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Effects of BMOV on protein-serine kinase activities in skeletal muscle of diabetic rats Girn, Jaspal S. 1998

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EFFECTS OF BMOV ON PROTEIN-SERINE KINASE ACTIVITIES IN SKELETAL MUSCLE OF DIABETIC RATS by JASPAL S. GIRN B.Sc, The University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences, Div. of Pharmacology and Toxicology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1998 S) Jaspal S. Girn In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of fxWi/>r>^c£xott<^fJo /4-CAM^X^& The University of British Columbia Vancouver, Canada Date <\%-0<i-0H DE-6 (2/88) ABSTRACT The in vivo effects of bis (maltolato) oxovanadium (IV) (BMOV) on the activity of various protein kinases in two cascades that are implicated as u p s t r e a m mediators of the biological effects of insulin were investigated. The integrity of the MAP kinase pathway was evaluated by studying the activation of ERKl , ERK.2 and p90 r s . T h e activity of p70 6 was also evaluated as a possible downstream enzyme in insulin action . T h e intact rat served as a convenient animal model system to s t u d y the mechanism of insulin action. Hind limb muscle extracts were p repared and subjected to specific immunoprecipi tat ion experiments for the kinases ment ioned above. T h e first part of the study examined the effects of B M O V on p ro te in - se r ine kinases in Zucker Diabetic Fatty (ZDF) rats. The Zucker Diabetic Fatty (ZDF) provided an ideal model to study the activity of protein kinases in noninsulin-dependen t diabetes mellitus (N1DDM), as the development of the disease in t he se rats closely parallels N I D D M in humans. BMOV treatment for 8 weeks significantly (p < 0.05) decreased the basal activity of p70S6K by 30% when compared to the un t rea ted group. Futhermore , following B M O V treatment the basal kinase ac t iv i ty of ERK-1 and ERK-2 significantly decreased by 40% and 35%, respect ively, when compared to the unt rea ted group. p90rsk is postulated to be downstream of ERK-2 and any changes in ERK-2 could be reflected in the activity of p90 r s . B M O V t rea tment decreased p90 r s activity by approximately 75% when compared to the un t rea ted group. The activity of protein kinases was also investigated in the St reptozotocin (STZ) Diabetic Wistar rat. This model is representa t ive of insulin-dependen t diabetes melliuts ( IDDM), because the chemical, S T Z destroys the pancreat ic (3-cells. p70S6K activity was altered basally and following 5 U/kg insulin ii stimulation in STZ-diabetic rats and BMOV treatment for 8 weeks was unable to restore the activity to control values. In addition, ERK-1 and ERK-2 are markedly active in the diabetic state following stimulation with 5 U/kg insulin when compared to control. The activity of both ERK-1 and ERK-2 was 5 fold greater than basal control. Chronic BMOV treatment was able to restore the activity of ERK-2 in the diabetic treated animals to normal, whereas the activity of ERK-1 was unaffected. Finally, there appears to be a dissociation between ERK-2 and p90rsk. 5 U/kg insulin was required to activate ERK-2 to its maximal level, whereas 10 U/kg was required to achieve maximal activity of p90rsk. In the diabetic state, ERK-2 activity was markedly active in response to insulin stimulation, whereas p90rsk showed no change. Finally, BMOV treatment decreased the activity of ERK-2, whereas p90rs activity was increased with BMOV treatment. in TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES v i i i LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS xiii l. INTRODUCTION 1.1 DIABETES MELLITUS l 1.2 CLASSIFICATION OF DIABETES MELLITUS I 1.2.1 Type I: Insulin-Dependent Diabetes Mellitus 2 1.2.2 Type II: Noninsulin-Dependent Diabetes Mellitus 3 1.3 VANADIUM AS AN INSULIN MIMETIC 5 1.3.1 The Insulin-Mimetic Effects of Vanadium In Vitro 6 1.3.2 The Insulin-Mimetic Effects of Vanadium InVivo 8 1.3.3 Organic Vanadium In Vivo 10 1.4 INSULIN SIGNAL TRANSDUCTION 12 1.4.1 The Insulin Receptor 13 1.4.2 Insulin Receptor Substrates 15 1.4.3 The MAP Kinase Pathway 16 1.4.4 The PI-3 Kinase Pathway 21 1.5 THE MECHANISM OF ACTION OF VANADIUM 26 1.6 EXPERIMENTAL MODELS 29 1.6.1 The Zucker Diabetic Fatty (ZDF) Rat 30 iv 1.6.2 The Streptozotocin (STZ) Diabetic Wistar Rat 31 1.7 RATIONALE, RESEARCH OBJECTIVE AND HYPOTHESES 35 1. MATERIALS AND METHODS 2.1 MATERIALS 38 2.2 EXPERIMENTAL PROTOCOL 39 2.2.1 ZDF Study 39 2.2.2 Wistar Study 39 2.3 PREPARATION OF TISSUE EXTRACTS 40 2.4 PROTEIN QUANTITATION 41 2.5 ANION-EXCHANGE CHROMATOGRAPHY 41 2.6 DETERMINATION OF PHOSPHOTRANSFERASE ACTIVITIES 42 2.7 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS 42 2.8 IMMUNOBLOTTING 43 2.9 IMMUNOPRECIPIATION STUDIES 44 2.10 DENSITOMETRY 45 2.11 STATISTICAL ANALYSIS 46 3. RESULTS 3.1 EFFECTS OF BMOV ON PROTEIN-SERINE KINASES IN ZUCKER DIABETIC FATTY RATS 47 3.1.1 Plasma Glucose Levels in the Zucker Diabetic Fatty Rat 47 3.1.2 Basal MBP Phosphotransferase Activity and Effect of BMOV Treatment 47 3.1.3 Basal S6-10 Peptide Phosphotransferase Activity and Effect of BMOV Treatment 49 3.1.4 p70 6 Immunoprecipitation Study 49 3.1.5 ERK-1 and ERK-2 Immunoprecipitation Study 52 3.1.6 p90rs Immunoprecipitation Study 54 3.2 EFFECTS OF BMOV ON PROTEIN-SERINE KINASES IN STZ-DIABETIC WISTAR RATS 55 3.2.1 Plasma Glucose Levels in the STZ-Diabetic Wistar Rat 55 3.2.2 Basal and Insulin Stimulated MBP Phosphotransferase Activity and the Effect of BMOV Treatment 59 3.2.3 Basal and Insulin Stimulated S6-10 Peptide Phosphotransferase Activity and the Effect of BMOV Treatment 62 3.2.4 p70S6K Immunoprecipitation Study 65 3.2.5 ERK-1 Immunoprecipitation Study 67 3.2.6 ERK-2 Immunoprecipitation Study 69 3.2.7 p90rs Immunoprecipitation Study 70 DISCUSSION 4.1 OVERVIEW 75 4.2 THE ROLE OF P70S 6 K IN INSULIN ACTION 76 4.2.1 Basal p70S6K Activity and the Effect of BMOV in ZDF Rats 78 4.2.2 P70S6K Activity and the Effect of BMOV in STZ-Wistar Rats 79 4.3 THE MAP KINASE (MAPK) PATHWAY 80 4.3.1 Basal MAPK Pathway Activity and the Effect of BMOV in ZDF Rats 81 4.3.2 MAPK Pathway Activity and the Effect of BMOV in STZ-Wistar Rats 83 4.4 LIMITATIONS OF THE ZDF AND STZ-WISTAR RAT STUDIES 88 4.5 FUTURE DIRECTION 89 4.6 CONCLUSIONS 91 5. REFERENCES 92 6. APPENDIX 107 V l l LIST OF FIGURES Figure 1.1 The structure of Bis(maltolato)oxovanadium(IV) (BMOV) 11 Figure 1.2 The structure of the insulin receptor 14 Figure 1.3 The signal transduction pathways through which insulin mediates its metabolic effects 17 Figure 1.4 The structure of OC-streptozotocin 32 Figure 1.5 Postulated mechanism of action of streptozotocin on pancreatic (3-cells 35 Figure 3.1 The plasma glucose levels in the ZDF rats at the beginning of the treatment period (initial) and at termination (final) 48 Figure 3.2 Mono Q profiles of MBP and S6-10 peptide in the ZDF rats 50 Figure 3.3 p70 6K immunoprecipitation study in the ZDF rats 53 Figure 3.4 ERK-1 immunoprecipitation study in the ZDF rats 56 Figure 3.5 ERK-2 immunoprecipitation study in the ZDF rats 57 Figure 3.6 p90rs immunoprecipitation study in the ZDF rats 58 Figure 3.7 Plasma glucose levels in the Wistar rats 60 Figure 3.8 MonoQ profiles of MBP phosphotransferase activity in the Wistar rats 63 Figure 3.9 MonoQ profiles of S6-10 peptide phosphotransferase activity in the Wistar rats 64 Figure 3.10 p70S6K immunoprecipitation study in the STZ-diabetic rats 66 Figure 3.11 ERK-1 immunoprecipitation study in the STZ-diabetic rats 68 Figure 3.12 ERK-2 immunoprecipitation study in the STZ-diabetic rats 73 vui Figure 3.13 p90rsk immunoprecipitation study in the STZ-diabetic rats 74 Figure 4.1 The regulation of glycogen synthase activity by the MAPK pathway and RSK-3 in skeletal muscle by insulin in vivo 86 IX LIST OF ABBREVIATIONS BB BMOV BSA cAMP D N A EGF ERK GSK-3 IDDM IgG IGT IP IRS kDa kg M MAPK MBP MEK biobreeding bis(maltolato)oxovanadium(IV) bovine serum albumin cyclic adenosine 3',5'-monophosphate deoxyribonucleic acid epidermal growth factor extracellular signal-regulated protein kinase glycogen synthase kinase-3 insulin-dependent diabetes mellitus immunoglobulin G impaired glucose tolerance immunoprecipitate insulin receptor substrate kiloDalton kilogram molar MAP kinase myelin basic protein MAP kinase kinase microgram microgram microlitre \iM micromolar ml mM NIDDM N O D p70S6K P90rsk PDGF PDKl PH PI PI3K PKB PPG-1 PTK SOS STZ U/kg ZDF millilitre millimolar noninsulin-dependent diabetes mellitus non-obese 70 kDa ribosomal S6 kinase 90 kDa ribosomal S6 kinase platelet derived growth factor 3-phosphoinositide-dependent protein kinase pleckstrin-homology phosphatidyl inositol phosphoinositide-3-OH kinase protein kinase B protein phosphatase-1 protein tyrosine kinase son-of-sevenless streptozotocin units/kilogram Zucker Diabetic Fatty XI Single and three letter codes for amino acids: A R N D C Q G H I M F P S T W Y V Ala Arg Asn Asp Cys Gin Gly His He Met Phe Pro Ser Thr Trp Tyr Val Alanine Arginine Asparagine Aspartic Acid Cysteine Glutamic Acid Glycine Histidine Isoleucine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Xll ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. John H. McNeill, for his encourgement, patience and independence during my research and thesis writing. I would also wish thanks to Dr. Sanjay Bhanot for his support and constructive critiscm of my work, and for the guidance during this study. I would also like to thank Kinetek Pharmaceuticals Inc. for allowing me to conduct my experiments at their company site. I appreciate Violet G. Yuen for her help in the statistical analysis of my data and her expert technical assistance during preparation of the tissue extracts, Erika Vera for performing the treatment and monitoring of the ZDF rats, Dr. Patrick Pouchert for the treatment and monitoring of the Wistar rats, Mary Battell for managing the lab and providing me with assistance when I needed it and Sylvia Chan for her secretarial assistance. Last but not least, I wish to acknowledge and thank the many friends at Kinetek Pharmaceutical Inc. and Dr. Steven Pelech's lab for their support and good times. xm CHAPTER 1. INTRODUCTION 1.1 DIABETES MELLITUS Diabetes mellitus is the fourth leading cause of mortality in Western populations at present. Due to a changing demography, and an increase in the elderly population, diabetes is likely to remain a major health problem. It is a genetic disorder in which environmental factors cause the phenotypic expression of the disease (Fajans, 1997). It is characterized by increased fasting and postprandial blood glucose concentrations, insulin deficiency and/or decreased insulin action, and abnormalities of glucose, lipid and protein metabolism (Horton, 1995). If left uncontrolled, diabetes causes blindness, kidney failure, neuropathy, and cardiovascular complications leading to stroke, amputations, heart attacks, and impotence (Hentzen, 1997). In the United States today, sixteen million Americans, or about 1 in 20 people, have diabetes with an estimated 8 million people ye t undiagnosed. The cost of diabetes in the United States is over $92 billion per year (Hentzen, 1997). 1.2 CLASSIFICATION OF DIABETES MELLITUS In 1979, an international workgroup, sponsored by the National Diabetes Data Group (NDDG) of the National Institutes of Health published a "Classification and Diagnosis of Diabetes Mellitus and Other Categories of Glucose Intolerance" (Harris and Cahill, 1979). The present classification includes three clinical groups: (1) Diabetes mellitus is characterized by either fasting hyperglycemia or plasma glucose levels exceeding defined limits during a glucose tolerance test. (2) Impaired glucose tolerance is characterized by plasma glucose levels during a glucose tolerance test that exceed normal values, but are below those defined as diabetes. (3) The third 1 class is gestational diabetes (Fajans, 1997). Diabetes mellitus can be subdivided into two major types of diabetes that differ in etiology and pathogenesis. 1.2.1 Type I: Insulin-Dependent Diabetes Mellitus Type I or insulin-dependent diabetes mellitus (IDDM) occurs in approximately 10% of all diabetic patients in the Western world. IDDM results from a chronic autoimmune destruction of the pancreatic P-cells, probably initiated by exposure of a genetically susceptible individual to an environmental agent (Bennett et al.,1997). Potential environmental factors include specific drugs or chemicals, nutritional constituents, and viruses. The development of IDDM can be divided into six stages: (1) genetic susceptibility,- (2) triggering events,- (3) active autoimmunity,-(4) gradual loss of glucose-stimulated insulin secretion,- (5) appearance of overt diabetes, with some residual insulin secretion,- and (6) complete (3-cell destruction (Eisenbarth, 1986). In susceptible strains of mice, the (3-cell damage induced by multiple subdiabetogenic doses of streptozotocin appears to elicit (3-cell autoimmunity, which contributes to loss of (3-cells. Consequently, this model provides evidence that a primary (3-cell insult can result in secondary (3-cell autoimmunity (Palmer and Lernmark, 1997). The possible role of proteins in cow's milk has received considerable attention as a "trigger" for IDDM over the last few years. In both the non-obese (NOD) mouse and the Biobreeding (BB) rat (animal models of Type I diabetes mellitus), the elimination of cow milk proteins from the diet at an early age protects against subsequent diabetes, and increased antibodies to bovine serum albumin (BSA) have been reported in diabetic NOD mice and BB rats 2 compared to controls (Elliott and Martin, 1984 and Martin etal., 1991). In a variety of animal species, viral infections are able to cause diabetes. Some of these include encephalomyocarditis (EMC) virus, coxsackie B viruses, and reovirus types 1 and 3 (Yoon, 1990). Although it is clear that viruses can cause diabetes in certain animal species, the situation in humans is far more controversial. 1.2.2 TYPE II: NONINSULIN-DEPENDENT DIABETES MELLITUS Type II or noninsulin-dependent diabetes mellitus (NIDDM) is present in approximately 90% of diabetics in the Western world. In Type II patients, there are at least two pathological defects. One is the decreased ability of insulin to act on peripheral tissues to stimulate glucose metabolism or inhibit hepatic glucose output, a condition known as insulin resistance (Kahn, 1994). The second defect is the inability of the [3-cells to compensate for insulin resistance, i.e., relative insulin deficiency (Kahn, 1994). These two fundamental defects in the pathogenesis of Type II diabetes are caused by a combination of genetic and environmental factors that lead to the progression from normal glucose tolerance to diabetes. A small proportion of the cases of NIDDM are caused by well defined abnormalities, such as mutations in glucokinase (the first rate-limiting enzyme for glucose metabolism within the (3-cell), insulin receptor, insulin genes or specific mitochondrial gene mutations (Bennett, 1997). NIDDM may also be caused by other endocrine disorders such as acromegaly and Cushing's syndrome. These specific types of NIDDM, however, are rare and account for no more than 1-2% of the total cases. 3 The contribution of genetic factors to the etiology of NIDDM is well accepted and is demonstrated by greater than 90% concordance rate in identical twins (O'Rahilly etal, 1988). In addition, the incidence of NIDDM is much higher with first degree family relatives with the disease (Permutt, 1990). Since NIDDM has a strong genetic component, one can assume that insulin resistance and/or p-cell malfunction is an inherited initial lesion in most patients. The result is compensatory hyperinsulinemia sufficient to maintain euglycemia or impaired glucose tolerance (IGT). However, as time progresses, this compensatory mechanism fails in some subjects, because P-cell function declines. Decreased insulin secretion, superimposed on the preexisting background of insulin resistance, leads to the development of hyperglycemia seen in NIDDM patients. The most common and leading cause of insulin resistance is obesity. In the absence of carbohydrate intolerance, compensatory hyperinsulinemia is present (Kreisberg et al., 1967). This adaptive response effectively maintains euglycemia in more than 85% of obese individuals, but these elevated insulin levels may contribute to alterations in insulin action. It has been shown that with a mild degree of obesity, the predominant change is a reduction in tissue insulin binding (Kashiwagi et al, 1984). As body weight and fat cell size increase further, a proportional increase in basal insulin secretion occurs. (Krotkiewski etal., 1983). These changes are associated with the development of postreceptor defects (Kolterman et al, 1980). The pattern of fat distribution also appears to be an important determinant of the insulin resistance associated with obesity. In particular, insulin resistance is associated with an increased waist/hip ratio, in which fat is deposited in the abdominally localized adipose tissue (Weir and Leahy, 1994). 4 Other factors that influence insulin sensitivity are physical activity and age. Studies in patients with NIDDM have shown that an exercise training program can cause an increase in glucose tolerance and lower basal and glucose stimulated insulin levels. Likewise, insulin sensitivity varies with age. Many studies have provided evidence for resistance to the metabolic effects of insulin in aging animals and humans. However, age related decrease in mobility and diminished physical activity make it difficult to determine how much of the insulin resistance is caused by old age (Hogikyan and Halter, 1997). 