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Glucocorticoid and its effect on cardiac glucose utilization Puthanveetil, Prasanth Nair 2008

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GLUCOCORTCOID AND ITS EFFECT ON CARDIAC GLUCOSE UTILIZATION by PRASANTH NAIR PUTHANVEETIL B.Pharm., Dr. MGR Medical University, India 2002 M.Pharm., Manipal Academy of Higher Education, (Manipal), India 2005 A thesis submitted in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE in The Faculty of Graduate Studies (Pharmaceutical Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August, 2008 ©Prasanth Nair Puthanveetil, 2008 ABSTRACT Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (DEX) treated animals, glycogen accumulation was enhanced. We examined the influence of DEX on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. Following DEX, cardiac tissue had limited contribution towards the development of whole body insulin resistance. Measurement of GLUT4 at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated to an increased GLUT4 mR.NA. Both total and phosphorylated AMPK increased following DEX. Immunoprecipitation of AS 160 followed by Western blotting demonstrated no change in Akt phosphorylation at Ser473 and Thr308 in DEX treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr172, which correlated well with AS 160 phosphorylation. In DEX hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas GSK-3-f3 phosphorylation was augmented. Our data suggest that AMPK mediated glucose entry, combined with activation of glycogen synthase and reduction in glucose oxidation (Qi, D., et al. Diabetes 53:1790, 2004), act together to promote glycogen storage. Our data suggest that in the presence of intact insulin signaling, AMPK mediated glucose entry, combined with activation of glycogen synthase and the previously reported reduction in glucose oxidation, act together to promote glycogen storage. Should these effects persist 11 chronically, they may explain the increased morbidity and mortality observed with long term excesses in endogenous or exogenous glucocorticoids. 111 TABLE OF CONTENTS ABSTRACT.ii TABLE OF CONTENTS iv LIST OF FIGURES vi LIST OF SYMBOLS vii LIST OF ABBREVIATIONS viii ACKNOWLEDGMENTS x DEDICATION xi 1.0 Introduction I 1.1 Glucocorticoids I 1.1.1 Endogenous release of glucocorticoids 1.1. 2 Activation of released glucocorticoids 2 1.1.3 Systemic functions of cortisol 1.1.4 Cellular mechanism of action of cortisol 1.1.5 Use of synthetic glucocorticoids 1.1.6 Complications associated with excess glucocorticoids 5 1.2 Insulin Resistance 7 1.3 Glucose Transporters 9 1.3.1 Signaling regulating glucose transport 10 1.3.2 Role of cytoskeleton in glucose transport 12 1.3.3 Factors regulating GLUT4 expression 12 1.4 Glucose metabolism and glucocorticoids 13 1.5 AMP-activated protein kinase 14 1.5.1 Structural basis of AMPK 15 1.5.2 Factors involved in AMPK activation 15 1.5.3 Role of AMPK in the body 15 1.5.4 Effect of AMPK activation in major tissues 16 1.5.5 .. .Metabolic complications associated with abnormal AMPK activity 17 1.6 Glycogen 18 iv 1.6.1 .Glycogenin 19 1.6.2 Enzymes regulating glycogen formation 20 1.6.3 Factors leading to glycogen storage 21 1.6.4 Cardiac complications linked to excess glycogen 21 2.0 Research rationale and hypothesis 23 3.0 Materials and methods 25 3.1 Experimental Animals 25 3.2 Euglycemic hyperinsulinemic clamp 25 3.3 Tissue specific response to insulin 26 3.4 Myocardial glycogen content 26 3.5 Subcellular compartmentalization of GLUT4 27 3.6 Immunoprecipitation and Western blotting 27 3.7 Measurement of mRNA 27 3.8 G-6-P content 28 3.9 Glucose uptake in cardiomyocytes 28 3.10 Materials 28 3.11 Statistical analysis 29 4.0 Results 30 4.1 Cardiac tissue has limited influence on whole body insulin resistance induced by DEX 30 4.2 Buildup of cardiac glycogen following DEX is coupled to GLUT4 translocation 30 4.3 DEX augments both total and phosphorylated AMPK 31 4.4 Phosphorylation of Akt substrate of 160 kDa is mainly regulated by AMPK 32 4.5 Glycogen synthase undergoes robust dephosphorylation with acute administration of DEX 32 5.0 Discussion 34 6.0 Figures 39 7.0 References 46 8.0 Appendix and future direction 57 V LIST OF FIGURES Figure-I Dexamethasone effects on whole body and tissue specific insulin 39 resistance Figure-2 Cardiac glycogen accumulation following dexamethasone 40 Figure-3 Subcellular localization of GLUT4 protein 41 Figure-4 Changes in AMPK protein (total and phosphorylated) and gene expressions in hearts isolated from DEX treated animals 42 Figure-5 AMPK regulation of Akt substrate of 160 kDa (ASI6O) 43 Figure-6 Enzyme regulation of cardiac glycogen synthesis subsequent to 44 administration of DEX Figure-7 Schematic mechanism of how DEX regulates cardiac glycogen 45 through AMPK vi LIST OF ABBREVIATIONS ABC ATP-binding cassette family ACC Acetyl coenzyme A carboxylase ACTH Adrenocorticotrophic hormone AMP Adenosine monophosphate AMPK AMP activate protein kinase ASI6O Akt substrate of 160 kDa ATP Adenosine triphosphate AV Arginine Vasopressin cAMP Cyclic adenosine monophosphate CBG Corticosteroid binding globulin CRH Corticotrphic releasing hormone CPT-1 Carnitine palm itoyl transferase-1 CS Cushings syndrome DEX Dexamethasone eNOS Endothelium derived nitric oxide synthase GC Glucocorticoid GLUT Glucose transporter GLP Glucagon like peptide G-6-P Glucose-6-phosphate GP Glycogen phosphorylase GR Glucocorticoid receptor GRE Glucocorticoid response elements GS Glycogen synthase GSK-3- Glycogen synthase kinase -3-beta HMG C0A 3-hydroxy-3-methyl-glutaryl-CoA red uctase HPA Hypothalamic pituitary axis l1--HSD 11--f3-Hydroxysteroid dehydrogenase HSP Heat shock proteins IL Intralipid vII IR Insulin resistance IRS Insulin receptor substrate IR Insulin resistance IRS Insulin receptor substrate H MG C0A 3-hyd roxy-3-methyl-g Iutaryl-C0A red uctase MR Mineralocorticoids receptor mRNA Messenger ribonucleic acid NAD Nicotinamide adenine dinucleotide NADP Nicotinamide adenine dinucleotide phosphate PDC Pyruvate dehydrogenase complex PDK Pyruvate dehydrogenase kinase PKC Protein kinase C PP Protein phosphatase PPAR Perioxisome proliferator activated receptor RNA Ribonucleic acid RT-PCR Real time-polymerase chain reaction SDS Sodium dodecyl sulphate SEM Standard error of means UDP Uridine diphosphate VAMP Vesicle associated membrane protein WPW Wolff-Parkinson-White syndrome vI11 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 evety member of my committee starting from Dr. Chang, Dr. Bandiera, Dr Marzban and Dr.Laher 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 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; Girish, Kim, Fang and Elham for providing a wonderful support and friendly lab atmostphere. I sincerely acknowledge the support and encouragement provided to me by Dr. Pulinnilkunnil. I express my deep love and gratitude for Manuettan, Achemma and my dear ones for being with me all time and for their sincere prayers. I thank William for being an inquisitive summer student who worked with me during my training program. 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. I would like to sincerely acknowledge the collaborative efforts of Dr. Michael Allard for helping me in performing some of the critical experiments in this thesis. 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 and Achan. Without my Amma’s prayers, wishes and support this would not have been possible. tx 1.0 Introduction 1.1 Glucocorticoids Although the term glucocorticoid (GC) represents cortical steroids released from the adrenal cortex, it also includes the synthetic analogues developed for various inflammatory and immune disorders. The human GC is known as cortisol, where as its rodent counterpart is known as corticosterone (18). 1.1.1 Endogenous release ofglucocorticoids Endogenous hormones are secreted in response to different stresses like physical, emotional and environmental factors. Their secretion has characteristic diurnal patterns which reach peak levels during the late sleep phase, and declines by early morning (113). On experiencing stress, the hypothalamus gets activated and releases two major initiators of GC synthesis, corticotrophic releasing hormone (CRH) and arginine vasopressin (AV) from the paraventricular nuclear region (80). These secreted hormones facilitate secretion of adrenocorticotrophic hormone (ACTH) by stimulating the anterior pituitary. The pituitary secretion then acts on the adrenal cortex to stimulate both the secretion and release of cortisol (110). Under normal conditions, our body is well equipped with a defense mechanism that prevents excess secretion of cortisol through a negative feed back mechanism on the hypothalamic-pituitary-axis-(HPA) (81, 110). In people suffering from Cushing’s (CS), this negative feed back defense mechanism is lost, likely as a consequence of a single nucleotide polymorphism of GR(95, 129). Under these conditions, the enhanced uncontrolled release of cortisol accelerates the process of adipogenesis contributing to obesity in these patients (104, 131). The augmented level of cortisol is also known to increase the expression of angiotensin-Il and endothelin-I, and 1 decrease the expression of eNOS leading to atherogenesis (31, 133). In addition, due to its antagonizing action on other steroids like growth and sex hormones, physical and mental retardation and reduced sexual activity are also apparent (51). Finally, the increased release of cortisol is also associated with osteoporosis and reduced bone development (71). 