plasma membrane through re-arrangement of the actin cytoskeleton. CD36 mediated lipid influx, accompanied by a decrease in PGC-1α activity, contributes towards lipid accumulation in the heart (Figure 33).  The resulting increase in  reactive oxygen/nitrogen species can induce multiple changes including apoptosis, hypertrophy, and heart failure. These outcomes emerge through activation of various signaling pathways (e.g., PKCs, p38, ASK-1, NF-kB) or nitration of tyrosine residues of different proteins (e.g., IRS, Cyt-c) that hamper insulin signaling, cause mitochondrial dysfunction, loss of structural 121  integrity, and DNA strand breaks.  Interestingly, models of T1D and T2D also showed  amplification in the nuclear content of FoxO1 along with an increase in plasma membrane CD36. Thus, impeding this FoxO-iNOS-CD36 pathway could decrease the unregulated FA supply and lipid accumulation in the heart, limit oxidative/nitrosative stress and help ameliorate the cardiovascular complications associated with excess lipids during obesity and diabetes. Finally, under conditions of hyperglycemia, FoxO1 had a role in inducing iNOS mediated nitrosylation of proteins which was associated with cardiac cell death (Figure 33). This may provide a pharmaceutical target for preventing the cardiac damage and cardiac cell loss under conditions of hyperglycemia, hyperlipidemia and sepsis. As diabetes is accompanied by turning on of different cell death mechanisms that can result in cardiomyopathy2, shedding light on this novel-cardiac cell death signalling axis could offer resistance to impairments and functional loss associated with diabetic cardiomyopathy.  It should be noted that even though FoxO1 has  independent roles in regulating cardiac glucose metabolism, lipid metabolism and cell death during insulin resistance, nutrient excess and diabetes, altered glucose and fatty acid metabolism can themselves lead to cell death and cardiac damage when extended for long durations (Figure 34).  122  Figure 33. Diagram describing the consequences of FoxO1 induction under conditions of nutrient excess, insulin resistance and diabetes.  123  Figure 34. Summary showing different mechanisms by which cardiac FoxO1 can bring about cell death following its activation.  124  References 1.  Stolar M. Glycemic control and complications in type 2 diabetes mellitus. Am J  Med;123:S3-11. 2.  An D, Rodrigues B. Role of changes in cardiac metabolism in development of diabetic  cardiomyopathy. Am J Physiol Heart Circ Physiol 2006;291:H1489-1506. 3.  Goudy KS, Tisch R. Immunotherapy for the prevention and treatment of type 1 diabetes.  Int Rev Immunol 2005;24:307-326. 4.  Alexandraki K, Piperi C, Kalofoutis C, Singh J, Alaveras A, Kalofoutis A. Inflammatory  process in type 2 diabetes: The role of cytokines. Ann N Y Acad Sci 2006;1084:89-117. 5.  Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interventions  in type 1 diabetes. Nature;464:1293-1300. 6.  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