1.3 VANADIUM AS AN INSULIN MIMETIC There is considerable interest in compounds that could be used as possible therapeutic agents in the management of diabetes. Current available drug therapy has not been completely successful in restoring abnormal glucose metabolism in diabetic patients and in eliminating the long-term complications associated with diabetes. The primary oral glucose lowering agents currently on the market include the sulfonylureas, the biguanides, the (X-glucosidase inhibitors and the thiazolidinediones. However, about 20-40% of the patients on these drugs are primary failures and about 10% of the patients are secondary failures. Primary failures are patients that do not respond to oral hypoglycemics. Some of these patients are likely to be unrecognized Type I diabetes patients. In the majority of primary failures, the precise mechanism for the drug failure is unknown. Secondary failure, refers to an initial successful response for at least 6 months followed by a subsequent failure to have an adequate response. The only alternative to those patients that do not respond to current oral hypoglycemics is exogenous insulin therapy. Besides the risk of hypoglycemia associated with insulin therapy, 5 considerable evidence indicates that insulin resistance and hyperinsulinemia are associated with cardiovascular complications such as hyperlipidemia, hypertension and coronary heart disease (DeFronzo and Ferrannini, 1991; Bhanot and McNeill, 1996). Therefore, there is a need to develop compounds that either mimic or enhance the glucoregulatory effects of insulin, but are not associated with the cardiovascular complications. Recent discoveries indicate that the trace element vanadium could be used as a possible agent in the management of this disease (Shechter, 1990). Vanadium is a group V transition element that is ubiquitous in nature and is traditionally used in the manufacture of alloy steels. It has an extremely complex chemistry,- vanadium can easily change its oxidation state and take an anionic or a cationic form (Brichard and Henquin, 1995). Under physiological conditions, in the +5 oxidation state (Vv), it predominantly exists in an anionic form that resembles phosphate, namely metavanadate ( V 0 3 ) or orthovanadate ( H 2 V 0 4 ) . In the +4 oxidation state (V ), vanadium exists in a cationic form, vanadyl (V0 2 + ) , and resembles Mg2+. In 1977, Cantley et al. (1977) demonstrated for the first time that vanadium could influence a biological system. They showed that vanadium could inhibit the Na+/K+ -transporting system in vitro. It was subsequently been shown that vanadium also inhibits other transport ATPases as well (Nechay etal., 1986). 1.3.1 THE INSULIN-MIMETIC EFFECTS OF VANADIUM IN VITRO A large body of in vitro evidence demonstrates the insulin-like actions of vanadium. In 1979, the first indication of the insulin-like effects of vanadium in vitro were reported (Tolman et al, 1979). Vanadate stimulated glucose uptake and oxidation in rat adipocytes, glycogen synthesis in the rat diaphragm and liver, and inhibited hepatic gluconeogenesis. Subsequently, two independent groups 6 recognized that the effects of vanadium on glucose transport and metabolism in intact adipocytes were not linked to its well established effect on NaVK+-ATPase (Shechter and Karlish, 1980; Dubyak and Kleinzeller, 1980). Vanadate also inhibits glucose output from perfused liver (Bruck etal, 1991) and accelerates glycolysis by affecting a variety of enzymes involved in the glycolytic pathway (Rodriguez-Gil et al, 1991). Vanadium compounds have also been shown to inhibit lipolysis (Duckworth et al., 1988), stimulate lipogenesis (Shechter and Ron, 1986), and stimulate glycogen synthase (Tamura et al., 1983) in rat adipocytes. In muscle, vanadate enhances glucose uptake, glycogen synthesis and glycolysis to a lesser extent compared to insulin, but causes a greater stimulation of lactate and glucose oxidation (Clark et al., 1985). However, unlike insulin, vanadate does not modify muscle protein synthesis or degradation (Clarke etal., 1985). A major effect of vanadate may be improved glucose transport in peripheral tissues. Vanadate's enhancement of glucose transport has been demonstrated in rat adipocytes (Dubyak and Kleinzeller, 1980) and in rat skeletal muscle (Okumura and Shimazu, 1992), an effect that could be attributed to an enhanced translocation of the insulin-regulatable transporter (GLUT-4) to the plasma membrane (Paquet et al., 1992). Vanadate also caused increased glucose transporter expression in vitro in NIH 3T3 mouse fibroblasts (Mountjoy and Flier, 1990). Finally, vanadate mimics the secondary actions of insulin, such as an increase in calcium influx, inhibition of Ca2+/Mg2+-ATPases in plasma membranes, stimulation of potassium uptake and elevation of cytoplasmic pH (Shechter, 1990). 7 1.3.2 THE INSULIN-MIMETIC EFFECTS OF VANADIUM IN VIVO The insulin-like properties of vanadium prompted in vivo investigations of the anti-diabetic activity of this compound. The demonstration of its efficacy by Heyliger, McNeill and coworkers (Heyliger etal, 1985) opened a new era of studies of vanadium in animal models of Type I and Type II diabetes. Heyliger et al. (1985) demonstrated that oral administration of vanadate to streptozotocin-treated diabetic rats, a representative model of Type I diabetes, lowered their high levels of blood glucose to normal values. Both control and STZ-diabetic rats received sodium metavanadate at a maximal concentration of 0.8 mg/ml in the drinking water. In addition to the glucose lowering effect, vanadium also restored heart function to normal. Heart function was evaluated using the isolated working heart preparation. The antihyperglycemic effect of vanadium does not result from a rise in circulating insulin levels. Plasma insulin levels are either not affected or are lower than controls in treated diabetic and nondiabetic rats (Sekar et al., 1990,- Cam et al., 1993). Several vanadium compounds at oral doses of between 0.1 and 0.7 mmol/kg/day allow moderate-to-good diabetic control in STZ-diabetic rats (Sekar et al., 1990; Blondel et al, 1989,- Pederson et al, 1989). In addition, the time of the start of vanadium treatment after STZ induction of diabetes (3,10 and 17 days) does not affect the effectiveness of vanadyl treatment. The findings support the concept that the efficacy of vanadyl treatment may be unrelated to protection from the acute toxic effects of streptozotocin on pancreatic (5-cells (Cam et al, 1993). Chronic treatment with vanadium has also been shown to result in sustained anti-diabetic effects in STZ-diabetic animals long after treatment has ceased (Cros etal, 1995). Thus, at 13 weeks after withdrawal from treatment, treated animals had normalized glucose and weight gain, and improved basal insulin levels. Because vanadium can accumulate in tissues 8 such as bone and kidney, it is possible that stored vanadium could be responsible for maintaining near-normal glucose tolerance in the short-term following withdrawal. The BioBreeding (BB) Wistar rat is a spontaneous model of Type I diabetes. It is characterized by rapid onset of hyperglycemia and by loss of pancreatic insulin production. Death usually results due to ketoacidosis, unless insulin is administered. Because the STZ model of diabetes is not completely insulin deficient, the efficacy of vanadium in controlling glucose levels in BB rats was studied. When vanadyl sulfate was administered in the drinking water at a maximal concentration of 0.75 mg/ml for 6 months, the dose of insulin required by diabetic BB rats was reduced, but vanadium could not completely replace insulin in this model (Battell etal., 1992). The insulin-lowering effect of vanadium compounds also has great importance to Type II diabetes. In the fa /fa rat, (pre-Type II diabetes animal model characterized by obesity, hyperinsulinemia and glucose intolerance, but not hyperglycemia) vanadate treatment decreased food and fluid intake, reduced weight gain, and attenuated hyperinsulinemia and improved glucose tolerance (Brichard etal., 1989). Vanadium treatment also reversed a number of secondary complications related to diabetes. STZ-diabetic rats commonly develop cataracts by 8 weeks following induction of diabetes and this complication was prevented with vanadyl treatment (Dai et al., 1994; Ramanadham ef al., 1989). Furthermore, vanadyl sulfate treatment improved or prevented the tissue damage seen in the kidney of diabetic animals. Vanadyl treatment normalized serum transaminases and urea nitrogen and creatinine levels in diabetic treated animals (Dai et al., 1994,- Dai and McNeill, 1994). Vanadyl treatment also resulted in near normal organ/body weight ratios of lung, heart, liver, kidney and adrenal glands as compared to the significantly elevated ratios observed in untreated diabetic rats (Dai etal, 1994,- Dai and McNeill, 1994). 9 1.3.3 ORGANIC VANADIUM IN VIVO The toxicity often associated with oral vanadium treatment is gastrointestinal, indicated by diarrhea and subsequent dehydration (Heyliger etal., 1985,- Meyerovitch et al, 1989). Several reports from Domingo and colleagues present an unfavorable toxicological profile of vanadium, regardless of the salt administered and show an increased incidence of mortality and the accumulation of vanadium in tissues (Domingo et al, 199lA ; Domingo et al, 199lB; Domingo et al., 1992). A one-year toxicology study involving vanadyl sulfate at doses of 0.16-0.71 mmol/kg/day has shown not only normalized plasma glucose and lipid levels in treated STZ-diabetic rats, but no acceleration in development of morphological abnormalities in a variety of organs (by histopathological tests) and no changes in haematological parameters (Dai etal., 1994,- Dai and McNeill, 1994). Since inorganic vanadium is poorly absorbed from the gastrointestinal tract (Gl) (Conklin et al, 1982,- Llobet and Domingo, 1984) and some GI difficulties have been reported with both vanadyl and vanadate (McNeill et al, 1992; McNeill et al, 1995), a series of organic vanadium compounds were synthesized and tested for their in vivo efficacy and safety. One of these compounds, bis(maltolato)oxovanadium(IV), BMOV, (Figure 1.1) was synthesized by complexing one molecule of vanadyl sulfate with two molecules of the common food additive maltol (McNeill et al, 1992). BMOV is a potent compound which is designed to be orally absorbed by passive diffusion as a result of its properties of water solubility, electrical neutrality and low molecular weight. 10 Figure 1.1 The structure of Bis(maltolato)oxovanadium(lV) (BMOV) During a 6-month treatment period, BMOV (0.4 mmol/kg/day) decreased blood glucose and lipid levels to near normal with no diarrhea and no mortality (Yuen et al., 1993). The glucose lowering properties of BMOV and vanadyl sulfate have also been compared in a number of experiments (Yuen et al., 1995). In one study, BMOV and vanadyl sulfate were administered by oral gavage at a concentration of 175 mg/kg (0.55 and 0.82 mmol/kg, respectively) or by intraperitoneal injection at a concentration of 20 mg/kg (0.063 and 0.091 mmol/kg, respectively). BMOV was found to be 2-3 times as potent as vanadyl sulfate by either route of administration. BMOV has also been used in fa/fa (fatty) Zucker rats to examine the effectiveness of organic vanadium in Type II diabetes (McNeill et al., 1995). At a maximum concentration of 0.5 mg/ml for 14 weeks of treatment, BMOV was able to reduce plasma insulin levels from 180 fxU/ml to normal (50 (xU/ml) by week 4. It was also 11 found at these concentrations, that BMOV did not affect body weight gain in lean controls, but significantly reduced body weight in the fatty treated group. Finally, the effects of BMOV have been studied in the Zucker Diabetic Fatty (ZDF) rats (Yuen etal, 1997). In the acute study, BMOV was administered to ZDF rats (9 to 11 weeks of age), when the ZDF rats demonstrate hyperglycemia, hyperinsuHnemia, and hyperlipidemia. Doses greater than 0.2 mmol/kg resulted in euglycemia (plasma glucose level <9 mmol/1). In the chronic study, BMOV was administered to ZDF rats (15 weeks of age), when the ZDF rats demonstrate hyperglycemia and hyperlipidemia in the presence of low-normal levels of insulin. After the 10-week treatment period, plasma glucose levels were significantly reduced, while the plasma insulin levels were maintained. 1.4 INSULIN SIGNAL TRANSDUCTION Great progress has been made in understanding the biochemistry and physiology of insulin since its discovery 75 years ago. In addition to its primary effects on glucose homeostasis, insulin promotes a number of other cellular events including regulation of ion and amino acid transport, lipid metabolism, glycogen synthesis, DNA synthesis, gene transcription, mRNA turnover, protein synthesis and degradation (Cheatham and Kahn, 1995). Therefore, insulin plays a key role in the storage of ingested fuels and in cellular growth and differentiation. To fully understand the events leading to insulin-resistant states and the pathophysiology of insulin deficiency, it is necessary to identify and understand, at the molecular level, the key components in the insulin-signalling pathways. 12 1.4.1 THE INSULIN RECEPTOR The insulin receptor is a large heterotetrameric transmembrane glycoprotein that is expressed in nearly all vertebrate tissues at levels ranging from as few as 40 receptors per cell in circulating erythrocytes to over 200,000 receptors per cell in adipocytes. The insulin receptor is composed of two a-subunits and two B-subunits covalently linked through disulfide bonds to form an a2B2-heterotetramer (Kasuga et al., 1982,- Cheatham and Kahn, 1995). The extracellular a-subunits contain the insulin binding domain, and the transmembrane B-subunits contain the insulin regulated tyrosine kinase domain. The binding of insulin to its receptor occurs with a stoichiometry of between 1 and 2 insulin molecules per receptor (DeMeyts et al., 1973). Although, the identification of the exact residues involved in ligand recognition and high affinity binding has been difficult and quite controversial, the ligand-binding domain has been broadly mapped to the first 500 amino acids of the a-subunit (Yip, 1992,- Schumaker et al., 1991). The N-terminal domain of the insulin receptor is necessary for high-affinity insulin binding (Yip, 1992). A chimeric IGF-1 receptor containing the N-terminal residues (1-68) from the insulin receptor resulted in a hybrid receptor that binds insulin with high affinity (Kjeldsen et al., 1991). In addition, regions more C-terminal have also been described as being involved in ligand recognition. Lys460—»Glu (in exon 6) is a naturally occurring mutation found in a patient with severe insulin resistance (Taylor et al., 1990). Once insulin is bound to the receptor, the B-subunits undergo rapid autophosphorylation on tyrosine residues in the 13 intracellular juxtamembrane domain, the regulatory region with the kinase domain, and the carboxyl terminus. Figure 1.2 shows the structure of the insulin receptor. alpha subunit intracellular beta subunit -ss-•ss-phosphorylated 1162 ~7~ tyrosines ^ 1163 1326 1334" -SS-Insulin Binding domain 1030 Lya Juxtamembrane ATP Binding Regulat ory C-Terminal Tyrosine Kinase domain Figure 1.2 The structure of the insulin receptor (Flier, 1996). Hormone binding appears to relieve an inhibitory constraint imposed by the extracellular domain of the receptor on its tyrosine kinase activity. The autophosphorylation of the regulatory region increases the activity of the receptor tyrosine kinase 10 to 20-fold, leading to increased tyrosine phosphorylation of cellular proteins. The receptor is also phosphorylated on both serine and threonine residues by cellular kinases in the basal state and after stimulation by phorbol esters, cAMP analogues, and prolonged insulin treatment (Stadtmauer et al., 1986; Takayama et al., 14 1988). These phosphorylation events are often inhibitory to the receptor tyrosine kinase activity (Takayama et al., 1988), and provide an additional level of control that may play an important role in altering receptor activity in both physiologic and pathologic states. 1.4.2 INSULIN RECEPTOR SUBSTRATES A great deal of effort has been concentrated on elucidating the molecular mechanisms responsible for translating the tyrosine kinase activity of the insulin receptor into downstream activation of serine/threonine kinases and phosphatases. Early theories of insulin receptor signal transmission focused on intracellular substrate proteins as candidate second messengers, but the identification of these substrates progressed slowly (Cheatham and Kahn, 1995). Recent advances including the identification of the substrate insulin receptor substrate-1 (IRS-1), has shifted the focus toward the association model of protein-protein interaction for signal transduction (Sun et al., 1991). In this model, the autophosphorylated receptors or their substrates associate through protein-protein interactions with other signalling molecules forming a large, multicomponent signalling apparatus. Several endogenous substrates for the insulin receptor tyrosine kinase have been identified in insulin responsive tissues, such as muscle, adipose tissues, and liver. The proteins that are tyrosine phosphorylated in response to insulin include the insulin receptor substrate-1 (IRS-1), the related IRS-2, She, and GABl. IRS-1 was initially identified as a 185 kDa phosphoprotein present in antiphosphotyrosine immunoprecipitates from insulin-stimulated Fao hepatoma cells (White et al., 1985). Subsequent experiments indicated that a second substrate (IRS-2) comigrates with 15 IRS-1 during SDS-PAGE (Sun et al, 1991). Furthermore, very recently a 60 kDa insulin receptor substrate (pp60IRS3) was identified and characterized in rat adipocytes (Smith-Hall, etal., 1997). Together, these IRS proteins define a family of multipotential signalling proteins. The most prominent feature of the IRS proteins is the presence of tyrosine phosphorylation sites,- they have a specialized function role in the recognition of a specific protein domain homologous to a region of the Src tyrosine kinase referred to as the Src homology (SH)2 domain. SH2 domains are present in many intracellular signaling molecules, and bind with high affinity to specific phosphotyrosine motifs, thus creating the basis for specific protein-protein interactions. IRS proteins bind directly to several enzymes and adapter proteins, including PI-3 kinase, SHP2, Fyn, Grb-2, nek and crk (Yenush and White, 1997). Based on the results of various studies, two kinase cascades appear to carry the signal initiated by insulin (Figure 1.3). 