1.1.2 Activation ofreleased glucocorticoids Once released from the adrenal cortex, more than 90% of cortisol binds to corticosteroid binding globulin (CBG) (1, 68). However, the effect of cortisol is only apparent when it is in the free form, as only free cortisol is able to permeate through cell membrane(68). Inside the cells, the concentration and fate of cortisol is also determined by efflux transporters like P-glycoproteins, that belong to the ATP-binding cassette family (ABC) (130). For example, in endothelial cells of the blood brain barrier and neurons, P glycoproteins are present in abundance, and are suggested to contribute towards drug resistance in the brain (55). 11-13-hydroxysteroid dehydrogenases (1 1-J3 HSD) are major enzymes which activate or deactivate GCs. Two major isoforms of this enzyme exist; 1 1-13 HSD1 and 11-13 HSD2. 11-13 HSD1 functions as a reductase and is NADP-dependent where as 11-13 HSD2 has a dehydrogenase function and is NAD-dependent (2, 49). The reductase activity is responsible for increasing cortisol action in both muscle and adipose tissue (by converting the GC into the more active cortisol) whereas the dehydrogenase activity plays an important role in deactivating cortisol through its conversion to an inactive form cortisone, there by facilitating its elimination from the kidneys (56). The presence of these two enzymes in various tissues is specific (123). 11-13 HSD1 is predominantly 2 present in liver, adipose and muscle tissue, where as 11-13 HSD2 is mostly present in the kidney (56). Interestingly, the heart demonstrates an increased presence of 11-13 HSD 1 in both cardiomyocytes and fibroblasts, and reduced content of 11-13 HSD2. 11-13 HSD1 has become a pharmacological target for use in conditions like obesity and the metabolic syndrome, conditions that are accompanied by increased cortisol activity. Currently, 11- 13 HSD inhibitors like glycerrhitinic acid and adamantyl triazole are under investigation in these conditions (57, 120). 1.1.3 Systemic functions ofcortisol Once activated, cortisol is known to have numerous important roles within the body, controlling glucose, salt and water homeostasis in addition to blood pressure. GCs favor pathways that promote plasma glucose elevation (41). In the systemic circulation, they inhibit glucose uptake and utilization in skeletal muscle there by reducing its peripheral consumption. In addition, GCs affect carbohydrate and lipid metabolism through their ability to up or down regulate many metabolic genes. For example, gluconeogenesis in the liver is enhanced through increased expression of phosphoenol pyruvate carboxykinase (PEPCK) (41, 144). In the liver, they are also known to enhance the activity of enzymes that convert glucose-6-phophate to free glucose which is then released into the systemic circulation (144). Their ability to maintain salt and water balance is thought to be due to their ability to activate the mineralocorticoid receptor (MR) as GCs have a high affinity towards this receptor (40). 1.1.4 Cellular mechanism ofaction ofcortisol Cortisol, on attachment to GR, release the heat shock (Hsp-90) and FK-binding proteins (FKBP) that act as molecular chaperones remaining attached to the receptor complex in 3 the resting state through protein-protein interactions (75). The formation of the receptor- chaperone protein complex is important to keep the receptor in the inert state, thereby preventing it from getting activated automatically in the absence of any ligand (13, 52). Once the ligand displaces the HSPs and FKBP from the complex, migration of the receptor-ligand complex into the nucleus brings about its actions. Thus, in the nucleus, they activate transcription of genes through direct interaction with DNA. This is possible either through glucocorticoid response elements (GRE) present in DNA or through a direct transactivation process that requires the involvement of their co-activators (30). Through their effects on GRE, they bring about either gene activation or gene repression (105). The transactivation process is mediated by cofactors attached to glucocorticoid receptors, thus bringing about repression of inflammatory cytokine expression through which they exhibit their anti-inflammatory and immmunosupressant activity (8, 9). The down stream effects of all these processes include either activation or suppression of protein expression. Apart from this, they are also known to directly regulate the post translational process without having any effects on the respective genes. In addition to this conventional pathway, they have also been known to act through specific membrane bound and non-specific membrane bound receptors by which they activate certain G protein coupled receptor systems thereby activating the downstream protein kinase-A and mitogen activated protein kinase pathways (69, 91). It is believed that through this membrane bound receptor activation, they bring about their rapid non-genomic effects, but this conclusion has yet to be validated. Because of these complexities of actions, GCs are known to play an important role in maintaining the body’s metabolic balance, 4 and irregular activity has been associated with metabolic complications like insulin resistance, CS and heart disease. 1.1.5 Use ofsynthetic glucocorticoids Administration of synthesized GCs like dexamethasone, prednisone, and prednisolone are in wide spread use for different conditions. They are mostly used during cortisol deficiencies like Addison’s disease, organ transplants, inflammatory (arthritis) and allergic (chronic asthma, obstructive pulmonary disease) conditions, malignancies, and severe pathogenic infections (39, 108, 122, 127). The biological half life of these synthetic GCs varies and they are classified accordingly as short, intermediate and long acting. The naturally occurring cortisol has a very short biological half life (8h). Conversely, the biological half life of dexamethasone is between 36-54h, whereas prednisolone has a half life between these two (1 8-36h) (24). As discussed earlier, GCs have a non-specific action on the MR, with cortisol having the most and dexamethasone having the least influence. Regarding potency the synthetic GCs have greater potency than the naturally occurring cortisol (24)and among the synthetic GCs, dexamethasone heads the list. All of these qualities makes it the drug of choice among the synthetic counterparts in clinical use. 1.1.6 Complications associated with excess glucocorticoids High levels of GCs leads to “Cushing’s syndrome” (CS) that is associated with both an increased secretion in addition to an augmented tissue uptake of cortisol. Clinically, patients suffering from CS share a close similarity to people with Metabolic Syndrome (M5). The major symptoms include central obesity, moon face (a characteristic feature), erectile dysfunction in men, menstrual disturbances and hirsuitism in women, 5 hypertension, decreased sexual activity, muscle weakness, insulin resistance, and diabetes (34, 35, 97). Cushing’s syndrome can either be ACTH induced or non-ACTH induced. ACTH induced CS arises due to carcinomas in pituitary regions, whereas non-ACTH induced CS arises due to adenomas in the adrenal cortex (88, 89). For ACTH induced CS, pituitary surgery is recommended, and for non-ACTH induced CS, adrenectomy is the preferred solution (7). Pharmacotherapy is also available for both of these disorders. Drugs like valproic acid and bromocriptine, which can act at the neuronal level, stabilize pituitary function, and are used for ACTH induced CS (63, 93). For non-ACTH induced CS, drugs that act directly on adrenal glands, and bring about a decline in both the synthesis and release of cortisol (ketoconazole, metyrapone), are used. Glucocorticoid receptor antagonists like mifepristone (RU-486) has also been proved to be an effective strategy to treat this complex syndrome (54). PPAR-’y agonists and dopaminergics have also been shown to be effective against CS, but are still under clinical evaluation (25, 47). Chronic therapy with GCs is also associated with adverse effects similar to that seen in patients with CS. In children, these adverse effects include a higher incidence of cardio and cerebrovascular problems, cerebral palsy and reduced brain development (33, 38, 132). In addition, long term dexamethasone use in infants with chronic lung disease has also been shown to result in hypertension. Children suffering from pulmonary dysplasia exhibit a reduction in heart rate along with hypertension with chronic dexamethasone administration. In adults, the use of GCs in patients with rheumatoid arthritis or chronic pulmonary disorders increases their risk of developing ischemic heart disease, cerebrovascular complications and heart failure (79, 121, 138). For example, data from clinical studies conducted in UK with more than 50,000 subjects receiving oral GC 6 therapy on a daily basis, were at a higher risk of developing heart failure, myocardial infarction and stroke (121). Surprisingly, in a different study, all of these complications were not seen in the control population taking GCs but were only observed in a population suffering from inflammatory complications. After analyzing the serum, it was concluded that only subjects with positive rheumatic factor developed the risk of cardiovascular and cerebrovascular complications. Chronic high doses of GCs increase the incidence of osteoporosis, resulting in fractures (14, 143). In rodents, just few days treatment with dexamethasone caused a reduction in insulin levels, hyperglycemia, hyperketonemia, and dyslipidemia (72). 