1.4.3 THE MAP KINASE PATHWAY The MAP kinase pathway begins with the insulin receptor which phosphorylates intracellular docking proteins, such as IRS-1 or She (Figure 1.3). As a result, the adaptor protein GRB-2 (growth factor receptor binding protein 2) is recruited to the tyrosine phosphorylated IRS-1 or She protein. GRB-2 is a 23 kDa protein, devoid of catalytic activity and is comprised of a single SH2 domain, flanked by two SH3 domains. SH3 domains recognize proline-rich sequences, thus mediating specific protein-protein interactions (Ren etal., 1993). GRB-2 is thought to stimulate p2Inact ivi ty through a noncovalent interaction of its SH3 domain with the recently identified p21ras GDP/GTP exchange factor, mSOS (the mammalian homolog to the Drosophila son-of-sevenless protein) (Li et al., 1993,- Skolnik etal., 1993). She also acts 16 Figure 1.3 The signal transduction pathways through which insulin mediates its effects. The two main pathways that are implicated are (1) the MAP kinase pathway and (2) the PI3/PKB pathway. Activation of the insulin receptor by insulin causes the receptor to phosphorylate itself at several tyrosine residues. This leads to docking of insulin receptor substrate-1 (IRS-1) and IRS-2 and their phosphorylation on tyrosine residues by the insulin receptor. These phosphorylated tyrosine residues on IRS-1 and IRS-2 then interact with SH2 domain containing proteins, such as PI3K and Grb2. The activation of PI3K may lead to the activation of protein kinase B (PKB). PKB has been shown to phosphorylate and inactivate GSK-3 thus leading to glycogen synthesis. The MAP kinase pathway begins with the interaction of Grb-2 and mammalian son of sevenless (mSOS) with She or IRS-l/IRS-2, resulting in the activation of Ras. This triggers a cascade involving the sequential activation of the protein kinases, Raf, MAP kinase kinase (MEK), MAP kinase and p90 RSK. PPl may be phosphorylated and activated by p90 RSK in vitro, but evidence discussed in the text indicated that the MAP kinase pathway does not mediate the activation of glycogen synthase in response to insulin in skeletal muscle. Therefore, the main pathway that is involved in the activation of glycogen synthase is the PI3K—»PKB->GSK-3 pathway and not the MAPK pathway. (Solid arrows indicate direct effects and dotted arrows indicate that other components may be involved) 17 Glucose It P I a sm a m em bra n e Others? ( l R S - l / I R S - 2 ) <S> Glut4 18 as a docking protein (analogous to IRS-1) for the SH2 domain of the adapter protein GRB2. Evidence suggests the predominant pathway involved in the activation of Ras is through an insulin receptor-She interaction (Sasaoka et al., 1994 and Ouwens et al., 1994). Therefore, the binding of the GRB-2/mSOS complex to She after insulin stimulation links the insulin receptor Tyr kinase and the p21ras signalling pathways. Ras is a 21 kDa membrane associated GTP binding protein that possesses a GTPase activity. It is a member of a superfamily of small GTP binding proteins that is involved in a variety of cellular processes including cell growth, protein trafficking (molecular chaperons), and vesicular transport (endocytosis and exocytosis) (Chardin, 1991,- Macara, 1991). The activity of these proteins is regulated by their cycling between a GTP bound active form and a GDP bound inactive form. In unstimulated cells, most of the p21ras is found in the GDP bound inactive form. However, after stimulation by insulin, there is a rapid increase in the amount of GTP bound p21ras. The regulation of this cycle is governed in part by specific GTPase activating proteins (GAP) and guanine nucleotide exchange factors (Medema, 1993). An alternate and possibly redundant pathway that links insulin signalling with p21ras activation is through the She protein (Pelicci et al., 1992). In this model, insulin stimulates the Tyr phosphorylation of She, which provides a binding site for GRB-2, resulting in the formation of a Shc/GRB-2/mSOS complex. Furthermore, in C H O cells expressing the insulin receptor double point mutant, Tyr"62,1163—»Phe"62'"63, insulin induced the Tyr phosphorylation of She, formation of the Shc/GRB-2 complex, and an increase in GTP bound p21ras in the absence of any detectable T y r phosphorylation of IRS-1 (Cheatham and Kahn, 1995). The downstream target for p21ras is Raf-1, a 74 kDa serine/threonine kinase. It has been shown that p21ras, specifically in the GTP bound, form activates Raf-1 19 (Avruch etal., 1994). Mutations in the effector domain of p21ras that render it inactive in cells also abolish Raf-1 binding. The binding of Raf-1 directly to p21ras explains the translocation from the cytosol to the cell membrane that Raf-1 undergoes in response to growth factors such as insulin. Once bound to p21ras at the cell membrane, Raf-1 is activated. Raf-1, in turn, phosphorylates one or more protein kinases activating a cascade of serine and threonine kinases and phosphatases. The substrate specificity of Raf-1 is quite limited and the only known cellular target is the MAP kinase kinase (MEK). MEK is itself activated by phosphorylation on two serine residues. MEK, in turn, phosphorylates and activates MAP kinase,-activation requires the phosphorylation of both threonine and tyrosine residues in the sequence -TEY. Two very similar proteins, ERK-1 and ERK-2 are members of the MAP kinase family and are expressed in virtually all cells. They are activated by insulin and many growth factors such epidermal growth factor. The array of potential substrates for the MAP kinases is very extensive (Denton and Tavare, 1995) and includes upstream components of the insulin signalling pathway to MAP kinase, such as SOS and MEK, transcription factors (such as c-Jun and Elk-1), p90rsk and eIF-4E. The role of the MAP kinase cascade in insulin action is unproven. More specifically, the role of the MAP kinase pathway in the activation of glycogen synthase by insulin is debatable. Initial studies suggested that MAP kinase phosphorylated and activated p90rsk (Sturgill et al., 1988), which then phosphorylated the glycogen-associated protein phosphatase-1 (PPG-1) (Dent et al, 1990). PPG-1 regulates glycogen synthesis by inactivating phosphorylase kinase and activating glycogen synthase. However, recent studies provide strong evidence that this model is incorrect. Results obtained in adipocytes with epidermal growth factor (EGF) provided the first 20 evidence that the MAP kinase cascade was not involved in the activation of glycogen synthase by insulin (Robinson etal, 1993 and Lin and Lawrence, 1994). It was found that EGF was more effective than insulin in increasing MAP kinase and p90rs . If these pathways lead to glycogen synthase activation, EGF would be expected to activate the enzyme. However, unlike insulin, EGF did not activate glycogen synthase. Furthermore, with the development of the plasma membrane permeable inhibitor of MEK. activation, PD 098059, another approach in the investigation of the role of MAP kinase could be used (Dudley et al., 1995). When rat diaphragms were incubated with PD 098059, the activation of MAP kinase and p90rsk by insulin was blocked, however the inhibitor did not significantly decrease the activation of glycogen synthesis by insulin (Azpiazu etal., 1996). 1.4.4 THE PI-3 KINASE PATHWAY The discovery of phosphoinositide-3-OH kinase (PI3K) about nine years ago suggested that there was a new intracellular signalling system involving lipid second messengers (Figure 1.3). The PI3K kinase pathway begins with the insulin receptor which phosphorylates intracellular docking proteins, such as IRS-1. As a result, PI3K is recruited to the tyrosine phosphorylated IRS-1 protein. PI3K consists of two subunits, a regulatory subunit of molecular weight 85 kDa (p85) and a catalytic subunit with a molecular weight of 110 kDa (pi 10) (Cheatham and Kahn, 1995). The p85 subunit contains two SH2 domains that allow the association with tyrosine phosphorylated IRS-1. This interaction results in the stimulation of PI3K activity. It catalyzes the phosphoryation of phosphatidylinositol (PI) , PI-4P and PI-4,5P2 to PI-3P, PI-3,4P2 and PI-3,4,5P3 respectively (Toker and Cantley, 1997). PI3K also has a tightly associated serine kinase activity, the function of which is poorly understood 21 (Carpenter etal, 1993). Although the exact mechanisms remain unknown, activation of PI3K appears to be a critical upstream step for insulin stimulation of Glut 4 glucose transporter translocation and glucose uptake, as well as stimulation of some enzymes involved in glycogen synthesis and protein synthesis. The discovery of two relatively specific (but structurally dissimilar) inhibitors of PI3K, wortmannin (Ui et al, 1995) and LY 294002 (Vlahos et al, 1994) was critical in demonstrating that this enzyme plays an essential role in mediating nearly all the metabolic actions of insulin. Wortmannin, a fungal metabolite, inhibits PI3K at nanomolar concentrations by binding to the pi 10 subunit (Yano et al, 1993). The inhibitor, LY 294002 acts at micromolar concentrations by acting as a competitive inhibitor for ATP (Vlahos et al, 1994). These inhibitors have proved to be useful reagents for analysis of the downstream signalling components of this lipid kinase. One of the first downstream targets of PI3K to be identified was the Akt/PKB protein Ser/Thr kinase (also known as RAC-a) (Franke et al, 1995; Burgering and Coffer, 1995). It was identified in 1991 by three different groups independently. Two groups identified the approximately 60 kDa kinase as a result of its homology with both protein kinase C (73% similarity with the kinase domain of PKCe) and protein kinase A (68% similarity with the kinase domain of PKA). This gave rise to the names PKB (protein kinase B) (Coffer and Woodgett, 1991) and RAC-PK (Related to the A and C kinases) (Jones etal, 1991). At the same time, this kinase was identified as the product of the oncogene v-akt of the acutely transforming retrovirus AKT8 found in a rodent T-cell lymphoma (Bellacosa et al, 1991). PKB is activated by many mitogens and growth factors, including platelet derived growth factor (PDGF), epidermal growth factor (EGF), insulin, thrombin and nerve growth factor (NGF) 22 (Marte and Downward, 1997). In addition, PKB can also be activated by PI3K independent pathways (Konishi etal., 1996). Studies suggest that there are multiple levels involved in the regulation of PKB activity. PKB contains an amino terminal pleckstrin-homology (PH) domain, a central kinase domain and a carboxyl terminal regulatory domain (Marte and Downward, 1997). PH domains are about 100 residues long and are most likely involved in either protein-protein or protein-lipid interactions. Recent studies have indicated that the lipids produced by PI3K (PI-3,4P2 and PI-3,4,5P3) can bind to the PH domain of PKB with relatively high affinity and specificity (Franke et al, 1997A, Klippel etal, 1997,- Freeh etal, 1997). However, the binding of PI-3,4,5P3 to the PH domain does not activate its kinase activity in vitro (Klippel et al., 1997), whereas, PI-3,4P2 can directly stimulate PKB activity (Franke etal, 1997A). Furthermore, Franke et al. (1997A) also demonstrated that binding of PI-3,4P2 to the PH domain led to the dimerization of PKB and subsequent activation of the kinase. One major concern is the fact that in some systems the entire PH domain can be deleted without loss of stimulation of the kinase by growth factors such as insulin (Kohn etal, 1996). It is not yet known whether the regulation of PKB lacking a PH domain is still wortmannin-sensitive, as is the case for full length PKB. The most important method of regulation is likely to involve phosphorylation of PKB by other protein kinases. PKB becomes phosphorylated at two major sites, Thr-308 and Ser-473 in response to growth factors (Franke et al, 1997B). Both sites must be phosphorylated for PKB to be fully active (Alessi et al, 1996). A novel, 3-phosphoinositide-dependent protein kinase, PDKl, has recently been detected and purified (Alessi etal, 1997). PDKl phosphorylates PKB at Thr-308 and increases its activity 30-fold. This enzyme is only active in the presence of lipid vesicles 23 containing PI-3,4P2 or PI-3,4,5P3. These results disagree with reports that claim that PKB is activated directly by PI-3,4P2. It is possible that reports showing PKB activation directly by PI-3,4P2 may have been the result of contamination of PKB preparations with trace PDKl activity. Although PKB is not activated by PI-3,4P2 or PI-3,4,5P3, the interaction with these lipids may facilitate the activation of PKB by PDKl, either by a conformational change or by recruiting PKB to the plasma membrane (Cohen etal., 1997). A direct in vivo substrate for PKB is glycogen synthase kinase-3 (GSK-3) (Cross etal., 1995). GSK-3 is a serine/threonine kinase that was initially identified as a regulatory enzyme in intermediary metabolism (Rylatt etal., 1980). The activation of glycogen synthase in response to insulin involves inhibition of GSK-3, a kinase that phosphorylates sites 3a, 3b and 3c in glycogen synthase. Therefore, phosphorylation of GSK-3 results in its inactivation and the consequent activation of glycogen synthesis. The sites in GSK-3 phosphorylated by PKB in vitro are the same as those phosphorylated in response to insulin (Lawrence et al., 1996). More specifically, GSK-30C is phosphorlated at Ser-21 and GSK-3(3 at Ser-9. It is important to note that there is consistent evidence that PKB is involved in the activation of glycogen synthase. However, the inhibition of GSK-3 alone cannot account for the action of insulin on glycogen synthase, as GSK-3 does not phosphorylate glycogen synthase in site 2, which is dephosphorylated in response to insulin (Lawrence etal., 1996). GSK-3 also phosphorylates and inactivates the initiation factor eIF2B, and this inhibition is abolished with insulin treatment, leading to the onset of protein synthesis (Welsh and Proud, 1993). Another downstream kinase that may be important in protein synthesis is p70 6 . The S6 ribosomal protein, which is a component of the small (40S) subunit of 24 eukaryotic ribosomes, is phosphorylated by p70S6K. Therefore, the p70S6K plays a key role in cellular growth control mechanisms by coordinating protein synthesis via regulation of the S6 protein and the activity of 4E-BP1 (Proud, 1996). The p70S6K also plays an important role in the progression of cells from Gl to S phase of the cell cycle (Chung etal., 1992). The immunosuppressant drug rapamycin, a bacterial macrolide, has become one of the most useful tools for dissecting the pathway leading to p70 6 activation and its regulation. Rapamycin blocks activation of p70S6K by all known mitogens by preventing the phosphorylation of a specific subset of sites (Ferrari et al., 1993). Rapamycin does not interact with p70S6K directly, but rather it binds to a protein called FKBP12, and the rapamycin-FKBPl2 complex actually interacts with mTOR/FRAP upstream of P70S6K. mTor/FRAP is a member of the PIK-related family of protein kinases, which contain a C-terminal lipid kinase activity (Hunter, 1995). Recent data demonstrate that mTOR/FRAP is a rapamycin sensitive regulator of p70S6K in vivo and that mTOR/FRAP undergoes autophosphorylation, which is blocked by rapamycin (Brown et al., 1995). This suggests that the kinase activity of mTOR/FRAP is essential for its function in the activation of p70S6K. A growing body of evidence has implicated PI3K and PKB as upstream signalling molecules in p70S6K activation. Two different inhibitors of PI3K (wortmannin and LY294002) block the activation of p70S6K by insulin as well as PDGF (Chung et al., 1994). Whilst PKB has not been shown to directly phosphorylate p70S6K, overexpression of a ^^-fusion of PKB constitutively activates p70S6K (Burgering and Coffer, 1995). It is postulated that PKB is either upstream of, or acts in parallel to, the rapamycin-sensitive step in the activation of p70 6K. The latter is 25 likely, as wortmannin and rapamycin block p70 6K by inhibiting different inputs (Chung eta!., 1994). 1.5 THE MECHANISM OF ACTION OF VANADIUM Currently, the insulin signal transduction pathways are very complex and far from being completely elucidated. Therefore, much controversy is still present concerning the mechanism of action of vanadium. Both vanadyl (+4) and vanadate (+5) ions have been show to have insulin-mimetic effects, including inhibition of lipolysis and stimulation of glucose and fat metabolism (Goldfine et al., 1995). Initially it was thought that the insulin like effects of vanadate were related to the inhibition of the Na+, K+-ATPase (Cantley and Aisen, 1979). This however does not seem to be the case in intact cellular systems. Direct measurement of rubidium uptake, a measure for Na+, K+-ATPase activity, indicated that even in the presence of high (1 mM) concentrations of vanadate, Na+, K+-ATPase was not blocked (Dubyak and Kleinzeller, 1980). In analyzing the insulin stimulated signalling pathways, it is clear that protein phosphorylation is a key component leading to insulin's metabolic effects. Tamura et al., (1984) provided evidence that vanadate might directly stimulate insulin receptor (3-subunit tyrosine autophosphorylation. However, several other studies were unable to reproduce this finding (Strout et al., 1989; Green, 1986). Quercetin, a cell-permeable inhibitor of insulin-receptor tyrosine kinase (InsRTK), not only effectively blocked the bioeffects of insulin, but actually slightly enhanced the same bioeffects triggered by vanadate (Shisheva and Shechter, 1992). The best studied site of action for vanadium compounds is at the level of the phosphotyrosyl protein phosphatases (PTPases). There are two major classes of 26 PTPases: one class is represented by large, transmembrane molecules which resemble receptors and the other are smaller cytoplasmic enzymes (Fischer et al., 1991). Because vanadate is a well documented PTPase inhibitor, it was initially believed that vanadate blocks the PTPase that dephosphorylates the insulin receptor and therefore activates it in an insulin independent manner (Shechter et al., 1995). Two protein tyrosine kinases (PTKs) have been identified and partially characterized as being activated in response to vanadium. The first identified protein was a cytosolic (soluble) protein tyrosine kinase (CytPTK) with a molecular weight of 53 kDa (Shisheva and Shechter, 1991,- Shechter et al., 1995). When adipocytes were preincubated with vanadate (20-30 min at 37 °C), CytPTK was activated about 3-5 fold, but not by insulin treatment. The inhibitor staurosporine prevented the activation of CytPTK by vanadate. In intact adipocytes, at a concentration range of 0.1-0.2 |J,mol/l, staurosporine blocked the effects of vanadate in activating glucose metabolism (i.e., lipogenesis, glucose oxidation), but not in promoting hexose uptake or in inhibiting lipolysis (Shisheva and Shechter, 1993). In a cell free system, vanadyl was unable to activate CytTPK, unless the membrane fragments were solubilized with Triton X-100 (Li et al, 1996). However, in the intact cell, vanadyl sulfate was 100 fold more potent in activating CytPTK than vanadate. A second vanadate-activatable nonreceptor PTK has been identified in rat adipocytes (Elberg et al., 1997). The membranous nonreceptor protein tyrosine kinase (MembPTK) has an estimated molecular weight of 60 kDa. In cell-free experiments, vanadate activates MembPTK seven to nine fold. MembPTK is 60-fold less sensitive to staurosporine inhibition than CytPTK and therefore staurosporine has been used to define the different roles of the two PTKs. It is thought that 27 MembPTK participates in the plasma membrane events such as the activation of hexose uptake and the antilipolytic effect of vanadate (Elberg et al., 1997). MembPTK appears to be activated by autophosphorylation following vanadate treatment and has been suggested to interact with PI3 kinase (Elberg et al., 1997). This may explain how vanadate activates PI3K without involving receptor activation and IRS-1 phosphorylation. Vanadate and insulin have been shown to activate Pl3K; however, inhibition of PI3K by wortmannin, which blocks the antilipolysis effect of insulin, fails to block vanadate-mediated antilipolysis (Li et al., 1997). Therefore, the inhibition of lipolysis caused by vanadate is mechanistically distinct from that of insulin, independent of PI3K activation and independent of the cascade through which PI3K acts. (Li etal., 1997). The P13K pathway leads to the activation of protein kinase B (PKB) (Burgering and Coffer, 1995). When adipocytes were stimulated with insulin, vanadate or pervanadate, a decrease in PKB mobility was seen on sodium dodecyl sulfate-polyacrylamide gels (Wijkander et al., 1997). This is indicative of increased phosphorylation, which correlates with increased kinase activity. When comparing vanadate and peroxovanadate potencies, peroxovanadate was approximately 1000-fold more potent than vanadate and active at micromolar concentrations (Wijkander et al, 1997). PKB has been shown to mediate the insulin-induced phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) in myotubes, indicating an important role of PKB in the regulation of glycogen synthesis (Cross etal, 1995). Vanadium salts (sodium orthovanadate, sodium metavanadate and vanadyl sulfate) have also been shown to activate serine/threonine kinases - MAP kinase, p90rs and p70S6K in C H O cells overexpressing a normal human insulin receptor (Pandey et al, 1995). Among the three vanadium salts tested, vanadyl sulfate 28 appeared to be slightly more potent than the others in stimulating MAP kinases and p70S6K kinase. p70S6K lies downstream of the PI3K/PKB signalling cascade, leading to the stimulation of protein synthesis, whereas p90rsk is implicated to be directly downstream of MAP kinase (Pandey et al., 1995). Finally, vanadyl sulfate has been demonstrated to be involved in the modulation of adenylyl cyclase signalling through G-protein regulation (Anand-Srivastava et al., 1995). The liver of STZ-induced chronic diabetic rats showed enhanced levels of G-proteins and augmented GsOC activity. The levels were restored to normal with insulin or vanadyl sulfate treatment. Vanadyl sulfate was also more potent than insulin in restoring the altered levels of G-proteins (Anand-Srivastava et al., 1995). 1.6 EXPERIMENTAL MODELS Through the use of animal models of diabetes mellitus, our understanding of the complex pathogenesis of this disease has progressed greatly. The critical role of animal studies is illustrated by early studies of dogs over a century ago. The role of the pancreas in diabetes was realized when it was discovered that complete removal of this organ led to hyperglycemia. The eventual extraction of insulin from pancreatic extracts began the modern era of our understanding and treatment of diabetes. A large and diverse array of animal models exist for studying diabetes. Although all models exhibit hyperglycemia, the degrees of glucose tolerance and the etiologies differ. The animal models of diabetes can be classified according to causative factor, pathogenesis, nature of diabetes, or other characteristics of the disease. The major limitation of animal models is the lack of a perfect correlation 29 between animal and human disease. Animal models will never reproduce the characteristics of diabetes identical to those in humans. Although there may be a poor correlation between animals and humans, animals may be the best and only possible research model until a better and more accurate representation is found 1.6.1 The Zucker Diabetic Fatty (ZDF) Rat The Zucker rat, first described by Zucker in 1965, is a result of cross-breeding Sherman and Merck Stock M rats (Zucker, 1965). In this strain, obesity is inherited as an autosomal recessive mutation assigned the name fa. The Zucker rat is characterized by insulin resistance, hyperinsulinemia, glucose intolerance, hyperlipidemia and obesity (lonescu et al, 1985). The development of the obesity hyperinsulinemic syndrome remains unclear , but it has been postulated that a defect in the hypothalamus may be the primary defect that causes hyperinsulinemia, which in turn causes obesity and insulin resistance (Shafrir, 1992). Recently, it has been identified that the fa/fa Zucker rats have a gene defect in the leptin receptor and reduced leptin binding at the cell surface (Chen et al., 1996,- Chua et al., 1996). Leptin is a product of the obese (ob) gene, is secreted by adipocytes, and circulating leptin concentrations correlate positively with body mass index and percent body fat (Considine et al., 1996). The ja/ja Zucker rats would therefore represent a pre-diabetic state, since the glucose intolerance and insulin resistance appear to be compensated by stable endogenous insulin output. The Zucker Diabetic Fatty (ZDF) rat is a relatively new strain of obese rats derived from the Zucker rat (Peterson et al, 1990). The ZDF rat at 6-7 weeks of age demonstrates hyperinsulinemia, insulin resistance, glucose intolerance and hyperlipidemia, similar to those observed in the Zucker rat (Yuen et al, 1997). 30 Following this pre-diabetic state, hyperglycemia is manifested at 7 weeks of age and all obese male rats are fully diabetic by 10-12 weeks of age (Yuen et al, 1997 and Tokuyama et al, 1995). ZDF rats are diabetes prone, with approximately 100% of susceptible males developing NIDDM by 12 weeks of age. Homozygous fa/fa ZDF female rats are obese and insulin resistant but do not become diabetic. Blood insulin levels are also high between 7 and 10 weeks of age, but by 12 weeks there is a marked and rapid decline in plasma insulin levels as the (3-cells cease to respond to the glucose stimulus. During this stage of development, the pancreatic insulin stores are depleted and plasma insulin is reduced to the low-normal range (Yuen et al, 1997 and Tokuyama etal, 1995). While there is some functional (3-cell activity, plasma glucose levels cannot be maintained within the normal range. The development of diabetes is associated with changes in islet morphology, and the islets from diabetic animals are markedly hypertrophic and dysmorphic with multiple irregular projections invading the surrounding exocrine pancreas (Tokuyama et al, 1995). Furthermore, the islets of diabetic ZDF rats show changes in gene expression, with a decrease in insulin mRNA that is associated with reduced islet insulin levels (Tokuyama et al, 1995). Since the ZDF rat consistently progresses from the pre-diabetic to the diabetic state it is an ideal model for NIDDM. 1.6.2 The Streptozotocin (STZ) Diabetic Wistar Rat Chemical compounds that selectively damage pancreatic P-cells constitute a class of diabetogenic agents. Alloxan, a pyrimidine structurally similar to uric acid and glucose, was first reported to produce permanent diabetes in animals (Dunn et al, 1943). Streptozotocin (STZ), has replaced alloxan as the principle agent to produce 31 experimental diabetes. This is due to the greater P-cell cytotoxic effects as demonstrated by the wide range between diabetogenic dose and general toxicity (Junod etal., 1967), and its longer half-life (15 min.) compared to alloxan (1 min) after intravenous injection (Agarwal, 1980). Streptozotocin (STZ) (2-deoxy-2-(-methyl-3-nitrosourea) 1-d-glucopyranose) is a broad spectrum antibiotic which is isolated from Streptomyces achromocjenes. STZ can be deemed as glucose with a highly reactive nitrosourea side chain linked to position C-2 of D-glucose, as shown is Figure 1.4. CH2OH HO T pX)H HN I o=c I CH 3_N—NO Figure 1.4 The structure of a-s t reptozotocin. STZ destroys most islet (3-cells after a single injection. It is effective at many different, species-specific doses, ranging from 25 to 200 mg/kg in rats, dogs, mice, hamsters, monkeys, pigs, and rabbits (Rerup, 1970). A single large dose of STZ (100-32 150 mg/kg) causes intense (3-cell necrosis and hyperglycemia is evident within 24-72 hr. Disintegration and phagocytosis of necrotic cells is rapid, with relatively no evidence of debris or inflammation visible after 3 days. One complication with this dose of STZ is the occurrence of non-specific lesions in cells in close contact to the necrotic P-cells. These diabetic rats can survive between 4-7 days without exogenous insulin, but eventually die due to the development of a severe ketotic state. A dose of about 45-75 mg/kg STZ results in the most commonly used model of STZ-diabetes. After a single injection, P-cell necrosis can be detected within 2-4 hrs by ultrastructure examination and within 24 hours by light microscopy. A triphasic pattern of changes in blood glucose begins within 45-60 min. of STZ injection (Rerup, 1970,- Junod et al., 1967). The first phase is hyperglycemia which lasts for about 2 hr, probably due to liver glycogenolysis. This is followed by a period of marked hypoglycemia (lasting approximately 6 hr), which is brought about by massive P-cell degranulation and an enormous release of pancreatic insulin. Stable hyperglycemia develops within 24-48 hours and glucose levels remain 3-4 times higher than normal rats. These animals, though insulinopenic, retain some insulin secretion capacity, are not ketotic, and do not usually require exogenous insulin for survival. Rats treated with STZ display many of the features seen in human subjects with uncontrolled diabetes mellitus, including hyperglycemia, polydipsia, polyuria, and weight loss. Insulinopenia appears to be responsible for the metabolic and growth disturbances, because hormone replacement, islet cell, or pancreatic transplantation rapidly reverses the disease. Some of the endocrine abnormalities seen include high circulating levels of glucagon, somatostatin, vasopressin, 33 corticosterone, atrial natriuretic peptide, and reduced levels of renin, angiotensin II, aldosterone, and thyroid hormones T4 andT3 (Tomlinson etal., 1992) A wealth of knowledge on the mechanism of STZ diabetogenicity has accumulated. Its nitrosourea moiety is responsible for [3-cell toxicity, while the deoxyglucose moiety facilitates transport across the cell membrane. The OC-anomer of STZ shows higher potency, in parallel with the greater effect of the Ot-glucose anomer on insulin secretion, suggesting the involvement of a membrane glucoreceptor in (3-cell penetration (Rossini etal., 1977). On the molecular level, the deleterious effects of STZ result from the generation of highly reactive carbonium ions (CH3+), formed during the decomposition of STZ. The CH3+ ions cause D N A breaks by alkylating DNA bases at various positions, resulting in the activation of nuclear enzyme poly (ADP-ribose) synthetase as part of the cell repair mechanism (Uchigata et al., 1983). As cellular pyridine nucleotides, particularly NAD+ are utilized as substrates for the nuclear enzyme, which is involved in the excision and repair of broken DNA strands, a profound decline in NAD+ occurs (Wilson et al., 1984). In effect, an abrupt and irreversible NAD+ exhaustion leads to cessation of NAD+-dependent energy and protein metabolism, ultimately leading to cell death. (3-cells are particularly vulnerable as their NAD content is lower than in other tissues. Inhibition of poly (ADP-ribose) synthetase by benzamides, such as nicotinamide and picolinamide prevent the STZ-induced DNA damage in vivo and help maintain the NAD content and insulin biosynthesis in vitro (Uchigata etal. 1982). Figure 1.5 shows the mechanism of action of STZ in (3-cells. 34 Streptozotocin (STZ) CH3+ (?) fc z ^ BREAKS I activation . poly(ADP-ribose) synthetase chromatin I (ADP-ribose)n 4 * NAD y nucleus ^ ^ ^ ^ ^ B-cell function y (proinsulin synthesis) islet B-cell Figure 1.5 Postulated mechanism of action of streptozotocin on pancreatic (3-cells. Streptozotocin induces diabetes through the following biochemical events,- islet DNA strand breaks —> stimulation of nuclear poly(ADP-ribose) synthetase —> depletion of intracellular NAD —» inhibition of proinsulin synthesis. Adapted from Uchigata, et ctl. 1982. 1.7 RATIONALE, RESEARCH OBJECTIVE AND HYPOTHESES Our laboratory has previously determined MAP and S6 kinase activities in rat skeletal muscle under diabetic conditions as well as after oral vanadium treatment. Recently, Hei et al. (1995), demonstrated that the activity of these kinases is markedly altered in streptozotocin (STZ)-diabetic rats and that vanadium treatment restored the activity to those observed in control rats. More specifically, basal MAP kinase activity in 6-month diabetic rats was significantly decreased (30%) and BMOV treatment completely normalized the kinase activity. Likewise, basal S6 kinase activity was decreased (40%) and BMOV treatment was able to restore the kinase activity. In these studies the kinases were resolved by MonoQ chromatography and 35 evidence for activation of MAP and S6 kinases was obtained using specific antibodies and Western blotting. Just recently, Hei et al., 1998, used isolated rat adipocyte preparations to study the effects of vanadium and selenium on MAP kinase and S6 kinase activation. Vanadyl sulphate was shown to stimulate the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Likewise, significant stimulation of both kinases was observed by selenium. However, it is now known that many protein kinases are present in each phosphotransferase activity peak and therefore it is difficult to assess changes in specific kinases. For example, myelin basic protein (MBP) is often used as a substrate to test for MAP Kinase activity. However, since MBP is used as a substrate for many different protein kinases, such as ERK-1, ERK.-2, PK.B and PKC, the results obtained are not specific. Therefore, in my thesis I will further characterize the changes in specific protein kinases by conducting immunoprecipitation experiments. Extensive work on the role of protein kinases in insulin signalling has been obtained from cultured cells or isolated tissues, while a few studies have investigated these kinases in pathological conditions such as diabetes. To gain a more complete insight into the physiological regulation of these kinases (and their possible role in diabetes) it is necessary to elicit in vivo activation of these kinases in model systems of diabetes. Finally, the mechanism of action of vanadium remains uncertain in spite of extensive research efforts. It is possible that vanadium may act on certain steps in the post-receptor signalling cascade to exert insulin-like effects. Exploration of the effects of vanadium on the kinase cascades would provide additional insights into the molecular mechanism of action of vanadium. Thus, the research objective and hypotheses proposed are outlined accordingly. 36 The objective of my thesis is to examine the in vivo effects of bis(maltolato)oxovanadium (IV) (BMOV) on two kinase cascades that are implicated as upstream mediators of the biological effects of insulin: (1) the MAP kinase cascade and (2) the PI3K/PKB pathway. The specific hypotheses are: ZDF study 1) In ZDF rats, the basal activity of serine/threonine protein kinases is altered and contributes to defective glucose metabolism 2) BMOV exerts its insulin-mimetic effect by affecting the basal activities of protein kinases and thus improves the defective glucose metabolism Wistar study 1) In STZ-diabetic Wistar rats, the activity of serine/threonine protein kinases is altered in the basal and insulin stimulated states when compared to control Wistar rats. 2) BMOV treatment restores the activity of the serine/threonine kinases in the STZ-diabetic Wistar rat to control values. 