1.2 Insulin Resistance Insulin resistance (IR) is a growing global epidemic. Some of the major reasons for its occurrence include stress, unhealthy dietary habits, sedentary life style, lack of exercise, genetic mutations and chronic use of drugs which can induce it. It can normally lead to complications like Type 2 diabetes, obesity, hypertension, metabolic syndrome, non alcoholic steatohepatitis and polycystic ovarian syndrome (102). Although JR forms the maj or backbone for all these complications, the actual cause of its occurrence is still not clear and is under extensive investigation. One of the major issues with JR is that there is no easy, cheap and direct measure to effectively diagnose this condition. Obesity and stress are considered to be the major culprits who initiate JR. During obese conditions, there are high amounts of circulating free fatty acids which can stimulate the release of inflammatory cytokines. A similar situation is prevalent during stress, with resultant activation of the adrenergic and HPA system, accompanied by excessive secretion of 7 catecholamines and cortisol. Increased activity of 11.43 HSD1 in inducing insulin resistance in these conditions cannot be excluded (96, 106). Normal insulin signaling is always accompanied by auto-desensitization through down stream kinases. During IR, IRS-i tyrosine phosphorylation is accompanied by phosphorylation of Akt and finally S6 Kinasel. S6 Kinasel phosphorylates serine/threonine of IRS, thereby destabilizing the IRS complex, changing its configuration, and reducing phosphorylation at tyrosine to bring about JR. The high fat induced JR follows a different pattern (128). In normal subjects, insulin maintains blood glucose and plasma triglycerides in equilibrium with its ability to uptake glucose to peripheral tissues, and inhibits lipolysis from tissues of fat storage. During obesity the circulating free fatty acids, due to their ability to activate certain G-protein coupled receptors like GPR-40, stimulate the protein kinase-Ce and C-Jun-N-terminal kinase-l pathway, resulting in increased serine phosphorylation of IRS-i instead of normal tyrosine phosphorylation. During stress, activation of NF-icB stimulates the release of cytokines like TNF-c and interleukins. These are known to increase the expression and activity of protein-tyrosine-phosphatase lB and phosphatase and tensin homologue on chromosome 10 (PTEN) (73), resulting in dephosphorylation of IRS at tyrosine and Akt respectively . These different mediators, by obstructing the normal insulin signaling pathways, hinder insulin action on peripheral and fat tissues, there by raising blood glucose and triglycerides and aggravating the condition of JR. In addition to this, TNF-cL induces IR through PKCö by its effect on phosphorylating Ser/Thr residue on IRS (84). In IR, the body responds by increasing insulin secretion, thereby maintaining blood glucose. Chronically, the ability of the pancreas to secrete this excess insulin to meet the 8 higher demand of the body is lost, and Type 2 diabetes develops. More recent findings suggest that increased insulin secretion, in the presence of IR, can further aggravate the condition of JR. GCs have been very closely associated with insulin resistance. We have recently shown that a single dose of dexamethasone (1 mg/kg) produced whole body JR using the euglycemic-hyperinsulinemic clamp. In these animals, there was no change in blood glucose or plasma insulin levels (101). Measurement of cardiac and skeletal muscle insulin signaling only showed a decline in basal and insulin stimulated Akt levels in skeletal, but not cardiac tissue. This was also shown under in vitro conditions using isolated skeletal muscles treated with GCs for 12-days. In pancreatic 3 cells, GCs have been shown to cause a decrease in GLUT2 mRNA expression. When combined with an increased fatty acid oxidation, there is a resultant decrease in glucose stimulated insulin secretion (43) 1.3 Glucose transporters There are multiple types of glucose transporter isoforms in different tissues playing specific roles. For example, GLUT 1 is the transporter protein located at the plasma membrane, and is not regulated by the normal insulin signaling pathway. They are responsible for basal glucose uptake. GLUT3 and GLUT12 are transporters present in fetal heart, but as cardiac development occur, its expression decreases (136). GLUT3 and GLUT8 are predominantly present in the brain. GLUT2 acts as the major glucose transporter in pancreatic f cells. GLUT1O and GLUT1 1 are widely distributed in placenta, pancreas, kidney and muscle tissues. GLUT5 is the transporter primarily responsible for fructose uptake. In adipose tissue and cardiac and skeletal muscle, 9 GLUT4 is the major glucose transporter under the control of insulin, and is involved in the continuous supply of substrate for storage and generation of energy (136). 1.3.1 Signaling regulating glucose transport Among the glucose transporter isoforms, GLUT4 is the integral transporter involved with insulin stimulated glucose uptake in muscle and adipose tissue. Present predominantly in an intracellular pool, there are many signals which regulate the translocation of GLUT4 from this intracellular compartment to the plasma membrane. The most important signal is the insulin stimulated IRS-PI3K-PDK1-Akt pathway. Apart from this conventional pathway, current research has also implicated PKC isoforms as playing a role in insulin stimulated glucose transport (53). A unique feature of GLUT4, which demarcates it from other GLUT isoforms, is its ability to undergo recycling within the cell. Insulin stimulation is not only required for translocation of GLUT4 to the plasma membrane but also to recycle between the intracellular compartments (107). GLUT4 vesicles normally has two areas of existence; either they remain attached to the plasma membrane or they are found in the recycling compartments within the cytosol like recycling endosomal compartment (ERC), specialized compartments (SC) and GLUT4 storage vesicles (GSV). Insulin signaling is mainly responsible for exocytosis of these vesicles but is only partially responsible for endocytosis (126) There are many vesicular proteins that co exist with GLUT4 during its journey to the surface and vesicle associated membrane proteins (VAMP) are the major ones. Among VAMPs, VAMP-2 is the one that is closely associated with insulin mediated translocation to the surface (62). Clarithrin coated pits and caveolea are the major proteins involved with GLUT4 during its internalization process. The Rab family of proteins are the ones involved with GLUT4 trafficking 10 within the cytosolic compartments. Once budded out from transgolgi network, GLUT4 proteins are carried to ERC from where they undergo dynamic cycling between the GSV and SC (111). Rab proteins play a significant role in the mediation of this recycling process only in the presence of an intact insulin signaling. Rab-4 and Rab.-l 1 are the major isoforms involved. Rab is active only in the GTP bound form and is in an inactive state in the GDP bound form. AS-160, which is known as Akt substrate of 160 kDa maintains Rab in a GDP bound form through its GTPase activity there by acting as a breaking system for GLUT4 trafficking. Once insulin signaling is switched on, Akt comes and phosphorylates AS-160 . Once phosphorylated, it loses its ability to maintain Rab in a GDP bound form, there by allowing GLUT4 movement to the membrane surface. Like the Rab family, Rho proteins also play a similar role, whose major upstream regulators are still under investigation. TC1O and Cdc42 have been identified as the putative proteins involved in trafficking via Rho (137). Apart from these main stream regulators, AMPK has also been identified as an important player in GLUT4 translocation. Using PAS (phosphorylation of Akt substrate at serine/threonine sites), multiple serine/threonine sites have been identified in AS- 160 that can be phosphorylated not only by Akt but also by AMPK (124). Muscle contraction is accompanied by increased AS-160 phosphorylation with resultant increase in glucose uptake through the mediation of kinases that are still under investigation. During insulin resistance, there is a reduced translocation of GLUT4 that accompanied by a lower uptake of glucose into the peripheral tissues (48). As discussed, exercise and drugs which stimulate AMPK like biguanides and thiazolidinediones are known to stimulate the uptake process and help to maintain the plasma glucose under control. 11 1.3.2 Role ofcytoskeleton in glucose transport Stimulation of the IRS-PI(4,5)P2pathway can lead to a decrease in fibrillar actin length, which is the major constituent of cytoskeletal proteins. A depolymerization of fibrillar actin has also shown to be associated with decreased glucose uptake. For GLUT4 vesicles to be correctly docked onto the membrane surface, cytoskeletal proteins require rearrangement for which insulin signaling needs to be intact. During high fat induced insulin resistance states, excess free fatty acids leads to ceramide synthesis. This facilitates the increased expression of protein phosophatases (PP) especially PP2A, leading to impaired phosphorylation of IRS-i at tyrosine, and its subsequent down stream signaling (114). These effects hinder cytoskeletal rearrangement and prevent the translocation of GLUT4. 1.3.3 Factors regulating GL UT4 expression Fasting for long durations leads to a down regulation of GLUT4 mRNA and protein content. Factors which lead to an increase in PPARcL expression like excess fatty acids are also known to decrease the GLUT4 mRNA content. Insulin has always been shown to positively regulate GLUT4 mRNA and protein. Other positive influences include AMPK through its ability to augment the myocyte enhancement factor-2 (MEF-2) in the nucleus. Hormones like GCs and thyroid hormones are also known to regulate GLUT4 in a differential manner in different tissues (145). In C2C12 myocytes, GCs increased GLUT-4 mRNA expression with no effect on the uptake process. In the developing intestine, chronic dexamethasone along with GLP-2 have shown to increase the glucose uptake process (50). Interestingly, dexamethasone treatment impairs the glucose uptake process in obese high fat fed mice. 12 1.4 Glucose metabolism and glucocorticoids The glucose molecule entering the cells gets converted into glucose-6-phosphate in the presence of glucokinase. The formed phosphate entity either goes for storage as glycogen in the presence of glycogen synthase or undergoes glycolysis through mediation of phosphofructokinase, the rate limiting enzyme. The steps involved with glycolysis culminate in the formation of pyruvate through the mediation of pyruvate kinase. During anaerobic conditions like hypoxia and ischemia, pyruvate cannot be metabolized and gets converted to lactate in presence of lactate dehydrogenase (LDH). The entire process of glycolysis generates four molecules of ATP along with two molecules of NADH. Under aerobic conditions, pyruvate is taken into the mitochondria for generation of ATP, a process catalyzed by the pyruvate dehydrogenase complex (PDC). The activity of PDC is determined by pyruvate dehydrogenase kinase (PDK) (101). There are different isoforms of PDK (1-4), that are tissue specific with regard to their expression and activity. Cardiac tissue has predominantly PDK-2 and PDK-4 (101). The expression and activity of PDK-4 is mostly determined by circulating insulin levels. Insulin negatively regulates PDK-4 expression and activity (64). During diabetes and insulin resistance, when there is a decreased influence of insulin on tissues, PDK-4 expression and activity is enhanced, thereby inhibiting PDC and bringing about reduced glucose utilization. PPAR-y agonists are known to have a similar action to that of insulin on PDK4 in muscle tissue. This permits its use during conditions like insulin resistance and diabetes, to enhance glucose metabolism and maintain the plasma glucose at normal levels. In the heart, increased PDK-4 activity can lead to pathologies like lipotoxic induced cardiomyopathy. Fatty acids are known to up regulate the expression and activity of this 13 enzyme causing high fat induced metabolic complications. Administration of insulin has been known to reverse this condition (64). GCs are known to decrease glucose utilization by their ability to enhance PDK-4 expression and activity, in addition to their role in elevating blood glucose by promoting gluconeogenesis through their action on PEPCK. We have previously reported that a single dose of GC can bring about an alteration in cardiac metabolism, along with whole body insulin resistance. The changes in cardiac metabolism included an increased expression of PDK-4, with no change in PDK-2. There was a reduction in rate of glucose oxidation, with no changes observed in glycolysis (101). In tissues other than the heart, GCs show varied effects. For example, in the liver, GCs are essential for 6- phosphofructo-2 kinase, fructose-2,6-biphosphatase, PEPCK and phosphofructokinase gene expression. In thymocytes, dexamethasone exhibited a reduction in the protein expression of phosphofructokinase-2. In peritoneal macrophages, GCs have an inhibitory action on both phosphofructokinase-1 and 2, enzymes that determine glycolytic rates (45). 1.5 AMP-activated protein kinase (AMPK) AMPK is known as an energy sensor, and gets activated by cellular stress, including hypoxia, ischemia, and exercise which cause an elevation in the AMP/ATP ratio. In the human body, there exists equilibrium between the activity of ATP synthases and ATPases. When this equilibrium is disturbed and ATP levels decrease below the homeostatic limits of the body, the increase in AMP activates AMPK, thereby shutting off energy consuming pathways and turning on energy producing pathways, thus allowing the body to regain the lost ATPs (4). 14 1.5.1 Structural basis ofAMPK AMPK is a heterotrimeric complex comprising of (X, 13 and ‘y subunits. These subunits are again subdivided into (X1, ct2, 13i, 132, i, y and y3. The ct-subunits have catalytic or auto-regulatory action whereas f3 and ‘ have a regulatory function. In this heterotrimeric structure, the ct and f3 subunits are closely bound and bridged to the ‘y subunit through a 13 subunit. The y subunit consists of four cystathione-beta synthase units. Three AMP units are always linked to the AMPK, and two of these bound AMP units competitively bind to Mg:ATP complex. Thus, any change in the AMP ratio is closely linked to AMPK activation (20). 1.5.2 Factors involved in AMPK activation The activation of AMPK can occur either through an allosteric modification by AMP or due to activation by upstream kinases like LKB 1 (tumor suppressor factor) or a Ca2 mediatedCa2/Calmodulin dependent kinase kinase pathway. These upstream kinases, by a covalent modification, phosphorylate AMPK resulting in its increased activation (140). 1.5.3 Role ofAMPK in the body AMPK is a major metabolic enzyme that has similar properties to insulin in many of its actions. It has been identified as a major target for insulin resistance and Type 2 diabetes. Studies in rodent models have revealed multiple functions of AMPK including its role in increasing glucose uptake by increasing translocation of glucose transporter (GLUT4) to the plasma membrane, thereby improving glucose tolerance. AMPK enhances the rate of glycolysis through its influence on the rate limiting enzyme phosphofructokinase-2. AMPK also has a role in regulating fatty acids metabolism (22). It promotes uptake 15 through increased translocation of fatty acid transporter proteins and increases their utilization by phosphorylating acetyl CoA carboxylase (ACC). This action on ACC reduces its activity and prevents its ability to convert acetyl CoA to malonyl CoA. Thus, the inhibitory effect of malonyl CoA on carnitine palmitoyl transferase-1 (CPT1) is removed, promoting mitochondrial influx of acylated long chain fatty acids, promoting their oxidation. The role of AMPK in decreasing cholesterol synthesis is expected due to their inhibitory action on HMG CoA reductase, a rate limiting enzyme in cholesterol synthesis (4). Apart from its ability to regulate the activity of metabolic enzymes, AMPK is also known to regulate the expression levels of certain metabolic and transporter proteins. These include the upregulation of GLUT4 protein through its nuclear interaction with MEF-2, in addition to its ability to enhance hexokinase and mitochondrial enzyme expression along with mitochondrial biogenesis. Increased AMPK expression is also known to down regulate the expressions of ACC and fatty acid synthase. 1.5.4 Effect 0fAMPK activation in major tissues In different organs, AMPK exhibits diverse actions. In liver, it is known to inhibit fatty acid and cholesterol synthesis. In adipose tissue, it mimics the role similar to insulin by inhibiting lipolysis and fatty acid synthesis. This property of AMPK is made use of in combating insulin resistance due to excess free fatty acids in the plasma. Even though it is an insulin mimetic in many of its actions like glucose uptake and inhibition of lipolysis, it is known to inhibit insulin secretion in pancreatic f3-cell. By permitting lower secretion, it helps in sensitizing tissues to the actions of insulin during insulin resistance and hyperinsulinemic conditions. In skeletal muscle, AMPK promotes the translocation 16 of both fatty acid and glucose transporter (GLUT4) (109). In muscle tissue, it is also known to promote mitochondrial biogenesis that is required for ATP generation, for muscle to perform a high level of activity. In cardiac tissue, it also increases the uptake process of both glucose and fatty acids, by allowing their respective transporters to move to the membrane surface. Apart from this, AMPK increases fatty acid utilization through an ACC mediated pathway, and also stimulates phosphofructokinase-2 and glycolysis, an immediate source of ATP during conditions of reduced oxygen availability like hypoxia and ischemia (4). AMPK inhibits other pathways that lead to energy conservation (eg., glycogen formation). 