37 CHAPTER 1. MATERIALS & METHODS 2.1 MATERIALS Regular insulin for intravenous injections was from Eli Lilly Co. (Indianapolis, IN),- B-glycerophosphate, EGTA, EDTA, MOPS, B-methylaspartic acid, sodium orthovanadate, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, benzamidine, dithiothreitol, soyabean trypsin inhibitor, pepstatin A and the peptide inhibitor of cAMP-dependent protein kinase (PKI) were from Sigma Chemical Co. (Oakville, Canada). Myelin basic protein (MBP) was purified from bovine brain and was obtained from Kinetek Phamaceuticals (Vancouver, Canada). The S6-10 peptide (AKRRRLSSLRASTSKSESSQK) is based on a portion of the S6 protein, which is a part of the 40S ribosome. [y-32P]ATP was purchased from Amersham Pharmacia Biotech (Oakville, Canada) or ICN Pharmaceuticals Inc. (Montreal, Canada). The anti-Rsk2-PCT antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). The monoclonal antibody raised against the 85-kDa subunit of rat PI3-K. The monoclonal anti-phosphotyrosine antibody (4G10) was also purchased from Upstate Biotechnology Inc (Lake Placid, NY). The anti-p70 S6K, anti-ERK 1 and anti-Erk 1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Acrylamide, Bis-acrylamide, p-Mercaptoethanol were purchased from ICN Pharmaceuticals Inc. (Montreal, Canada) or Sigma Chemical Co. (Oakville, Canada). Nitrocellulose membrane and 3mm filter paper were from VWR Canlab (Mississauga, Canada) or Gelman Sciences (Montreal, Canada). Alkaline phosphatase-conjugated goat anti-rabbit and goat anti-mouse IgGs and horse radish peroxidase-conjugated goat anti-mouse IgG were obtained from Bio-Rad Laboratories (Mississauga, Canada). Protein A-Sepharose CL-4B and the HR5/5 Mono Q column 38 were purchased from Pharmacia Biotech (Baie d'Urfe, Canada). Whatman P-81 phosphocellulose filter paper was purchased from VWR Canlab (Mississauga, Canada). All other chemicals and reagents were of the highest grade commercially available. 2.2 EXPERIMENTAL PROTOCOL 2.2.1 ZDF STUDY Eighteen (18) male Zucker Diabetic Fatty (ZDF) rats were received from Genetic Models Inc., (Indianapolis, Indiana) at 7 weeks of age. The rats at 15 weeks of age were then randomly divided into two groups: untreated (n=10) and BMOV treated (n=8). BMOV treatment was initiated at a concentration of 0.2 mg/ml in the drinking water and increased weekly in increments of 0.1 mg/ml to a maximum concentration of 0.8 mg/ml. The total treatment period was for 10 weeks. All animals were monitored weekly for body weight gain. Food and fluid intake were measured twice a week. Once a week, following a 5-hour fasting period, blood was collected for determination of plasma glucose, triglycerides, cholesterol, and insulin levels. At the end of the treatment period, the rats were fasted overnight (16 hours) and hind leg skeletal muscle was removed and a crude extract was prepared, as described below. 2.2.2 WISTAR STUDY Sixty-two male Wistar rats (Charles River) weight approximately 200 g were used. Streptozotocin (STZ) dissolved in 0.9% saline was intravenously injected through the tail vein at a dose of 60 mg/kg in 32 rats. Thirty animals were identified as diabetic with glucose levels over 14 mmol/1 (measured in blood with a glucometer) 7 days after STZ injection. Thirty age matched control rats were injected with saline. 39 The rats were then randomly divided into four groups of 15 animals: control untreated (C), control treated (CT), diabetic untreated (D), and diabetic treated (DT). Seven days after STZ injection, the treated groups received BMOV in the drinking water at a concentration of 0.75 mg/ml for 8 weeks (2 months). Blood glucose levels were monitored throughout the study by sampling the blood from the tail vein and measuring glucose with an enzymatic colorimetric test. At the end of the eight weeks of treatment, the rats were fasted overnight. Each group of 15 rats was divided into 3 subgroups of 5 animals each which were injected with 0 (saline), 5 or 10 Units/Kg (U/Kg) of insulin through the tail vein. Hind leg skeletal muscle was removed and a crude extract prepared 15-20 minutes after insulin injection, as described below. 2.3 PREPARATION OF TISSUE EXTRACTS The procedure described by Gregory et al. (1989) and Pelech and Krebs (1987), was modified and used to prepare tissue extracts. The rats were killed by pentobarbital overdose and the skeletal muscles from the hind legs were removed. The muscles were immediately homogenized in ice-cold MOPS buffer (25 mM, p H 7.2) containing 5 mM EGTA, 2 mM EDTA, 75 mM B-glycerophosphate, 1 mM sodium orthovanadate, 2 mM dithiothreitol (DTI) and various protease inhibitors (1 mM phenylmethylsulfonylfluoride, 3 mM benzamidine, 5 (J.M pepstatin A, 10 (iM leupeptin and 200 |Ig/ml trypsin inhibitor). Sodium orthovanadate was added to the buffer as a phosphatase inhibitor. The affect of sodium orthovanadate was accounted for by including it in the buffer of both control and diabetic extracts. The homogenate was spun at 10,000g for 25 min at 4°C (Beckman J2-21) and the pellet was discarded. The supernatant was spun at 100,000g for 60 min (Beckman L8-60M) and 40 the resultant supernatant was stored at -70°C until further analysis. Plasma samples were collected at the time of sacrifice for subsequent insulin and glucose assays. 2.4 PROTEIN QUANTITATION Protein concentration was quantitated by the method of Bradford (1976). A series of protein standards were prepared ranging from 0-25 fig BSA with 2.5 ml of Bradford reagent (100 mg Coomassie Blue G, 50 ml ethanol, 100 ml H 3 P 0 4 , 850 ml d H 2 0 ) . The extract to be quantitated was diluted by 10 fold with d H 2 0 to within 5-20 |Ig/5 fll. The appropriate diluted sample was added to 2.5 ml Bradford reagent and mixed. After 5-10 minutes incubation, the absorbances of the solutions were measured at 595 nm and the protein concentration of the samples calculated through linear regression plotting of the standards. 2.5 ANION-EXCHANCE CHROMATOGRAPHY Due to the existence of a multitude of protein kinases and other proteins in crude skeletal muscle extracts that could interfere with the various enzyme assays, the muscle extracts were fractionated to partially purify the various MBP kinases before conducting specific immunoprecipitation assays. A fast protein liquid chromatography system was used for all the chromatographic fractionations of muscle extracts, as previously described (17). Briefly, samples containing 5 mg of the protein were applied at a flow rate of 0.8 ml/min to a Mono Q anion exchange column equilibrated with buffer A (10 mM MOPS, pH 7.2, 25 mM B-glycerophosphate, 5 mM EGTA, 2 mM EDTA, 2 mM sodium orthovanadate and 2 mM DTT). The column was developed at the same flow rate with a 15-ml linear NaCl 41 gradient (0-800 mM) in buffer A. Fractions (0.25 ml) were collected for assaying protein kinase activities, for Western Blots and for specific immunoprecipitation assays. 2.6 DETERMINATION OF PHOSPHOTRANSFERASE ACTIVITIES MBP phosphotransferase activity from the Mono Q fractions was measured by employing an assay kit developed by Kinetek. The reaction mixture comprised of 25 [Lg substrate, 10 |ll Mono Q fraction, 0.5 |iM PKI, 50 |lM [y-32P]ATP (specific activity -2000 cpm/pmol) and assay dilution buffer, pH 7.2 (20 mM MOPS, 25 mM B-glycerophosphate, 20 mM MgCl^ 5 mM EGTA, 2 mM EDTA, 1 mM DTT, 5 mM B-methylaspartic acid and 1 mM sodium vanadate). The reaction was allowed to proceed for 20 min at room temperature (20-25°C) and then terminated by spotting 15 |i,l of the reaction mixture onto P-81 phosphocellulose paper located in the spotting plate. The papers were washed 5 times with 1% phosphoric acid to remove the free [y-^PJATP and were then counted for radioactivity. Ribosomal S6 kinase phosphotransferase activity was determined by following the same protocol used for MBP. However, the substrate used was the peptide S6-10. 2.7 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS Proteins were separated using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). Proteins were boiled for 5 min with a volume of 4X sample loading buffer (125 mM Tris-HCl (ph 6.8), 4% SDS (w/v), 20% glycerol (v/v), 0.3 M B-mercaptoethanol, 0.01% bromophenol blue (w/v)). Proteins were subjected to electrophoresis on 1.5 mm thick polyacrylamide gels with 4% 42 stacking gels and 10% separating gels. The gels were electrophoresed in running buffer (25 mM Tris, 192 mM glycine, 3.5 mM SDS) at 10 mA overnight, or until the dye front reached the bottom of the gel. 2.8 IMMUNOBLOTTINC The gel was first equilibrated for 5 min in transfer buffer (20mM Tris, 120 mM glycine, 20% methanol (v/v), pH 8.6). The nitrocellulose membrane was hydrated in transfer buffer for at least 1 min before the transfer. The gel and membrane were assembled into a transfer apparatus between 2 pieces of 3mm filter paper. The proteins were electrophoretically transfered in a Hoefer transfer cell at 4°C for 3 hr at 300 mA. The transferred proteins were first visualized using Ponceau S dye by incubating the membrane in the dye for approx. 1 min at room temp., then washing the excess dye away with water. Membranes were blocked in 5% skim milk in TBS (50 mM Tris base, 150 mM NaCl, pH 7.5) for 4 hrs to overnight at room temp.. Subsequently, the membranes were rinsed with TTBS (0.05% Tween-20 in TBS) to remove the excess blocking solution. Then the primary antibody (anti-rabbit polyclonal) was incubated with the membrane for 4 hrs to overnight with agitation. The primary antibodies (anti p70 S6K-NT, ERK-l-CT and RSK-2-PCT) were diluted to 1/1000 in TTBS with 0.1% azide. The blots were washed again in TTBS and then incubated with the appropriate secondary antibody (goat anti-rabbit, polyclonal) for 1.5 -2 hrs at room temp, with agitation. Alkaline phosphatase conjugated secondary antibodies were diluted 1/2000. Excess secondary antibody was removed by washing with TTBS. Alkaline phosphatase immunoblots were developed in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) development 43 solution (a mixture of 3% NBT in 1 ml 70% dimethylformamide (DMF) and 1.5% BCIP in 1 ml 100% DMF with 100 ml of AP buffer (0.1 M Tris, pH 9.5, 0.1 M NaCl, 5 mM MgCl2)). When the band intensity reached desired levels (10-15 min., depending on the enzyme), the development was stopped by soaking the membranes in d H 2 0 . 2.9 IMMUNOPRECIPITATION STUDIES For all immunoprecipitation assays, 1 mg of the crude muscle cytosolic extracts was incubated with an equal volume of 3% NETF (100 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl pH 7.4, 50 mM NaF, 5% glycerol and 3% Nonidet P-40) and 35 |J,1 of protein A-Sepharose beads and 5 |ll of antibody. For ERK 1,2 immunoprecipitation assays, the anti-Erk 1 (carboxy terminal epitope corresponding to amino acids 352-367 of rat ERK 1 encoded MAP kinase p44) or the anti-Erk 2 (carboxy terminal epitope corresponding to amino acids 345-358 of ERK 2 encoded MAP kinase p42 of rat origin) rabbit IgG antibodies were used. For p70 S6 kinase immunoprecipitation assays, anti-p70 S6 kinase (epitope corresponding to amino acids 485-502 mapping at the carboxy terminus of rat p70 S6 kinase) rabbit polyclonal IgG antibody was used. With p90 immunoprecipitation assays, anti-RSK2-PCT (CNRNQSPVLEPVGRS) rabbit IgG polyclonal antibody was used. It is based upon residues 602-615 of mouse RSK2 S6 kinase, which are located near the carboxy terminus of the protein. All the immunoprecipitation assays were performed simultaneously for all groups and after a 3 h incubation period at 4°C, the protein A-Sepharose beads were pelleted by centrifugation at 13000 rpm for 2 min. The beads were washed twice with 3% NETF and then twice with KII buffer, pH 7.2 (12.5 mM MOPS, 12.5 mM B-glycerophosphate, 20 mM MgCh, 5 mM EGTA, 0.25 mM DTT, 50 mM sodium fluoride), following which kinase assays were performed. For the 44 ERK 1,2 immunoprecipitations, the reaction was initiated by the addition of 35 jll KII buffer, 5 |ll of 200 mM MgCh, 5 [L\ MBP (5 mg/ml) and 50 |lM [y-32p]ATP (specific activity -2000 cpm/pmol). The reaction was allowed to proceed for 30 min at 30°C after which it was stopped by the addition of 20 |ll of 4X sample buffer. The reaction contents were boiled and loaded onto 10% SDS polyacrylamide gels. After transferring, the MBP bands were Ponceau stained, cut and counted. The membrane was then probed with specific antibodies to visualize the protein of interest using immunoblotting procedures described above. For the Rsk2 and p70 S6K immunoprecipitation assays, the kinase reaction was performed in a manner identical to that of ERK-1,2 except, 20 \i\ of KII buffer was used and the S6-10 peptide was used as the substrate. The reaction was allowed to proceed for 30 min at 30 °C after which it was stopped by spotting 30 JLLl onto P-81 phosphocellulose paper. The papers were washed 5 times with 1% phosphoric acid to remove the free [y-32p]ATP and were then counted for radioactivity. At the same time, 20 Jll of 4X sample buffer was added to the reaction mixture. The reaction contents were boiled and loaded onto 10% SDS polyacrylamide gels. After transferring, the membrane was probed with specific antibodies to visualize the protein of interest. 2.10 DENSITOMETRY The computer program NIH Image 1.61 was used to quantify the band intensity for each kinase immunoprecipitated. The activity levels were then adjusted for, based upon the amount of protein present for each group. A sample calculation is in Chapter 6: Appendix. 45 2.10 STATISTICAL ANALYSIS The data is expressed as the means +/- SEM. Comparisons between means were performed using a two-sample t-test or general linear model analysis of variance (GLM-ANOVA) followed by Neuman-Keuls multiple comparison test as appropriate. Results were considered significantly different if p < 0.05. 46 CHAPTER 3. RESULTS 3.1 EFFECTS OF BMOV ON PROTEIN-SERINE KINASES IN ZUCKER DIABETIC FATTY RATS This study was designed to investigate the activity of protein kinases thought to be central in insulin signalling in diabetic rats. Furthermore, the effects of chronic BMOV treatment on basal kinase activity were also investigated. 3.1.1 PLASMA GLUCOSE LEVELS IN THE ZUCKER DIABETIC FATTY RAT The plasma glucose levels in ZDF rats are shown in Figure 3.1. Before the initiation of BMOV treatment, the plasma glucose level was 29.35 +/- 0.52 mmol/1 for the untreated group and 28.65 +/- 0.36 mmol/1 for the BMOV treated group. Following chronic (10 weeks) BMOV treatment, the plasma glucose level in the untreated group remained unchanged and measured 28.71 +/- 0.6 mmol/1. However, the plasma glucose was significantly lower in the BMOV treated group, measuring 13.25 mmol/1 +/- 1.43. Therefore, chronic BMOV treatment was able to significantly lower plasma glucose levels when compared to the untreated group. 3.1.2 BASAL MBP PHOSPHOTRANSFERASE ACTIVITY AND EFFECT OF BMOV TREATMENT The muscle extracts were fractionated by anion-exchange chromatography using a MonoQ column and the resultant fractions were examined for phosphotransferase activity toward myelin basic protein (MBP). To produce a column profile that was more representative of each group (untreated and BMOV treated), muscle extracts from rats in each group were pooled separately and applied to the column. Several peaks of MBP phosphotransferase activity were observed and are numbered in Figure 3.2A. Peak I represents the MBP phosphotransferase 47 o E o u o n £ m Initial Final Untreated (n= 10) Initial Final Treated (n=8) Figure 3.1 The plasma glucose levels in ZDF rats at the beginning of the treatment period (initial) and at termination (final). BMOV treatment was initiated at a concentration of 0.2 mg/ml in the drinking water and increased in increments of 0.1 mg/ml to a maximum concentration of 0.8 mg/ml over 10 weeks. * denotes value is significantly different from all other groups (p < 0.05, ANOVA followed by Neuman-Keuls). Note: The error bars on the columns representing the "initial" treatment period were too small and did not appear on the graph. 48 activity in the flow through column fractions. The flow through fractions contain proteins that do not bind to the MonoQ column. Using a 15 ml linear NaCl gradient (0-800 mM), four major MBP phosphotransferase activity peaks were observed, numbered II, III, IV and V. Small changes were observed in peaks II and IV between the two groups. However, only peak I (flow through) demonstrated a large difference between the two groups such that BMOV treatment was able to produce an increase in the MBP kinase activity of peak I. 3.1.3 BASAL S6-10 PEPTIDE PHOSPHOTRANSFERASE ACTIVITY AND EFFECT OF BMOV TREATMENT The muscle extracts were again fractionated by anion-exchange chromatography and the fractions were examined for phosphotransferase activity toward the peptide S6-10. The S6-10 peptide is based upon a portion of the S6 protein, which is a part of the 40S ribosome. Pooled muscle extracts were again applied to the Mono Q column. Several peaks of S6-10 peptide phosphotransferase activity were observed and are numbered in Figure 3.2B. Two peaks, numbered II and III, contained the majority of the S6-10 peptide phosphotransferase activity. On comparison of the untreated and the BMOV treated column profiles no large difference was observable. A small change was observed in peak IV, such that the untreated group had higher activity than the BMOV treated group. 3.1.4 P70S6K IMMUNOPRECIPITATION STUDY p70 6K was chosen as a marker enzyme to assess the integrity of the PI3 kinase/PKB pathway. It is evident from Figure 3.3A that BMOV treatment had a significant effect on p70 6K phosphotransferase activity. The untreated group was 49 Figure 3.2 Representative Mono Q profiles of MBP and S6-10 peptide phosphotransferase activity in the ZDF rats. (A) MonoQ profile of MBP phosphotransferase activity in the Zucker Diabetic Fatty (ZDF) rats. (B) MonoQ profile of S6-10 peptide phosphotransferase activity in the ZDF rats. The pooled muscle extracts containing 5 mg of protein were subjected to MonoQ chromatography. Column fractions of 0.25 ml each were collected and assayed for phosphotransferase activity. 50 S6 phosphotransferase activity (pmol/min per ml) U\ o 3 O o o 3 3 o MBP phosphotransferase activity (pmol/min per ml) o o o o 0\ o o taken to represent 100 percent activation of p70S6K. Following BMOV treatment, the activity of p70S6K decreased to approximately 70 percent when compared to the untreated group. Therefore, BMOV treatment was able to decrease the activity of p70S6K by about 30 percent. Figure 3.3B shows a representative immunoblot of anti-p70S6K with the amount of immunoprecipitated protein for each group. With immunoblotting data, the activity of the kinase can be correlated with protein levels. Following densitometry of the anti-p70S6K immunoblots the activity of p70S6K was adjusted based upon the amount of protein immunoprecipitated. Since similar amounts of protein were immunoprecipitated for each group, the decrease in p70 6 activity after BMOV treatment is due to a decrease in p70S6K activity and not due to lower amounts of protein. 3.1.5 ERK-1 AND ERK-2 IMUUNOPRECIPITATION STUDY ERK-l/2 were used as indicators for the integrity of the MAP kinase pathway, since they are thus far the best characterized kinases in the cascade. Figure 3.4A provides the results of the ERK-1 immunoprecipitation experiments. Following BMOV treatment, the activity of ERK-1 was significantly decreased to approximately 60 percent of the untreated group. The untreated group was taken to represent 100 percent activation and the activity of ERK-1 was assessed by MBP phosphotransferase activity. Figure 3.4B shows a representative immunoblot of ERK-1. The antibody, ERK-l-CT was used to blot for ERK-1. Following densitometry of the ERK-1 immunoblots, the activity was adjusted based upon protein levels. Therefore, the 40 percent decrease in ERK-1 activity is attributable to lower enzyme activity and not due to a change in the levels of protein. 52 p70 S6K Immunoprecipitation > ro C S 2 •K c O u O CL, a vo t/1 -I c E ra — ZD -00 -75 -50 -25 -0 -A * T 1 B . m _ . . ^ ^ . ^ ^ — _ , ^ -. 69 kDa p70 S 6 K •Antibody Band (49 kDa) Untreated BMOV treated Figure 3.3 p70 immunoprecipitation study in the ZDF rats. (A) The effect of BMOV treatment on basal p70S6K activity in the ZDF rats. The ZDF rats were chronically treated with BMOV for 10 weeks. Results are expressed as a percentage of the control (untreated) value. (B) Western blot of anti-p70S6K immunoprecipitates from control (untreated) and BMOV treated. Each group was done in triplicate. Antibody band indicates the position of the IgG present in the antibody. * denotes value is significantly different from the untreated value (p < 0.05, two-sample t-test). Note: the error bar on the column representing the "untreated" group was too small and did not appear on the graph. 53 Figure 3.5A shows the results of the ERK-2 immunoprecipitation experiments conducted. Similar to the results for the ERK-1 immunoprecipitation experiments, ERK-2 activity was significantly decreased by 35 percent following BMOV treatment when compared to the untreated group. Figure 3.5B presents an immunoblot of ERK-2. The antibody ERK-l-CT was used to blot for ERK-2. It is well established (data not shown), that the ERK-l-CT antibody recognizes both isoforms of ERK and ERK-l-CT is routinely used to blot for ERK-2 in our laboratory. Following densitometric analysis of the ERK-2 immunoblots the activity of ERK-2 was adjusted based upon the amounts of protein immunoprecipitated. Once again BMOV treatment decreased kinase activity. Furthermore, ERK-2 is present as a doublet (2 bands present) in the untreated group and following BMOV treatment the top band of the doublet disappears. This observation, strengthens the hypothesis that BMOV treatment decreases kinase activity. The band shift present in the untreated group is probably due to increased basal phosphorylation of ERK-2. When proteins are phosphorylated, they become heavier and thus migrate slower on SDS-polyacrylamide gels. Following BMOV treatment the band shift disappeared, suggesting that the levels of ERK-2 phosphorylation had decreased. 3.1.6 pgcf* IMMUNOPRECIPITATION STUDY p90rsk is postulated to be directly downstream of ERK-1/2. Therefore, any changes in ERK-1/2 should be reflected in p90rsk. Figure 3.6A shows the data for the p90rsk immunoprecipitation study. p90rsk activity decreased by approximately 75 percent when compared to the untreated group. The change in p90ksk activity was as expected, since it is directly downstream of ERK-1/2, but the changes in p90rsk were more dramatic. Following BMOV treatment, ERK-1/2 activity decreased by 35-40 54 percent when compared to the untreated group, whereas, the activity of p90rsk decreased by 75 percent. Figure 3.6B shows an immunoblot for p90rsk. The antibody anti-RSK2-PCT was used to blot for p90rsk. As before, the activity of p90rsk was adjusted for based upon the amount of protein present for each group. Therefore, the decrease in p90rsk activity is due to decreased enzyme activity. 3.2 EFFECTS OF BMOV ON PROTEIN-SERINE KINASES IN STZ-DIABETIC WISTAR RATS With the ZDF study, various kinases were analyzed in the basal state and then the effect of BMOV on basal kinase activity was examined. With the Wistar study, both basal and insulin stimulated samples were available and thus allowed us to examine protein kinases in the basal and insulin stimulated states from diabetic animals. Finally, the effects of BMOV on insulin stimulated kinase activities were investigated in the diabetic state. 3.2.1 PLASMA GLUCOSE LEVELS IN THE STZ-DIABETIC WISTAR RAT The plasma glucose levels in the Wistar rats are shown in Figure 3.7. One week following BMOV treatment (Figure 3.7A), the plasma glucose levels in the control animals ranged from 7.42 +/- 0.16 to 7.50 +/- 0.18 mmol/1 for the 3 groups (A,B,C). Likewise, the plasma glucose levels for the control treated animals were similar to control animals, with the levels ranging from 6.55 +/- 0.17 to 7.00 +/- 0.15 mmol/1 for all groups. However, the plasma glucose levels were significantly greater in the STZ-diabetic rats when compared to the control animals. The plasma glucose levels were approximately 22.44 +/- 0.26 to 23.04 +/- 0.49 mmol/1 for the three groups. BMOV treatment for only one week already started to significantly lower the plasma glucose levels in all groups (A,B,C); the levels ranged from 19.03 +/- 0.66 to 18.61 +/- 0.89 55 > y oj o ra * ; t. c Ji o C • * . 10 o C 4-i o c Q. fcf n u Q Q_ & 125 100 75 -50 -25 -n A ERK-1 Immunoprecipitation * T 1 - Antibody Band (49 kDa) -44.5 kDa ERK-1 Untreated BMOV treated Figure 3.4 ERK-1 immunoprecipitation study in the ZDF rats. (A) The effect of BMOV treatment on basal ERK-1 activity in the ZDF rats. The ZDF rats were chronically treated with BMOV for 10 weeks. Results are expressed as a percentage of the control (untreated) value. (B) Western blot of anti-ERK-1 immunoprecipitates from control (untreated) and BMOV treated. Each group was done in triplicate. Antibody band indicates the position of the IgG present in the antibody. * denotes value is significantly different from the untreated value (p < 0.05, two-sample t-test). Note: the error bar on the column representing the "untreated" group was too small and did not appear on the graph. 56 ERK-2 Immunoprecipitation 125 -Antibody Band (49 kDa) -44.5 kDa - ERK-2 Untreated BMOV treated Figure 3.5 ERK-2 immunoprecipitation study in the ZDF rats. (A) The effect of BMOV treatment on basal ERK-2 activity in the ZDF rats. The ZDF rats were chronically treated with BMOV for 10 weeks. Results are expressed as a percentage of the control (untreated) value. (B) Western blot of anti-ERK-2 immunoprecipitates from control (untreated) and BMOV treated. It is established in our laboratory that the ERK-l-CT antibody recognizes both ERK-1 and ERK-2. The arrow indicates the position of phosphorylated ERK-2 (band shift present). Each group was done in triplicate. Antibody band indicates the position of the IgG present in the antibody. * denotes value is significantly different from the untreated value (p < 0.05, two-sample t-test). Note: the error bar on the column representing the "untreated" group was too small and did not appear on the graph. 57 D C 1 / p90 ° immunoprecipitation & 100 -> E g 75 -g ° •&y 50 -O Q-J= w a *o 25 -o-l h £j rs o 00 3 Pi E i E c ' ra A * - r B «*«.«» D I M * <»••«(§ «•<•(» « M — » mmm> *w& ^m aa .-^^ ML, -•-• 94 kDa • p 9 0 RSK - Antibody Band (49 kDa) Untreated BMOV treated Figure 3.6 p90rs immunoprecipitation study in the ZDF rats. (A) The effect of BMOV treatment on basal p90rsk activity in the ZDF rats. The ZDF rats were chronically treated with BMOV for 10 weeks. Results are expressed as a percentage of the control (untreated) value. (B) Western blot of anti-p90rsk immunoprecipitates from control (untreated) and BMOV treated. Each group was done in triplicate. Antibody band indicates the position of the IgG present in the antibody. * denotes value is significantly different from the untreated value (p < 0.05, two-sample t-test). Note: the error bar on the column representing the "untreated" group was too small and did not appear on the graph. 58 mmol/1. Following BMOV treatment for 2 months the rats were terminated 15 minutes following 0,5 and 10 U/kg insulin injection. The plasma glucose levels in the control animals following insulin injection (5 or 10 U/kg) were significantly different (p < 0.05) from the basal control animal. BMOV treatment caused a basal decrease in plasma glucose levels, from 19.03 +/- 0.66 mmol/1 (one week following BMOV treatment) to 6.76 +/- 0.74 mmol/1 (at termination) in the diabetic animals. Following 5 U/kg insulin injection, the plasma glucose levels in the control and control treated animals decreased to 2.83 +/- 0.31 mmol and 3.06 +/- 0.17 mmol/1, respectively. However, the plasma glucose levels remained unchanged in the diabetic animals when compared to the basal diabetic group (15.35 +/- 2.55 mmol/1). BMOV treatment was able to significantly lower the plasma glucose levels in the diabetic treated animals injected with 5 U/kg insulin to levels comparable to basally treated diabetic animals (3.82 +/- 2.29). Finally, following 10 U/kg insulin injection, the plasma glucose levels were 3.80 +/- 0.33 mmol/1 for the control group, 2.61 +/- 0.25 mmol/1 for the control treated group, 17.41 +/- 0.44 mmol/1 for the diabetic group and 2.06 +/- 0.42 mmol/1 for the diabetic treated group. BMOV treatment in the diabetic treated animals with 10 U/kg insulin had significantly lower plasma glucose when compared to basal control. Furthermore, BMOV with 10 U/kg insulin was able to decrease plasma glucose further compared to BMOV and 5 U/kg insulin (diabetic treated animals with 5 U/kg insulin were no different statistically from the basal control). 3.2.2 BASAL AND INSULIN STIMULATED MBP PHOSPHOTRANSFERASE ACTIVITY AND THE EFFECT OF B M O V TREATMENT The MBP kinases were analyzed in an identical manner as before with the ZDF rats. Briefly, a MonoQ anion-exchange column was used to partially purify the 59 Figure 3.7 Plasma glucose levels in the Wistar rats. A concentration of 60 mg/kg of STZ was used to produce diabetes. Seven days after STZ injection, the treated groups received BMOV in the drinking water at a concentration of 0.75 mg/ml for 8 weeks. At the end of the treatment period, each group of rats (control (C) n=5, control treated (CT) n=5, diabetic (D) n=5 and diabetic treated (DT) n=5) were sub-divided and injected with saline 0 (saline), 5 or 10 U/kg of insulin. A. The plasma glucose levels of the Wistar rats following one week of BMOV treatment. * denotes value is significantly different from the control value (0 U/kg),- ** denotes value significantly different from the diabetic group (p < 0.05, ANOVA followed by Neuman-Keuls). B. The plasma glucose levels of the Wistar rats at termination following insulin injection. * denotes value significantly different from the control value (0 U/kg),- ** control with 5 U/kg and control with 10 U/kg insulin values were significantly different from the control value (0 U/kg) when analyzed separately from the other groups (p < 0.05, ANOVA followed by Neuman-Keuls). Note: the muscle was removed 15 minutes following insulin injection. 60 Plasma Glucose (mmol/l) ON + o c > era 13 + OQ W + O 0 1 _l_ O _ l _ kfej—i o CfQ ^ ^ ! i * _ l _ o _L_ H * as Plasma Glucose (mmol/l) ^ H o o n n muscle extracts and the fractions were analyzed for phosphotransferase activity towards myelin basic protein (MBP). To produce a kinase profile that was representative of each group (control, control treated, diabetic and diabetic treated), similar amounts of protein from rats in each group were pooled and then applied to the MonoQ column. Several peaks of MBP phosphotransferase activity were observed and are numbered in Figure 3.8. Only MBP kinases in peak III of the diabetic group showed activation following 5 U/Kg insulin stimulation. Whereas, peak V in the control treated and the diabetic treated groups showed increased basal activity when compared to the insulin stimulated peak. However, no general trend in MBP phosphotransferase activity was observed when comparing normal and diabetic rats and the effects of BMOV treatment. 3.2.3 BASAL AND INSULIN STIMULATED S6-10 PEPTIDE PHOSPHOTRANSFERASE ACTIVITY AND THE EFFECT OF B M O V TREATMENT Several peaks of S6-10 peptide phosphotransferase activity were observed and are numbered in Figure 3.9, following MonoQ purification. The majority of the S6-10 peptide phosphotransferase activity was observed in peak II for all the groups. Also, peak IV appeared to be increased in the basal state in the diabetic treated animals. Based upon the profiles, two general trends were observed after examining the kinase activity profiles of the different groups. First, the S6-10 phosphotransferase activity was much greater in peak II in the diabetic group when compared to the control group. Second, it appears that treatment with BMOV is able to increase the S6-10 phosphotransferase activity in peak II. The control treated and diabetic treated groups have greater S6-10 kinase activity when compared to the control group and the diabetic group respectively. As with the MBP kinase profiles, 62 Control Group 0 | • • • • | i • i • | i i i i | i i i i | n • • | n i i | • •• • ! 0 10 20 30 40 50 60 70 40-30-Diabetic Group - insulin + 5 U/Kg insulin 0 10 20 30 40 50 60 70 Mono Q traction no. Mono Q fraction i Control Treated Group i i i i 1 1 1 i i 1 1 1 i i 11 i i i 1 1 1 1 1 1 i*0i 11 i i i i 0 10 20 30 40 50 60 70 Mono Q fraction no. •2 40 3 30-20-10 Diabetic Treated Group -0 • insulin ••#• + 5 U/Kg insulin I I I 0 • • ^ • ^ • ^ • ^ • ^ r ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ H ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 10 20 30 40 50 60 70 Mono Q fraction no. Figure 3.8 MonoQ profiles of MBP phosphotransferase activity in the Wistar rats. Crude skeletal muscle extracts were prepared and samples containing 5 mg of protein were subjected to MonoQ chromatography. Column fractions of 0.25 ml each were collected and assayed for phosphotransferase activity. (A) Control, (B) Control Treated, (C) Diabetic and (D) Diabetic Treated. 63 200-100-0 -A Control Group t II I c S ^ l 1L • insulin + 5 U/Kg insulin ryOOOQO i 11 i 10 20 30 40 50 60 70 Diabetic Group Mono Q fraction no. 20 30 40 Mono Q fraction no. 70 200-100-o-B Control Treated Group 1 — O — -insulin t + 5 U/Kg insulin n 1 v" X « b lv Diabetic Treated Group 0 10 20 30 40 50 60 70 Mono Q fraction no. O • insulin • = 5 U/Kg insulin 10 20 30 40 50 Mono Q fraction no. Figure 3.9 MonoQ profiles of S6-10 peptide phosphotransferase activity in the Wistar rats. Crude skeletal muscle extracts were prepared and samples containing 5 mg of protein were subjected to MonoQ chromatography. Column fractions of 0.25 ml each were collected and assayed for phosphotransferase activity. (A) Control, (B) Control Treated, (C) Diabetic and (D) Diabetic Treated. 64 there appeared to be no observable trend in the S6-10 peptide kinases following 5 U/Kg insulin stimulation. 3.2.4 P70S6K IMMUNOPRECIPITATION STUDY Figure 3.10A shows the results of the immunoprecipitation experiments conducted to assess p70 6 activity. Basal control was taken to represent 100 percent activation of the kinase. Following 5 U/kg insulin stimulation, there was a significant increase in p70S6lc activity (6 fold) when compared to basal control. Surprisingly, following 10 U/kg insulin stimulation there was not a further activation of the kinase and was only activated 3 fold above basal control. A dose of 10 U/kg insulin was significantly different from 5 U/kg insulin in terms of p70S6K activity. Because maximum stimulation was achieved with 5 U/kg, the activity of p70 6 for the other groups (control treated, diabetic and diabetic treated) was assessed by injecting 5 U/kg and not 10 U/kg. The control treated group without insulin stimulation had activity levels similar to the basal control. Likewise, following insulin stimulation with 5 U/kg, the activity of p70S6K was similar to the control with 5 U/Kg. With the diabetic group, p70 6K was increased in the basal state, although this increase in activity was not statistically significant. Its activity was about 3-4 folds higher than the basal control group. Insulin stimulation did not activate p70S6K any further. BMOV treatment did not seem to correct this defective activity of p70 6 in the diabetic state in the basal or insulin stimulated states. Therefore, the major conclusion that can be drawn about p70S6K activity is that in the diabetic state, p70S6K activity is altered and BMOV treatment is unable to restore p70S6K activity to normal levels. 65 > >- O JJ u C O IS u J= c O U a Jj a p 7 0 S6K Immunoprecipitation 1000 750 _ 500 -250 -T 1 T T 1 rr T _ 69 kDa ' P 7 0 ^ 6 K •Antibody Band (49 kDa) Control Control Treated Diabetic Diabetic Treated Figure 3.10 p70S6K immunoprecipitation study in the STZ-diabetic rats. (A) The effect of BMOV on basal and insulin stimulated p70S6K activity in the Wistar rat. A dose of 60 mg/ml of STZ was given to the animals to make them diabetic. The rats were then treated with BMOV for 2 months. Results are expressed as a percentage of the control. (B) Western blot of anti-p70S6K immunoprecipitates from all the groups (control, control treated, diabetic, and diabetic treated). Antibody band indicates the position of the IgG present in the antibody. * denotes value significantly different from basal control (p < 0.05, ANOVA followed by Neuman-Keuls),- note: control with 10 U/kg insulin was not included in the ANOVA analysis. ** denotes value is significantly different from control with 5 U/kg insulin (p < 0.05, two-sample t-test). The error bars for the groups "basal control" and "basal control treated" were too small and did not appear on the graph. 