1.5.5 Metabolic complications associated with abnormal AMPK activity In conditions like insulin resistance, high fat diet induced obesity and metabolic syndrome, a decreased AMPK activity can result in decreased insulin sensitivity. In addition, there is reduced glucose and fatty acid uptake, together with a decreased metabolism of these substrates leading to an enhanced storage of glycogen and triglycerides. There is also unregulated increase in gluconeogenesis from liver, and lipolysis from adipose tissue. Apart from this, there is increased stimulation of hypertrophic and apoptotic pathways (87). Major metabolic abnormalities can also arise if AMPK is activated in an abnormal manner. In the heart, genetic mutation in the y subunit can lead to an increased ct2 activity, resulting in cardiac dysfunction and heart failure. One of the major consequences of an increased ct2 activity is excess glycogen deposition. The glycogen overload has been shown to hamper the normal functioning of cardiac tissue in three different ways. Hypertrophic cardiomyopathy, conduction system failure and Wolf 17 Parkinson White (WPW) syndrome are the three major drawbacks associated with AMPK activation and glycogen accumulation (94). Over activation of AMPK is related to hypertrophic cardiomyopathy in two different ways. One is the ability of excessive glycogen deposits to initiate hypertrophic signaling (12, 94). The other includes the ability of AMPK to increase fatty acid oxidation. The resultant accumulation of large amounts of fatty acid metabolites can lead to sarcomeric protein dysfunction and can initiate hypertrophic cardiac failure (12). Conduction system failure arises due to dramatic changes in pH as a consequence of excess lactate formation, which further aggravates the reduced impulse conduction. In WPW syndrome which is a phenotype of cardiac arrhythmia, glycogen deposition has been shown to play an important initiating role. The excess glycogen deposits on the accessory fibers (which are normally inert) allows irregular conduction, initiating ectopic impulses thereby causing fibrillation (12, 27). Some of the major mutations in the y subunit that can lead to increased AMPK activity are R53 1Q, R302Q, H383R, N4881 and R226Q mutations in the PRKAG2 gene. In all these studies, the mutations are accompanied by an abnormal AMPK activity with the resultant cardiac pathologies (16, 26, 27, 76, 146). 1.6 Glycogen Glycogen is a branched homopolysaccharide which is a storage form of carbohydrates in mammals (82). It is polymerized into a straight chain by an c(1—>4) linkage and branched chain by an c( 1 —>6) linkage at the reducing end. They are considered to be a reservoir and a quick supplier of glucose in times of energy shortage to a particular tissue (116). Major organs of storage include liver and muscle tissue, but the liver is an instant source of blood glucose by promoting glycogenolysis of this stored polysaccharide (32). 18 The glucose molecules brought into the cell get converted to glucose-6-phosphate (G-6- P) in the presence of glucokinase. In the liver, it is hexokinase that performs this particular function and the process is reversible so as to facilitate glucose release following glycogenolysis (32); this reaction is not reversible in muscle tissue. The G-6-P is then converted to glucose-i-phosphate in the presence of phosphoglucomutase. Glucose-i-phosphate in the presence of Uridine di phosphate (UDP)-giucose pyrophosphorylase and Uridine triphosphate gets converted to UDP glucose, which then donates glucose on to a primer-glycogenin on which the entire structure of glycogen is built. The primer glycogenin, apart from being the backbone of the glycogen structure, also has the ability to detach the glucose moiety from the UDP-glucose complex and this function requires the presence of manganese ion (Mn2). The UDP-glucose provides monomeric glucose units which keeps adding on to glycogenin in the presence of a rate limiting enzyme glycogen synthase to form a straight chain by means of c( i —*4) linkage. Another enzyme that plays an important role in the formation of this complex structure is the branching enzyme which helps in formation of the cc(1—*6) linkage (116). The formed complex polysaccharide undergoes breakdown, requiring glycogen phosphorylase and debranching enzyme (32). 1.6.1 Glycogenin Glycogenin is a backbone primer for the glycogen network, and has unique properties. The innate property of this primer is its ability to self glucosylate and start the polymerization process. For this glucosylating ability it requires a tyrosine residue at the 194 position. It was demonstrated that when this amino acid was mutated and replaced by other moieties, the primer lost the ability to self glucosylate (42). Apart from this, it 19 also has the property to hydrolyze UDP-glucose thereby adding the released glucose unit to itself. Olycogenin has a mutual existence with glycogen synthase when present alone, but when glucose monomers are provided, they detach from the complex and undergo self glucosylation. A recent target “glycogenin interacting protein”(GNIP) has been identified as a catalyst for the process of self glucosylation. There is also evidence that ONIP is the main regulator to determine the location of formation of glycogen within the cell by allowing excess transport of glycogenin to that particular region (37). The continued process of straight and branched chain growth leads to different structures of glycogen. Once the polysaccharide reaches a molecular weight of 1 O Da, they are called proglycogen, and as it advances and becomes a larger structure with molecular weight more than i07 Da, it is termed macroglycogen (118). 1.6.2 Enzymes regulating glycogenformation Glycogen synthase (GS) and glycogen phosphorylase (OP) are the two major rate limiting enzymes involved in glycogen synthesis and glycogen breakdown respectively. When phosphorylated, OS becomes inactivated whereas OP gets activated (74). These major enzymes are mainly regulated by allosteric and covalent modifications. Glucose-6- phosphate has been shown to be the major allosteric activator for OS and in activator for OP. Similarly, AMP levels have been known to allosterically regulate GP positively and OS in a negative manner (74, 82). OS has nine serine residues which are all prone to phosphorylation and deactivation. Some of the initiators of OS phosphorylation are Caseine kinase, AMP-activated protein kinase andCa2/calmodulin dependent kinase by which OS undergoes structural modifications in such a way that it gets further phosphorylated and inactivated by glycogen synthase kinase-3- (GSK-3-f3), a major 20 upstream covalent regulator of GS. Insulin regulates GS activity in a dual manner; one of which is activation of the IRS-Akt signaling pathway thereby increasing the phosphorylation of GSK-3-13 at the Serine-9 residue resulting in its inactivation (5). In this situation, GSK-3-13’s ability to phosphorylate and deactivate OS is lost. On the other hand, insulin can directly activate protein phosphate-i, a major enzyme involved with dephosphorylation and activation of GS. With GP, an increase in the AMP/ATP ratio allosterically activates this enzyme. Moreover, activators of the cAMP pathway are known to phosphorylate and activate OP, which explains the role of adrenergics and PKA stimulants in activating GP (74). Insulin plays an important role in deciding the activity of OP by its ability to activate PP 1, which dephosphorylates and inactivates OP. 1.6.3 Factors leading to glycogen storage Although glycogen is an immediate source of carbohydrate, abundant storage can lead to many disorders like hypertrophy, conduction system disease and WPW syndrome. Glycogen storage diseases are usually genetic in origin. In skeletal muscle, it occurs mainly due to deficiency of two major enzymes, c-1,4 glucosidase and lysosomal associated membrane proteins. The storage disorder due to deficiency of a-1,4- glucosidase is known as Pompe’s disease (67). In cardiac tissue, glycogen storage is accompanied by mutations in the regulatory Y2 subunit of AMPK leading to an increased AMPK activity (76). 1.6.4 Cardiac complications linked to excess glycogen Hypertrophic cardiomyopathy induced by glycogen is likely due to its ability to induce certain mitogenic signaling pathways including MAPK!ERK kinase. The conduction system disorder resulting from an increased glycogen is likely due to an excess lactate 21 formation, thereby reducing pH and promoting the existence of an acidic environment. The reduced pH hampers contractile ability due to its action on the sarcomeric proteins of myocardial tissue resulting in decreased rate and force of contraction (146). In WPW syndrome which is a type of cardiac arrhythmia, the accessory fibers in cardiac tissue develop a conduction ability due to excess glycogen deposition (76). These fibers start conducting impulses in an irregular pattern in the atrial region resulting in fibrillation. In summary, excess glycogen storage leads to hypertrophy, conduction system failure and arrhythmias resulting in heart failure (12). 22 2.0 Research rationale and hypothesis On metabolic demand, glycogen, a mobilized storage form of glucose, is readily broken down to yield glucose moieties. The major rate limiting enzymes involved with the metabolism of glycogen include glycogen synthase and phosphorylase. For its synthesis, the core protein glycogenin acts as a primer for the attachment of UDP-glucose moieties, promoting the growth of this polymer (82). Glycogen synthase catalyzes the attachment of UDP-glucose to the non reducing end of already formed glycogen (92). Glycogen breakdown is mediated by glycogen phosphorylase. The activity of these enzymes is regulated by both phosphorylation and allosteric stimulation (66). Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis (6). However, its excess has been suggested to bring about structural and physiological impairments including an ionic imbalance, a change in pH and stimulation of pathways leading to hypertrophic signaling. Thus, glycogen accumulation that is associated with mutation of AMPK has been reported to cause cardiac hypertrophy, conduction system failure and ventricular arrhythmias (3). In glycogen storage diseases like Pompe disease, characterized by deficiency of debranching enzyme function, an excessive accumulation of cardiac glycogen leads to left ventricular hypertrophy and subsequent failure (135). Glucocorticoids have widespread use as anti-inflammatory and immunosuppressive agents (112). However, both excess endogenous and exogenous glucocorticoids are known to contribute towards cardiovascular complications (96, 115). These cardiac abnormalities could be secondary to glucocorticoid induced insulin resistance and Type 2 diabetes, and alterations in cardiac metabolism. With the latter, we have previously 23 demonstrated that in hearts from dexamethasone (DEX) treated animals, amplification of lipoprotein lipase (LPL) provided the heart with excessive fatty acids (FA) that are known to induce cardiomyopathy (101, 132, 141). Glycogen accumulation was also enhanced in these hearts. We rationalized that this increase in glycogen was a consequence of compromised glucose oxidation observed in DEX treated hearts (101). In the present thesis, we hypothesize that in addition to DEK’s effect on glucose oxidation, additional factors related to glycogen synthesis like glucose entry and glycogen synthase also play a role in the accumulation ofthis storedpolysaccharide. 24 3.0 Materials and methods 3.1 Experimental animals 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. Adult male Wistar rats (260-3 00 g) were obtained from the UBC Animal Care Unit. The synthetic glucocorticoid hormone dexamethasone (DEX; 1 mg/kg) or an equivalent volume of ethanol was administered by i.p. injection to non-fasted rats, and the animals were euthanized at 4 h. In the human body, the basal daily secretion of cortisol is approximately 6-8 mg/rn2 (in a 70 kg adult male, this translates to approximately 0.2 mg/kg). In response to stress, cortisol release is increased up to 10- fold of the basal value (2 mg/kg) (28). For exogenous administration, the dosing with corticosteroids depends on the disease condition, and varies from 75-300 mg/day (approximately 1-4 mg/kg) clinically. Previous studies using the euglycemic hyperinsulinemic clamp have determined that this dose of DEX induces whole-body insulin resistance within 4 h (59, 101). 3.2 Euglycemic-hyperinsulinemic clamp Whole-animal insulin resistance was assessed using a euglycemic-hyperinsulinernic clamp, as described previously (101). This procedure involves the simultaneous intravenous infusion of insulin (HurnulinR; 3 mU/mm/kg) (to inhibit endogenous hepatic production) and d-glucose (50%) for 3 h; the quantity of exogenous glucose required to maintain euglycemia is a reflection of the net sensitivity of target tissues (mainly skeletal muscle) to insulin. At regular intervals, a small amount of blood taken from the tail vein 25 was analyzed for glucose (using a glucometer; AccuSoftTM AdvantageTM). Glucose infusion rate (GIR) was adjusted accordingly to maintain euglycemia. 3.3 Tissue specIc response to insulin To assess tissue specific insulin resistance, skeletal muscle (gastrocnemius and soleus from hind leg) and heart from control and 4 h DEX treated animals were evaluated for total and phospho IRS-i and Akt, prior to and after 15 mm of injecting a rapid acting insulin into the tail vein (8 U, HumulinR) using Western Blot (58, 65). At this early time point, there was no significant reduction in blood glucose with insulin. 3.4 Myocardial glycogen content Frozen cardiac tissue was powdered, weighed, incubated at 85°C (10 mm) with iN NaOH, and followed by neutralization with iN HC1. The neutralized sample was subjected to further acid hydrolysis with 6N HC1 (85°C, 2 h) (83). After neutralization with 5N NaOH, samples were subjected to glucose analysis using a glucokinase assay kit. Presence of cardiac glycogen was confirmed by visualization after periodic acid-Schiff (PAS) staining (60). Where indicated, animals were administered mifepristone (RU-486, 20 mg/kg), one hour prior to DEX. After 4h of DEX, hearts were isolated, frozen and powdered tissue used to measure cardiac glycogen. We have previously reported that phosphorylation of AMPK was inhibited by acute intralipid infusion (58). To determine the effect of AMPK inhibition on cardiac glycogen accumulation, animals were anaesthetized with sodium pentobarbital, and the left jugular vein cannulated. Intralipid (IL 5%; 1.2 ml/kg/h) or vehicle (saline-0.9%) was then infused over a period of 6h, following which hearts were removed for determination of glycogen. In some animals, DEX was administered 2h after the IL infusion was initiated. 26 3.5 Subcellular compartmentalization ofGL UT4 Membrane fractions were isolated by a previously described method using sucrose density gradient (29). With western blot, identification of GLUT4 protein was done by using rabbit polyclonal GLUT4 as the primary and mouse anti-rabbit horseradish peroxidase as the secondary antibody. NatK ATPase was used as a plasma membrane marker. 3.6 Immunoprecipitation and western blotting Following DEX treatment, heart homogenates (500 jig of protein) were immunoprecipitated using a rabbit monoclonal total AS 160 antibody (3 h, 4°C). The immunocomplex was pulled down with protein AIG-sepharose for 3 h, separated and boiled for 5 mm at 95°C in Laemmli buffer, and subjected to SDS-PAGE. Western blotting using antibodies against AS 160, PAS (Phospho (Ser/Thr) Akt substrate antibody, Phospho-Akt (Ser-473, Thr-308), total and Phospho-AMPKx (Thr-172) and Phospho ACC (Ser-79) was then performed as described previously (4). Measuring the phospho form of AMPK, ACC, glycogen synthase and glycogen synthase kinase-3-13 is a surrogate for estimation of their activities. This was done using Western blot. Reaction products were visualized using an ECL detection kit, and quantified by densitometry. 3.7 Measurement ofmRNA mRNA levels were measured using quantitative real time PCR. cDNA was synthesized from 1 jig RNA and purified using a sample purification kit (QIAGEN). RNA levels were determined from standard curves generated for each primer using picogreen assay. The oligonucleotide primers were as follows: GLUT4 mRNA, forward 5’- GGGCAAAGGAACACAACAGT-3’, reverse 5’ -TGGAGGGGAACAAGAAAGTG-3’; 27 AMPK o1 mRNA, forward 5’-GCAGAG AGA TCC AGA ACC TG-3’, reverse 5”-CTC CH TIC GTC CAA CCT TCC-3; AMPK a-2 mRNA, forward 5’- GCTGTGGATCGCCAAATTAT-3’, reverse 5 ‘-GCATCAGCAGAGTGG CAATA-3’ which are specifically used for rats. The sample run was carried out for 40 cycles. Sample amplifications were done with the help of a fluorescent SYBER green dye (Roche Applied Science) in a Roche Applied Science Light Cycler system. The values are expressed as a ratio to 102 copies of standard for all genes. 3.8 G-6-P content G-6-P was determined in perchloric acid extracts of frozen ventricular tissue using a standard spectrophotometric technique (11, 74). 3.9 Glucose uptake in cardiomyocytes Ventricular calcium-tolerant myocytes were prepared by a previously described procedure (99). Cells were plated on laminin-coated culture plates (60 mm; to a density of 300,000 cells/well). Glucose uptake was evaluated using radio labeled 2- deoxyglucose (DOG) (19). Briefly, myocytes were incubated in a glucose-free DMEM containing 0.2% FA-free BSA and pyruvate (1.0 mM) as the energy source. DEX (100 nM) was then added to the incubation media for 20 mm. A radioactive mixture (containing 5 .tCi of 2-deoxy-[3H1 glucose) was added to the plates, and incubation continued for another 10 mm. Following removal of buffer, cardiomyocytes were washed with cold PBS (2x), lysed using NaOH, and lysates used to determine the radioactivity. 2-DOG uptake is expressed as nanomoles per milligram per mm. 3.10 Materials 28 ECL® detection kit was purchased from Amersham Canada. Akt, phospho-Akt (Ser 273/Thr -308), AMPK, Phospho-AMPKa, glycogen synthase (GS), Phospho-GS, GSK-3- f3, Phospho-GSK-3-3, AS16O, PAS, Na-K ATPase and GAPDH antibodies were obtained from Cell Signaling (Danvers, MA). GLUT4 antibody was purchased from Abcam Inc (Cambridge, MA). All other chemicals were obtained from Sigma Chemical. 3.11 Statistical analysis Values are means ± SE. Wherever appropriate, one-way ANOVA followed by the Tukey or Bonferroni tests or the unpaired Student’s t-test was used to determine differences between group mean values. The level of statistical significance was set at P < 0.05. 29 4.0 Results 4.1 Cardiac tissue has limited influence on whole-body insulin resistance induced by DEX. We have previously reported that injection of DEX for 4 h was not associated with either hyperinsulinemia or hyperglycemia (101). Nevertheless, using the euglycemic hyperinsulinemic clamp, a direct measure of insulin sensitivity, DEX lowered the glucose infusion rate necessary to maintain euglycemia (Fig. 1 A). We assessed the effects of DEX on the responses of skeletal muscle and cardiac tissue to insulin. In skeletal muscle, both basal and insulin stimulated phosphorylation of IRS-i (Fig. 1 C) and Akt (Fig. 1 B) were reduced after 4 h of DEX. These effects were not observed in cardiac tissue, which demonstrated a normal response to insulin when IRS-i (Fig. lE) and Akt (Fig. 1D) phosphorylation were measured. Following 4h of DEX, total IRS-i and Akt did not change in skeletal and cardiac muscle when compared to control (Fig. 1 B-D). Thus, following DEX, cardiac tissue has limited contribution towards the development of whole body insulin resistance. 