66 Figure 3.10B shows a representative immunoblot for p70S6K using the anti-p70 S6K-NT antibody. Following quantitative analysis of the immunoblots, the activity of p70S6K in the different groups was corrected for based upon the amount of protein immunoprecipitated. Band shifts also appear to be present in the insulin stimulated groups. With the control group, band shift becomes more prominent as the dose of insulin is increased from 5 U/kg to 10 U/kg. However, even though the band shifts are most prominent in the 10 U/kg group, the activity is lower. Therefore, a correlation between the level of phosphorylation of p70S6K and S6 phosphotransferase activity does not exist for 10 U/kg. Likewise for the diabetic group, the immunoblot shows that p70S6K is being phosphorylated (band shift present) in response to insulin, but the activity of the kinase is unchanged above the diabetic basal group. 3.2.5 ERK-1 IMMUNOPRECIPITATION STUDY The results of the ERK-1 immunoprecipitation experiments are presented in Figure 3.11. ERK-1 activity was assessed by using MBP as the substrate, with basal control taken to represent 100 percent activation (Figure 3.11 A). Following 5 U/kg insulin stimulation, there was a significant increase (2 fold) in ERK-1 activity. With 10 U/kg, no further stimulation of ERK-1 activity was seen above 5 U/Kg. Therefore, as with p70S6K, 5 U/kg was used to produce stimulation of ERK-1 for other groups. The basal control treated group had ERK-1 activity comparable to control plus 5 U/kg insulin. Therefore, it appears that BMOV treatment was able to increase basal ERK-1 activity in the control animals. Following 5 U/kg stimulation, no further stimulation of ERK-1 was evident in the control treated group. This suggests that BMOV treatment was enough to activate ERK-1 to its maximum level without additional insulin stimulation. In the diabetic state, no differences were observable between the 67 > u o o U O 2 i JJ o C v i_ ra O is *•• o c 1* ERK-1 Immunoprecipitation 500 400 300 200 100 * 8 <* 3 UJ £ s E c — T JL 1 T 1 I 1 T -Antibody Band (49 kDa) .44.5 kDa •ERK) ra so ra DC ra 00 j2 OB Control Control Treated Diabetic Diabetic Treated Figure 3.11 ERK-1 immunoprecipitation study in the STZ-diabetic rats. (A) The effect of BMOV on basal and insulin stimulated ERK-1 activity in the Wistar rat. A dose of 60 mg/ml of STZ was given to the animals to make them diabetic. The rats were then treated with BMOV for 2 months. Results are expressed as a percentage of the control. (B) Western blot of anti-ERK-1 immunoprecipitates from all the groups (control, control treated, diabetic, and diabetic treated). Antibody band indicates the position of the IgG present in the antibody. * denotes value significantly different from basal control (p < 0.05, ANOVA followed by Neuman-Keuls),- note: control with 10 U/kg insulin was not included in the ANOVA analysis. ** denotes value is significantly different from basal control (p < 0.05, two-sample t-test). The error bar for the group "basal control" was too small and did not appear on the graph. 68 basal diabetic group and the basal control group. However, following insulin stimulation, ERK-1 was significantly increased with its activity approximately 5 fold greater than basal control. In the control animal, only a 2 fold activation was seen with a dose of 5 U/kg, but in the diabetic state, ERK-1 activity was 3 fold greater than control plus 5 U/kg insulin. Following BMOV treatment, the increased ERK-1 activity seen in the diabetic animals was not corrected. Therefore, BMOV treatment was unable to normalize the insulin stimulated activity of ERK-1 in the diabetic animals. The immunoblot data for ERK-1 is presented in Figure 3.1 IB. The antibody ERK-1-CT was used to probe for ERK-1. Using the immunoblot data, ERK-1 activity was adjusted based upon protein amounts and thus the changes in ERK-1 activity are attributable to kinase activity and not to protein expression. 3.2.6 ERK-2 IMMUNOPRECIPITATION STUDY Figure 3.12A shows the results of the ERK-2 immunoprecipitation experiments. As before, basal control represented 100 percent activation of the kinase. Following 5 U/kg insulin stimulation, there was approximately 2 fold stimulation of ERK-2 activity. Following 10 U/kg insulin, no further stimulation above 5 U/kg were seen. In the control treated animals, basal activity was 2 fold higher than basal untreated animals. The level of stimulation was very similar to control with 5 U/kg insulin. When insulin was injected into the control treated animals, a significant increase in ERK-2 activity were seen above basal untreated animals. In the diabetic state, ERK-2 activity was slightly greater than the control animals. However, following insulin stimulation with a 5 U/kg dose, ERK-2 activity showed a marked increase as compared to the control group. Its MBP 69 phosphotransferase activity was significantly increased approximately 5 fold above basal control. In control animals, 5 U/kg produced a 2 fold stimulation of ERK-2 activity, whereas in the diabetic state, the same dose of insulin produced a 5 fold stimulation of ERK-2 activity. BMOV treatment had a dramatic effect on ERK-2 activity in the diabetic rats. BMOV treatment completely normalized the increased activity of ERK-2. Following BMOV treatment, the activity of ERK-2 decreased from 5 fold to approximately 2 fold above basal control. Therefore, ERK-2 shows marked activity in response to insulin in the diabetic state and BMOV treatment tends to normalize this defective activity. Figure 3.12B shows the immunoblot for ERK-2. The antibody ERK-l-CT was used to probe for ERK-2. As stated above (in the ZDF study), ERK-l-CT has been shown to recognized both isoforms of ERK and is regularly used in our laboratory to probe for ERK-2. Using the immunoblot data, ERK-2 activity was corrected based upon protein amounts and thus the changes in ERK-2 activity are attributable to kinase activity and not to protein expression. 3.2.7 PQCf* IMMUNOPRECIPITATION STUDY The results of the p90rs immunoprecipitation experiments are presented in Figure 3.13A. The results are expressed as a percentage of the control. With 5 U/kg insulin, p90rsk was stimulated approximately 1.5 fold above control. With 10 U/kg insulin, a 3-3.5 fold activation of the S6 phosphotransferase activity of p90rsk was seen. The activity of p90rs was not at its maximum level with 5 U/kg insulin stimulation, as seen with ERK-1 and ERK-2, but further stimulation of p90rsk activity could be 70 obtained with 10 U/kg insulin. However, since 5 U/kg insulin was used for the previous immunoprecipitation experiments, 5 U/kg was used for the remaining groups in the p90rsk immunoprecipitation experiments. In the control treated animals, there was a small increase in p90rsk activity above control. There was no difference between the basal and insulin stimulated groups. In the diabetic animals, no difference was seen between the basal and insulin stimulated groups when compared to the control group. BMOV treatment had no effect on the diabetic treated animals in the basal state. However, with 5 U/kg insulin BMOV treatment tended to normalize the low activity of p90rsk. After BMOV treatment, p90rsk activity increased by 2-2.5 fold above control. Therefore, the major conclusion that can be drawn from the p90rs immunoprecipitation experiments is that in diabetic animals, insulin is unable to activate p90rsk above control levels and BMOV treatment tended to normalize the activity of p90rs when compared to diabetic rats stimulated with 5 U/kg insulin. Figure 3.13B shows a representative blot of anti-p90 RSK immunoprecipitates from all the groups. The antibody RSK.2-PCT was used to probe for p90rsk. The proper controls were also conducted in parallel with all the immunoprecipitation experiments for both the ZDF and Wistar studies. The control experiments that were conducted in parallel with the immunoprecipitation experiments are the (l) beads only and (2) antibody only (data not shown) immunoprecipitates. The beads only represents the phosphotransferase activity of non-specifically bound proteins to the protein A-Sepharose beads without the antibody being present. Whereas, the antibody only represents the phosphotransferase activity of the antibody without the presence of any crude muscle extract. The phosphotransferase activity of both controls were low for all the 71 kinases we examined. This suggests the increased activity is due to the immunoprecipitated complex (kinase-antibody complex bound to the beads) and not due to non-specifically bound proteins nor the antibody used. 72 ERK-2 Immunoprecipitation u v O i/> I -2 c ju o c «-ra O o c n u O Q_ a. Q_ 600 500 400 300 200 100 o ^ T * * T J . X 1 T h J. u s * g UJ g B E c — B "ra ra 3C 3 to + Control bo 3 o + 3 ra ; ; ; ;; 5c 3 >n + Control Treated ra -5£ 3 (n + Diabetic «M» "ra ra 3 in + Diabetic Treated • Antibody Band (49 kDa) •44.5kDa • ERK-2 Figure 3.12 ERK-2 immunoprecipitation study in the STZ-diabetic rats. (A) The effect of BMOV on basal and insulin stimulated ERK-2 activity in the Wistar rat. A dose of 60 mg/ml of STZ was given to the animals to make them diabetic. The rats were then treated with BMOV for 1 months. Results are expressed as a percentage of the control. (B) Western blot of anti-ERK-2 immunoprecipitates from all the groups (control, control treated, diabetic, and diabetic treated). It is established in our laboratory that the ERK-l-CT antibody recognizes both ERK-1 and ERK-2. Antibody band indicates the position of the IgG present in the antibody. * denotes value significantly different from basal control (p < 0.05, ANOVA followed by Neuman-Keuls),- note: control with 10 U/kg insulin was not included in the ANOVA analysis. ** denotes value is significantly different from basal control (p < 0.05, two-sample t-test). The error bar for the group "basal control" was too small and did not appear on the graph. 73 > u ^ ra"o U u ui *-• re c J s s ° I- -w O OJ JS y o o-(X p90 Immunoprecipitation 400 300 -200 -100 -# T 1 T T 1 -94 kDa • p 9 0 R S K •Antibody Band (49 kDa) basal U/Kg LO + Control SB 3 o •1-ra Ifl a Contro Treatec u 3 m + m SO 3 ITl t Diabetic basal U/Kg m + Diabetic Treated Figure 3.13 p90rsk immunoprecipitation study in the STZ-diabetic rats. (A) The effect of BMOV on basal and insulin stimulated p90rsk activity in the Wistar rat. A dose of 60 mg/ml of STZ was given to the animals to make them diabetic. The rats were then treated with BMOV for 2 months. Results are expressed as a percentage of the control. (B) Western blot of anti-p90rsk immunoprecipitates from all the groups (control, control treated, diabetic, and diabetic treated). Antibody band indicates the position of the IgG present in the antibody. * denotes value significantly different from basal control (p < 0.05, ANOVA followed by Neuman-Keuls),- note: control with 10 U/kg insulin was not included in the ANOVA analysis. ** denotes value is significantly different from basal control (p < 0.05, two-sample t-test). # denotes value is significantly different from control with 5 U/kg insulin (p < 0.05, two-sample t-test). The error bar for the group "basal control" was too small and did not appear on the graph 74 CHAPTER 4. DISCUSSION 4.1 OVERVIEW The main purpose of this study was to examine the in vivo effects of bis(maltolato)oxovanadium(IV) (BMOV) on the activity of various protein kinases on two cascades that are implicated as upstream mediators of the biological effects of insulin. The integrity of the MAP kinase pathway was evaluated by studying the activation of ERKl, ERK.2 and p90rsk. The activity of p70S6K was also evaluated as a possible downstream enzyme in insulin action . The intact rat served as a convenient animal model system to study the mechanism of insulin action. The Zucker Diabetic Fatty (ZDF) rat strain provided an ideal model to study the activity of protein kinases in NIDDM, because the development of the disease in these rats closely parallels NIDDM in humans. The activity of protein kinases was also investigated in the Streptozotocin (STZ) Diabetic Wistar rat. This model is representative of IDDM, because the chemical, STZ destroys the pancreatic (3-cells. Rat skeletal muscle was chosen to perform the studies because it is insulin sensitive, it represents a major site of insulin stimulated glucose utilization and a relatively large quantity of tissue can be obtained for the biochemical analysis. The role of protein kinases in insulin signalling is usually obtained through studies conducted in cultured cells or isolated tissues. The main advantage of in vitro studies is that the experimental conditions can be easily controlled and interference from unknown sources does not occur. However, to gain a more complete insight into the physiological regulation of these protein kinases, it is necessary to perform studies using an in vivo model. The approach used to examine kinase activity included MBP/S6-10 peptide phosphotransferase activity assays and specific immunoprecipitation experiments. 75 Before the results of each immunoprecipitation study are described, it is necessary to mention the drawback with studying kinase profiles. It is clear from the column profiles for both MBP and S6-10 peptide (Figures 3.2,3.8 and 3.9) that no observable effect of BMOV treatment alone was seen. Due to the existence of a multitude of protein kinases and other proteins in crude muscle extracts in each column fraction that could interfere with various enzyme activities, immunoprecipitation experiments were conducted to assess changes in individual protein kinases. Furthermore, changes in myelin basic protein (MBP) phosphotransferase activity peaks is commonly interpreted to represent changes in MAP kinases. This assumption is incorrect, as MBP is used as a substrate by many different kinases in vitro. The results indicate that the majority of the MBP phosphotransferase activity appears in peak II and III. It is known from Western blotting of column fractions that ERK-1 (42 kDa), ERK-2 (44 kDa), PKB and PKC isoforms appear in peak II and III (data not shown). Therefore, the kinase activities recorded on MonoQ profiles involve several enzyme activities and are not due to MAP kinase alone. The same holds true for the S6-10 peptide profiles. 4.2 THE ROLE OF P70S6K IN INSULIN ACTION At the beginning of this study published data indicated that PI3K activated PKB, because inhibitors of PI3K such as wortmannin inhibited the activation of PKB (Burgering and Coffer, 1995,- Cross et al, 1995). Furthermore, it was demonstrated that PI3K and PKB are upstream signalling molecules in p70S6K activation (Chung et al, 1994; Moule and Denton, 1997). Based upon that literature, we chose p70S6K as a marker enzyme to assess the integrity of the PI3K —> PKB —> p70S6K pathway. However, it is now known that the pathway may not be as simple as originally 76 thought. Recently, it was demonstrated that another enzyme called FRAP or mTOR lies upstream of p70S6K and downstream or parallel with PKB (Proud, 1996). Using the immunosuppressant drug rapamycin, the phosphorylation of a specific subset of sites on p70S6K was prevented (Ferrari et al, 1993). More specifically, rapamycin binds to a protein called FKBP12 and the rapamycin-FKBPl2 complex interacts with mTOR. Taken together, it appears that the activity of FRAP/mTOR is essential in the activation of p70S6K. Several enzymes, including the nonconventional PKC isoforms and members of the Rho family of G proteins (Cdc42 and Racl) have also been shown to activate p70S6K (Toker et al, 1994; Chou and Blenis, 1996). Just recently, two independent groups demonstrated that 3-phosphoinositide-dependent protein kinase 1 (PDKl) phosphorylates and activates p70S6K (Pullen et al, 1998,-Alessi etal., 1997). However, it still remains to be determined if any of these kinases activate p70S6K in diabetic animal models. The role of p70S6K in glucose utilization has been questioned recently, because rapamycin failed to inhibit glucose uptake in response to insulin (Chang etal., 1995,- Moule etal., 1995,- Cross etal., 1995). Glycogen synthase kinase-3 (GSK-3) is implicated in the regulation of several physiological processes, including the control of glycogen and protein synthesis (Cross etal., 1995,- Lawrence etal., 1996). Recent findings indicate that the inhibitory effects of insulin on GSK-3 may be mediated by the kinase, PKB (Cross et al., 1995). Several lines of evidence support the role of PKB in the inactivation of GSK-3. First, the half-time for activation of PKB (1 min) is consistent with it being an upstream activator of GSK-3 (Cross etal, 1995). Second, the activation of PKB by insulin, like the inhibition of GSK-3, is prevented by inhibitors of PI3K (Alessi et al, 1995; Kohn et al, 1995). Third, GSK-3 is inhibited when it is cotransfected with PKB in 293 cell 77 (Cross etal., 1997). Phosphorylation of GSK-3 by PKB results in its activation and the consequent activation of glycogen synthesis (Cross etal., 1997). 