4.2 Buildup ofcardiac glycogen following DEXis coupled to GLUT4 translocation. Even though DEX did not impede cardiac insulin signaling, we have previously reported a decline in cardiac glucose oxidation following DEX (101). In the presence of lower glucose oxidation, we hypothesized that glucose entering into the heart would be converted into glycogen. Indeed, DEX induced an approximately 2-fold increase in the cardiac glycogen content as measured enzymatically (Fig. 2A) or by histochemical 30 staining (Fig. 2B). This effect of DEX on glycogen was partially related to receptor activation as although RU-486 reduced the DEX induced increase in cardiac glycogen, the levels observed were still higher than control (CON 37.8±0.50, DEX 69.7±1.5, DEX+RU 44.6±2.1 jig/g dry weight; P<0.05). In addition to a reduction in glucose oxidation, it was unclear whether changes in glucose transport could also contribute towards the accumulation of glycogen. Interestingly, DEX treated hearts exhibited a higher G-6-P content compared to control (CON 2.67±0.16, DEX 3.3 1±0.20 jt mol/g dry weight; P<0.05). More importantly, after 4 h of DEX, measurement of GLUT4 at the plasma membrane revealed an excess presence of this transporter protein at this location (Fig. 3A). Usually, this observation is accompanied by a decrease in GLUT4 in the intracellular pool. However, in the present study, the DEX induced increase in plasma membrane GLUT4 was also accompanied by an increase in GLUT4 in the intracellular membrane fraction (Fig. 3B). This latter effect was well correlated to an increased GLUT4 mRNA (Fig. 3B, inset). DEX also increased glucose uptake directly in isolated cardiomyocytes (CON 4.2±0.85, DEX 9.86±1.74 n mol/mg/min; P<0.05). 4.3 DEX augments both total andphosphorylated cardiac AMPK. In addition to Akt signaling, cardiac GLUT4 translocation is also controlled by AMPK (109). In the presence of a normal Akt signal, we measured AMPK and report an increase in both total (Fig. 4C) and phosphorylated AMPK phosphorylation (Fig. 4A) following 4 h of DEX. This change in AMPK total protein was accompanied by a modest but insignificant increase in AMPKc-2 (Fig. 4E), but a significant increase in AMPKL-1 (Fig. 4D) gene expression. Once activated, AMPK phosphorylates and inactivates ACC, facilitating FA oxidation. We measured ACC phosphorylation as a 31 measure of AMPK activity. ACC phosphorylation was significantly increased in DEX hearts compared to CON (Fig. 4B). To substantiate the role of AMPK in DEX induced accumulation of cardiac glycogen, IL was used to inhibit AMPK phosphorylation. IL lowered cardiac glycogen accumulation that is observed after DEX (CON 37.4±1.8, DEX 66.2±2.0, DEX+IL 45.9±0.7, g/g dry weight; P<0.05). 4.4 Phosphorylation of Akt substrate of 160 kDa (AS16O) is mainly regulated by AMPK. AS 160 regulates GLUT4 translocation to the plasma membrane by retaining this transporter in intracellular membranes, a function that is lost upon its phosphorylation (61). The major upstream regulators of AS16O phosphorylation include Akt and AMPK. Immunoprecipitation of AS 160 followed by Western blotting demonstrated no change in Akt phosphorylation at Ser473 and Thr308 in DEX treated hearts (Fig. 5, A and B). However, there was a significant increase in AMPK phosphorylation at Thri 72 (Fig. SC), which correlated well with AS 160 phosphorylation, as reflected by an increase in PAS (measure of phosphorylation at Ser and Thr sites ofASl6O) (Fig. SD). 4.5 Glycogen synthase undergoes robust dephosphorylation with acute administration ofDEX In addition to the contributions by GLUT4 delivered glucose and reduction of glucose oxidation towards glycogen synthesis following DEX, enzymatic control of this stored polysaccharide is also an important factor that controls its accumulation. Glycogen synthase, the rate-limiting enzyme for glycogen synthesis, is activated upon dephosphorylation (74). In DEX treated hearts, there was a considerable reduction in the phosphorylation of glycogen synthase (Fig. 6, lower panel). The major upstream kinase 32 that regulates glycogen synthase phosphorylation is GSK-3-13, whose activity in turn is reduced by its phosphorylation. As GSK-3-13 phosphorylation was augmented (Fig. 6, upper panel), our data suggest that cardiac glycogen accumulation following DEX is also dependent on the phosphorylation states of these two rate-limiting enzymes. 33 5.0 Discussion Chronically, increased levels of endogenous glucocorticoids are known to cause Cushing’ s syndrome, a condition that is characterized by obesity, insulin resistance, increased lipid mobilization, and hypertension (77, 142). Exogenous delivery of glucocorticoids as anti-inflammatory and immunosuppressive agents is also associated with myocardial failure when administered chronically (90). We attempted to examine the acute effects of DEX specifically related to cardiac metabolism, given the injurious effects that excess triglyceride and glycogen (3) have on the heart. We reported an enlargement in the coronary LPL pool, with a subsequent increase in FA delivery and augmented cardiac TG accumulation (59, 100, 101). With carbohydrate metabolism, DEX promoted the expression of PDK4, which is known to inhibit PDH and pyruvate flux, and hence glucose oxidation (101). Under these circumstances, we proposed that glucose disposal occurred by its conversion to glycogen. In the present study, our data suggest that AMPK mediated glucose entry, combined with activation of glycogen synthase and the previously reported reduction in glucose oxidation (101), act together to promote glycogen storage. With insulin resistance, metabolism in multiple organ systems including the heart is altered, which is believed to be an important factor in increased morbidity and mortality (10, 15). Measurement of insulin sensitivity using the euglycemic-hyperinsulinemic clamp revealed the presence of whole body insulin resistance with acute DEX treatment, suggesting that any change in cardiac metabolism that is exhibited by DEX may be a consequence of the prevalent reduction of insulin sensitivity. Nevertheless, we also determined the responses of the skeletal muscle and the cardiac tissue to insulin and 34 unlike skeletal muscle, cardiac tissue responded normally to insulin. Under these conditions, it is possible that the effects of DEX on cardiac metabolism are also related to direct effects of this glucocorticoid on the heart. Glucocorticoids work through multiple mechanisms to bring about their desired effects. These include a specific cytosolic receptor mediated event, and specific and non-specific membrane bound receptor mediated effects through which glucocorticoids bring about genomic and non-genomic outcomes (17, 21, 23). Given our recently reported observation that DEX activated AMPK (a metabolic switch that plays an important role in maintaining cellular energy homeostasis) phosphorylation in isolated cardiomyocytes within 60 mm (59), and the use of RU486 in our current study to reduce cardiac glycogen accumulation, our data suggest that the effects of DEX on cardiac metabolism in vivo may be linked to its direct impact on the heart. At present the mechanism by which DEX increases phosphorylation of AMPK is unknown and could include changes in the AMP/ATP ratio (46) or activation of aCa2/calmodulin-dependent protein kinase kinase (117). AMPK is known to inhibit anabolic and promote catabolic processes leading to conservation of cellular ATP levels. It does so through multiple mechanisms including increased delivery and metabolism of both glucose and fatty acids (44, 78, 109). In this context, and related to glycogen, AMPK is known to inhibit the formation of this storage form of glucose in skeletal muscle (98, 119, 139). In the heart, both low flow ischemia (139) and exercise (86) increase AMPK activity, that is correlated to a reduction in glycogen content. Unexpectedly, our results suggest that accumulation of glycogen with DEX was associated with increased phosphorylation of AMPK, a phenomenon that is observed in transgenic models of AMPK activation. In these models, mutation of 35 regulatory gamma sub units (‘y1R7OQ, y2 N4881,72R531G) increased AMPK activation and facilitated glycogen accumulation (3, 27, 36, 76). It is possible that the intrinsic property of DEX to block glucose oxidation counteracts the ability of AMPK to prevent the storage of this carbohydrate. An additional explanation for this occurrence could be related to the effect of AMPK to decrease malonyl CoA, thereby removing its inhibition on CPT-1 and promoting fatty acid oxidation (100). The resultant blockade of glucose oxidation, as suggested by the Randle hypothesis (excess fatty acid utilization prevents glucose utilization), could explain the increase in glycogen storage (103). Whatever the mechanism, our data suggests that with carbohydrate metabolism, AMPK phosphorylation in the presence of DEX is associated with an anabolic function. Under basal conditions, only a small percentage of GLUT4 resides at the plasma membrane, with the remaining fraction being redistributed in endosomal recycling and GLUT4 storage compartments (70, 111). Translocation of this transporter protein from the intracellular pool to the plasma membrane is regulated largely by the P13 kinase-Akt pathway, in addition to AMPK. These kinases, by phosphorylating and inactivating AS 160 (which has multiple phosphorylation motifs at the serine and threonine residues), removes the constraint that AS 160 has on GLUT4, allowing the trafficking of this transporter to the membrane surface (61, 124, 125). Indeed, the increase in plasma membrane GLUT4 with DEX was well correlated to an increase in PAS, a measure of phosphorylation at Ser and Thr sites of AS 160. To determine the contribution of Akt and AMPK towards this increased AS 160 phosphorylation, we used immunoprecipitation to pull down the trimeric complex containing phospho-Akt, phospho-AMPK and AS-160, and immunoblotted for the respective phosphoproteins. The present study suggests that 36 following DEX, in the presence of normal insulin signaling, AMPK mediated phosphorylation of AS 160 is the predominant factor that controls cardiac GLUT4 movement. Increased substrate availability plays an important role in glycogen synthesis. Thus, the increase in glucose uptake and 0-6-P following DEX could be an important contributing feature in glycogen accumulation. Other factors include alterations in glucose oxidation (101) and changes in enzymes that control glycogen synthesis or breakdown. In skeletal muscle, glycogen regulation by GSK-3-3 and OS is well established. AMPK is known to phosphorylate OS making it prone to further phosphorylation by casein kinase-1 and GSK-3-f3 leading to its inactivation (98, 119). The relationship between AMPK, GSK-3-3 and glycogen has yet to be resolved in the heart. For example, Mora et al. showed that cardiac glycogen levels are regulated independently of insulin’s ability to phosphorylate GSK-3- and stimulate OS (85). In the present study, phosphorylation of OS after DEX administration decreased, an effect closely associated with an increase in GSK-3- phosphorylation. This effect of DEX on OS in the presence of increased AMPK phosphorylation was uncommon. As AMPK was recently shown to activate OS through a GSK-3- dependent pathway in HepG2 cells (134), our data suggest that through multiple mechanisms, AMPK activation with DEX is associated with glycogen storage. In summary, acute DEX administration was associated with significant accumulation of myocardial glycogen. One way by which this process is made possible is through an AMPK mediated augmentation of glucose uptake, coupled to its regulation of glycogen synthase, a key enzyme involved in glycogen synthesis. Given the contribution of 37 glycogen storage in eliciting cardiac hypertrophy, ventricular arrhytbmias and conduction system disorders, results from the present study could help in limiting the deleterious effects of long term excesses in endogenous or exogenous glucocorticoids on the heart. 38 6.0 Figures C C+I D D+I — — — — P.At 1-All — — — — GAPDH * 16 16 14 12 10 B 0 4 Fig. 1. Dexamethasone effects on whole body and tissue specflc insulin resistance. Following injection of vehicle or DEX for 4 h, whole-body insulin resistance was assessed using a euglycemic hyperinsulinemic clamp (A). Insulin (HumulinR; 3 mU/mm/kg) and d-glucose (50%) were continuously delivered (by a cannula inserted into the left jugular vein) for 3 h. At regular intervals, blood samples taken from the tail vein were analyzed for glucose using a glucometer. Glucose infusion rate (GIR) was adjusted accordingly to maintain euglycemia. To determine tissue specific insulin resistance, skeletal muscle (gastrocnemius and soleus from hind leg) (B and C) and heart (D and E) from control and 4 h DEX treated animals were evaluated for phospho IRS-i (Tyr-989) and Akt (Ser-473) and total IRS-l and Akt, prior to and after 10 mm of injecting rapid acting insulin into the tail vein (8 U, HumulinR) using Western Blot. Results are the means ± SE of 3-5 rats in each group. *Sjgnificantly different from control; Significantly different from all other groups; #Significantl different from control given insulin; @significantly different from respective basal (without insulin), P <0.05. A ::,E 0 60 60 80 120 160 180 lime (mine) B C C+I D 0+1 — -_- — P-All — — — T.Akl _—— GAPDH +# HJ C C+I 0 0+1 — — — T.IRS GAPDH C C+l 0 0+1 4 3 2 14 12 10 8 6 4 2 on :E• C C+l 0 0+1 5% I.. U’ 0 . a U’ 5.0 6 4 3 2 o o+i 0 0+1 W,w — - P-IRS — — T4RS __ GAPDH E C 0+1 0 0+1 39 A 80 0 60 a. 0 40 20 B Fig. 2. Cardiac glycogen accumulationfollowing dexamethasone. Cardiac glycogen was determined: a) as glucose residues using a glucokinase method after acid hydrolysis, and b) by histochemical analysis of cross sections of ventricular tissue using PAS-staining (B). Results are the means ± SE of 5 rats in each group. *Significantly different from control, P <0.05. 40 * CON CON lOx 5, u CON DEX — — — OLUT-4PM OLUT-41M IM PM — — Na-K ATPaS. A ____________ Fig. 3. Subcellular localization ofGLUT4 protein. Following DEX, heart homogenates were prepared and subjected to plasma (A) and intracellular (B) membrane separation using sucrose gradient. Identification of GLUT4 protein was carried out using rabbit polyclonal GLUT4 as the primary and mouse anti-rabbit horseradish peroxidase as the secondary antibody. Na’-K ATPase was used as a plasma membrane marker. Quantitative real-time PCR enabled determination of GLUT4 mRNA in hearts from control and DEX treated animals. Results are the means ± SE of 3-5 rats in each group. *Significantly different from control, P < 0.05. CON-control; DEX-dexamethasone; IM-intracellular membrane; PM-plasma membrane. 1’ 0’ I I CON DEX B 20 15 10 16 [I2.J 8- 0I CON DEX CON DEX 41 2 2 2 5 4 CON DEX — — — P.AMPK — T-IPK — p.ACC — •_. __j- GAPDH 12 10 C, C, .2 .2 D 2 0 8 6 4 2 CON DEX CON DEX OON DEX 0._. o .2 .4 — a a 2 .2 Fig. 4. Changes in AMPKprotein (total andphosphorylated) and gene expressions in hearts isolatedfrom DEK treated animals. Following DEX, total (C) and phosphorylated (A) AMPK-o and phosphorylated ACC (C) were measured using rabbit AMPK-ci, phospho-AMPK (Thr 172) and Phospho-ACC (Ser-79) antibodies respectively. AMPK-cd and AMPK-cQ gene expressions were measured using quantitative real-time PCR (D and E). Results are the means ± SE of 3-5 rats in each group. °Significantly different from untreated control, P <0.05. COI( DEX 42 IP:AS-1 60 P.AIt Ser473 P.Akt Thr308 T-Ai P-AMPK-Th,172 T.AMPK PAS AS.160 A B P C 0 16 12 8 CON Fig. 5. AMPK regulation ofAkt substrate of 160 kDa (ASJ6O). Animals were treated with DEX, and at 4 h, hearts from control and DEX treated animals were isolated. To examine the association between AS 160, Akt and AMPK, AS 160 was first immunoprecipitated using a total AS 160 antibody. The immunocomplex was then immunoblotted with anti phospho-Akt (Ser473, A; Thr308, B), anti phospho-AMPK (Thr 172, C), anti PAS (Ser/Thr, D), and anti-AS 160 (inset). Results are the means ± SE of 3-5 rats in each group. *Significantly different from untreated control, P < 0.05. IP-immunoprecipitation. 43 CON DEX — — — — — — a e — - — — — _ — — CON 16 12 8 0 7Fig. 6. Enzyme regulation of cardiac glycogen synthesis subsequent to administration of DEX Animals were killed 4 h subsequent to DEX injection and hearts isolated for determination of phospho (Ser 9) (top panel) and total GSK-3- and phospho (Ser 641) (bottom panel) and total glycogen synthase (GS) using Western Blot. Results are the means ± SE of 3-5 rats in each group. *Significantly different from untreated control, P < 0.05. 6 5 g 3 2 I 0 CON CON DEX — — — — P-GsK-3-p —— T-GSK4- — — — P-GS — T-GS —— —— GAPDH -I- 5 4 3 2 I CON DEX 44 GLUCOSE PM \ , AS -160 \ p UAPIC - G S K .3 .J3 G-6-P —- Oxidation GS I flEX GLYCOGEN Fig. 7. Schematic mechanism ofhow DEX regulates cardiac glycogen through AMPK. In the presence of intact insulin signaling (normal Akt function), DEX, through its effects in enabling AMPK phosphorylation, regulates GLUT4 translocation by its action on AS 160. The subsequent influx of glucose augments the cardiac content of G-6-P. This effect, combined with phosphorylation and inactivation of GSK-3-J3, together with dephosphorylation and activation of glycogen synthase, acts in unison to increase cardiac glycogen content. 45 7.0 References 1. Adcock RJ, Kattesh HG, Roberts MP, Carroll JA, Saxton AM, and Kojima CJ. 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Data are means ± SE. n5 myocyte preparations from different animals. *Significantly different from control, P < 0.05. These data suggest that the effects of DEX on AMPK phosphorylation are direct, and not mediated via its effects on whole body insulin resistance. This direct effect is currently under investigation. CON DEX ____ — P-AMPK — -E GAPDH CON DEX 57 A B Fig. A 2 Chronic effects ofDEX administration. Animals were treated with 1mg/kg of DEX for 7- days and the following parameters determined. There was a reduction in body weight (A) when compared to control, but other parameters like blood glucose (B) and plasma triglycerides (C) were elevated. In a separate group, a euglycemic-hyperinsulinemic clamp was performed (n=3), and it was noted that glucose infusion rate (GIR) was significantly lower compared to control (D). Finally a separate set of animals (n=3) were injected with rapid acting insulin into the tail vein (8 U, HumulinR, killed after 10 mm) and frozen cardiac tissues were used to measure basal and insulin stimulated Akt-phosphorylation using western blot (E). Results are the means ± SE. *Significantly different from control; #Significantly different from all groups, P < 0.05. These data suggest that whole body insulin resistance is still evident after chronic DEX treatment. However, in these animals, although cardiac Akt response to insulin is intact, the basal Akt response is lower and will be examined. CON DEX.7 14 12 _ 10 D 8 .Th.E fi 6 4 2 0 C D 16 140. E 12ca—. C .r 8 .x 6 — 4 2 350 300 250 0) 200 150 100 50 0 3.0 2.5 IEOE g E 35 .3) # 25 g11fl I. CON DEX-7 CON DEXJ C C+I D D+I — — — P-Akt — GAPDH D D+I 58 C C+I D D+I — — — * * -- •1• 25 2O I. 15 0 = 0 1o 0 0. (0 0 a 0 CON Fig. A 3 Effect of a lower dose of insulin on cardiac Akt in CON and DEX treated animals. We administered a lower dose of insulin equivalent to 10 mU/gram wt (compared to 20 mU/gram wt). Data are means ± SE. n=5 animals. *Siifictly different from control, P < 0.05. Even at this dose of insulin, cardiac Akt response remained unchanged in DEX treated animals. CON+I DEX DEX+I 59


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