4.2.1 Basal p70S6K activity and the effect of BMOV in ZDF rats BMOV treatment for 10 weeks decreased p70S6K activity by about 30% when compared to the untreated animals. As mentioned above, p70 6 does not appear to be involved in glucose utilization, but its activity may become altered in the diabetic state along with other protein kinases in other pathways. In the diabetic state, p70 6 may represent a redundant pathway that participates in the activation of glycogen synthase and thus glucose disposal. p70 6K has previously been shown to phosphorylate and inactivate GSK-3 in vitro (Sutherland et al., 1993). However, because p70S6K activity only decreased by 30%, and BMOV treatment had significant affects on blood glucose levels (Figure 3.1), it appears that other pathways are more important in glucose metabolism. Another possibility is that BMOV decreased the activity of an upstream kinase/regulator of p70S6K. If BMOV decreased the activity of an upstream regulator of p70S6K, then the activity of the kinase would be lower. An upstream kinase that phosphorylates and activates p70S6K is PDKl (Alessi et al, 1997). Co-expression of p70S6K with PDKl, resulted in strong activation of the S6 kinase in vivo. In vitro, PDKl directly phosphorylated Thr252 in the activation loop of the p70S6K catalytic domain, the phosphorylation of which is stimulated by PI3K in vivo (Alessi et al., 1997). PDKl could be the upstream kinase that is affected by BMOV treatment. Thus, if BMOV treatment can decrease PDKl activity, then p70 6 activity would be reduced as well. 78 4.2.2 P70S6K ACTIVITY AND THE EFFECT OF BMOV IN STZ-WlSTAR RATS In control Wistar rats, p70S6K activity increased by approximately 6 fold following 5 U/kg insulin stimulation. Interestingly, no further stimulation of the kinase was seen with 10 U/kg insulin stimulation, but the activity decreased by about 3 fold. The immunoblot shows that the band shift occurred to a greater extent with 10 U/kg, when compared to 5 U/kg. Band shift refers to a decrease in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels, which is indicative of increased phosphorylation. Taken together, it appears that with a very high dose of insulin (10 U/kg) the level of phosphorylation of p70S6K increases, but the activity of the kinase decreases. This suggests that with a high, non-physiological dose of insulin a kinase, which negatively regulates p70S6K activity, is being stimulated. The kinase phosphorylates p70S6K on specific residues to decrease its phosphotransferase activity. Downregulation of activity has also been seen with other proteins. For example, serine and threonine phosphorylation of IRS-1 may downregulate IRS-dependent signalling by inhibiting tyrosine phosphorylation during insulin stimulation (Tanti et a\.t 1994). In 3T3L1 adipocytes, treatment with okadaic acid increased the level of IRS-1 serine/threonine phosphorylation, while PI3K activation and deoxyglucose uptake decreased. This suggests that hyper-phosphorylated IRS-1 may be a poorer substrate for the activated insulin receptor tyrosine kinase (Tanti et al., 1994). Interestingly, in both the basal diabetic and basal diabetic treated groups no band shift was present even though the activity was increased above the basal control level. It is possible that the increased activity seen in the diabetic animals is a result of a genetic change in the structure of the kinase which causes it to be active without phosphorylation. 79 In the diabetic animals, p70S6K showed increased basal activity with its activity 3-4 fold higher than control animals. BMOV treatment did not decrease the high basal activity of p70S6K to levels found in control animals. This suggests that p70S6K is not a target of BMOV because the changes in blood glucose concentrations following treatment in the diabetic animals do not correlate with changes in p70S6K activity. This finding further supports the finding in the ZDF study (described above) and prevailing literature concerning the lack of a role of p70 6K in glucose utilization (Chang et al, 1995; M o d e et al, 1995,- Cross et al, 1995). In these studies, the immunosuppressant drug rapamycin (inhibits p70S6K) failed to inhibit glucose uptake in response to insulin. 4.3 THE MAP KINASE (MAPK) PATHWAY Activation of MAPK by insulin was first observed in 3T3-L1 adipocytes (Ray and Sturgill, 1987). Insulin injections into the portal vein of rats have been shown to increase the activity of both ERK-1 and ERK-2 in the liver (Tobe et al, 1992). Similarly, injection of insulin to either fed or starved rats with or without a glucose clamp has been shown to increase MAPK activity in skeletal muscle (Hei et al, 1993; Zhou et al., 1993). In these reports, the kinases were initially resolved by Mono Q chromatography and Western blotting was done to obtain evidence for ERK-1 and ERK-2 activation. The biological significance of MAPKs can be appreciated from its potential substrates, which include transcription factors, protein kinases, cytoskeleton proteins, and cytosolic phospholipase A2 (Johnson and Vaillancourt, 1994). An important effect of insulin action is to increase glycogen synthesis. The role of the MAPK pathway in glycogen synthesis is questionable. It has been 80 proposed that p90rsk (downstream target of MAPK) phosphorylates the glycogen-binding subunit of protein-phosphatase 1, which in turn dephosphorylates and activates the enzyme glycogen synthase to enhance glycogen synthesis (Dent et al., 1990; Lawrence et al., 1996). However, recent studies have challenged this notion. Results obtained with epidermal growth factor (EGF) provided evidence that the MAPK pathway was not involved in the activation of glycogen synthase (Robinson et al, 1993,- Lin and Lawrence, 1994). Similarly, the inhibitor of MEK activation, PD 098059 blocked MAPK and p90rsk but did not significantly decrease the activation of glycogen synthase (Azpiazu etal., 1996). Even though the role of MAPK in glycogen synthesis is doubtful, there is a need for studies to evaluate the extent of activation of MAP kinases in conditions of insulin resistance such as diabetes. Our laboratory has previously used an intact diabetic rat model to study MAP kinases and S6 kinases (Hei et al., 1993). Recently, Hei et al. (1995) showed that the activity of these kinases is significantly altered in STZ-diabetic rats and that vanadium treatment restored the activity to those observed in control rats. A similar approach was used to examine the MAP kinase pathway using the ZDF and the STZ-Wistar rat models in the present study. 4.3.1 BASAL MAPK PATHWAY ACTIVITY AND THE EFFECT OF BMOV IN ZDF RATS The basal activity of key enzymes in the MAPK pathway were altered in the Zucker Diabetic Fatty rats and BMOV treatment for 10 weeks was able to decrease the defective activity. The basal kinase activity of ERK-1 was decreased by 40% and ERK-2 activity was decreased by 35% when compared to the untreated group. p90rsk is postulated to be downstream of ERK-2 and any changes in ERK-2 could be 81 reflected in the activity of p90rsk. BMOV treatment decreased p90rsk activity by approximately 75% when compared to the untreated group. A role for the MAPK pathway in the activation of glycogen synthesis by insulin has been excluded by several findings, as stated above. Briefly, p90rsk is not activated by insulin in the skeletal muscle of transgenic mice expressing a mutated insulin receptor, whereas the activation of glycogen synthase is still insulin sensitive (Chang et al, 1995). Secondly, PD 98059 (inhibitor of MEK) has no effect on glycogen synthesis in primary adipocytes (Lazar et al, 1995). Lastly, epidermal growth factor (EGF) causes a stronger activation of MAPK than insulin in adipocytes, but does not stimulate glycogen synthesis (Lin and Lawrence, 1994). Since the MAPK pathway may not have a role in the activation of glycogen synthesis, it is therefore unlikely that the glucoregulatory effects of BMOV are due to the activation of the MAPK pathway. However, in the diabetic state, hyperglycemia and hyperinsulinemia could cause the activation of many different signalling cascades that may not be important in the normal state. These other pathways may not be important in glucose utilization, but they are activated none-the-less. The MAPK pathway could become aberrant, secondary to the development and/or progression of diabetes. Following BMOV treatment not only was there a significant decrease in blood glucose, but the activity of key enzymes in the MAPK pathway, such as both isoforms of ERK and p90rsk, was decreased as well. Furthermore, in the ERK-2 immunoblot (Figure. 5 A), ERK-2 is present as a doublet (2 bands present) in the untreated group and following BMOV treatment the top band disappeared. The band shift seen in the untreated group is due to increased basal phosphorylation of ERK-2. Usually as the level of phosphorylation of a protein 82 increases and thus its mass, it is sometimes possible to see the phosphorylated form of a protein (band shift). Following BMOV treatment the band shift disappeared, suggesting the level of ERK-2 phosphorylation had decreased. This observation, strengthens the hypothesis that the basal activity of ERK-2 is altered and BMOV is able to improve the defective activity of the kinase. 4.3.2 MAPK PATHWAY ACTIVITY AND THE EFFECT OF BMOV IN STZ-WlSTAR RATS The basal and insulin stimulated kinase activities of ERK-1, ERK-2 and p90rs were measured to assess the integrity of the MAPK pathway. Rat skeletal muscle was removed 15 minutes post insulin injection to evaluate insulin stimulation of the kinases. Previous work from our laboratory and a time course of activation for MAPK observed in cultured cells treated with insulin, indicated that maximal activation of MAPK occurs at about 10-15 min (Ray and Sturgill, 1987). ERK-1 and ERK-2 and p90RSK were activated by approximately 2 fold following 5 U/kg insulin stimulation in normal rats. There was no further increase in ERK-1 or ERK-2 activity following 10 U/kg insulin stimulation. This suggests that 5 U/kg insulin was sufficient to achieve maximal stimulation of ERK-1 and ERK-2. Surprisingly, there was a further increase in p90rsk activity following 10 U/kg. This indicates that at very high insulin concentrations other pathways are being activated andean feed into p90rs . Very recently, Bhanot et al., 1998, demonstrated that 2 U/kg insulin, a pharmacological dose of insulin, was not sufficient to activate the MAPK pathway. Activation could only be achieved by very high non-physiological insulin concentrations (5 and 10 U/kg). The main reason we chose 5 U/kg insulin to activate the kinase pathways was to ensure that activation would be achieved in the diabetic state. 83 In STZ-diabetic rats, both ERK-1 and ERK-2 activities showed a marked increase in response to 5 U/Kg insulin. ERK-1 and ERK-2 activities were approximately 5 fold above control. Because the STZ-diabetic rats represent a model of poorly controlled Type I diabetes, where the circulating insulin level is markedly reduced, it is not surprising that the signalling pathway overcompensates in response to insulin stimulation in an attempt to decrease the high blood sugar concentrations. p90rs , however, was surprisingly not hyper-responsive to insulin stimulation,- its activity was very similar to control. The activity of p90rsk was expected to be hyper-responsive to insulin stimulation, as seen with the ERKs, as it is postulated to be directly downstream of ERK. BMOV treatment had dramatic effects on ERK-2 activity. Following BMOV treatment the activity of ERK-2 was completely normalized to control levels. However, BMOV had no effect on ERK-1 hyper-responsiveness to insulin stimulation. Therefore, BMOV appears to have differential effects on the two isoforms of ERK. In skeletal muscle (the primary site where most of the insulin stimulated glucose utilization occurs by converting glucose to glycogen), ERK-2 has been reported to be physiologically important in activating p90rsk (Blenis, 1993 and Cobb et al, 1991). Thus, it is of no surprise that BMOV had an effect on ERK-2 activity and not on ERK-1. The activity of p90rsk increased following BMOV treatment in the diabetic treated animals. After treatment, the activity of p90rsk was increased to 2.5 fold above basal control, but it was not at the level seen in the control animals stimulated with 10 U/kg insulin. This suggests that in STZ-diabetic rats the activity of p90rsk is low and BMOV tends to normalize the kinase by increasing its activity. 84 Taken together, the results suggest that there is a dissociation between ERK-2 and p90rsk. It is commonly postulated that ERK-2 and p90rsk are in a linear pathway and any changes seen in ERK-2 should be reflected in p90rsk. However, the results imply that the regulation of p90rsk may be far more complex. First, 5 U/kg were required to activate ERK-2 to its maximal level, whereas 10 U/kg was required to achieve maximal activity of p90rsk. Secondly, ERK-2 activity was hyper-responsive to insulin stimulation in the diabetic state, whereas p90rsk showed no change. Lastly, BMOV treatment normalized the activity of ERK-2 by decreasing the hyper-responsiveness of the kinase, whereas p90rsk activity was increased with BMOV treatment. Accordingly, other pathways, distinct from the MAPK pathway, are being activated and feed into and modulate p90rsk activity. Recently, Moxham etal, 1996, demonstrated that an isoform of p90rsk, RSK3, is rapidly activated by insulin. The activation of RSK3 was mediated by a jun N-terminal kinase (JNK) in vitro. JNKs are additional members of the MAPK family that have been cloned. Furthermore, based upon the description of the temporal data provided, the authors suggested a linkage map in skeletal muscle in vivo that places JNK and RSK3 in a linear cascade promoting activation of glycogen synthase by insulin. RSK3 is expressed in skeletal muscle and can be activated by several growth factors including insulin when overexpressed in a variety of cell lines (Moller et al., 1994,- Zhao et al, 1995,- Bjorbaek et al, 1995). RSK-3 phosphorylates the regulatory subunit of glycogen-bound PP-1, a physiological regulator of glycogen synthase in vivo (Bjorbaek et al, 1995). Figure 4.1 shows the possible regulation of glycogen synthase by the MAPK pathway and RSK-3. 85 Figure 4.1 The regulation of glycogen synthase activity by the MAPK pathway and RSK-3 in skeletal muscle by insulin in vivo. The MAP kinase pathway begins with the interaction of Grb-2 and mammalian son of sevenless (mSOS) with IRS-l/IRS-2, resulting in the activation of Ras. This triggers a cascade involving the sequential activation of the protein kinases, Raf, MAP kinase kinase (MEK), MAP kinase and p90 RSK. PPl may be phosphorylated and activated by p90rsk in vitro, but data presented in the text indicated that the MAP kinase pathway may not mediate the activation of glycogen synthase in response to insulin in skeletal muscle. An isoform of p90rs , RSK-3, has been shown to phosphorylate the regulatory subunit of glycogen-bound PP-1, a physiological regulator of glycogen synthase. (Solid arrows indicate direct effects and dotted arrows indicate possible interactions). 86 tt Plasma Membrane (lRS-l/IRS-2 ) (ERKjJ) \ \ P; Glycogen ^ Synthase (less active) RSK3 / / PPi y Glycogen Synthase > (more active) 87 4.4 LIMITATIONS OF THE ZDF AND STZ-WISTAR RAT STUDIES One of the original objectives was to examine the MAP and S6 kinases in the ZDF rat. However, one of the limitations with the ZDF rat study was that the kinases could be only examined in the basal state. Since it is important to examine how kinases respond to insulin stimulation in the normal and diabetic states, as well as following BMOV treatment, it was decided to carry out a second study using Wistar rats. In the Wistar study both basal and insulin stimulated samples for normal, diabetic and BMOV treated rats were obtained. Another issue that deserves mention is the lack of a proper control in the ZDF rat study. In that study, only the effect of BMOV treatment on kinase activity could be examined, as only two groups were present (untreated vs treated). The altered kinase activity present in the ZDF rats could not be compared to any normal value and thus the level of alteration in kinase activity (i.e. fold change in activity) could not be realized. Furthermore, the major concern with in vivo studies is the possible interaction of many unknown factors. A large dose of insulin was injected into the Wistar rats producing hypoglycemia. It is known that physiological mechanisms exist to prevent or correct hypoglycemia (Gerich, 1988,- Cryer and Frier, 1997). Glucose-regulatory factors include hormones, neurotransmitters and metabolic substrates/intermediates (Lebovitz, 1995). Among the hormones, glucagon is the most potent and it is secreted from pancreatic A cells. It stimulates hepatic glucose production and thus tends to raise blood glucose concentrations. The mechanism of action of the adrenomedullary hormone, adrenaline (epinephrine) are more complex (Cryer and Frier, 1997). They involve both stimulation of glucose production and limitation of glucose utilization through direct ((32-adrenoceptor) actions on the liver and muscle, and through 88 indirect actions that include particularly (0C2-adrenoceptor) limitation of insulin secretion, but also ((32-adrenoceptor) stimulation of glucagon secretion and ((3,-,p2-,p3-adrenoceptor) stimulation of lipolysis (Clutter etal., 1988 and Cryer and Frier, 1997). Growth hormone and Cortisol are also secreted in response to falling glucose levels. The hyperglycemic effects of growth hormone and Cortisol are delayed (hours) compared to glucagon and adrenaline (min.). These particular hormones limit glucose utilization, but they also cause glucose production (De Feo etal., 1989A and De Feo et al, 1989B). These counterregulatory hormones could significantly affect the kinase activities that we chose to measure. Previous work from our laboratory has shown that general profiles of kinase activity remained largely the same when control animals where administered insulin under a euglycemic clamp (Hei, 1993). Additionally, the insulin-mediated signalling could also be affected by blood glucose concentrations. Taken together, we acknowledge that the results obtained may have been obscured to some extent by these factors that were not controlled during our studies. 4.5 FUTURE DIRECTION The ZDF rat is the ideal model to study NIDDM, because this model closely parallels the disease in humans. The ZDF rat is obese, hyperinsulinemic and also hyperglycemic. The present study allowed us to examine protein-serine kinase activity in the basal state and following chronic treatment with BMOV. However, it is also necessary to examine how protein kinases respond to insulin stimulation. Once the insulin stimulated activity of various kinases is established in the diabetic state, 89 the affect of BMOV can be assessed. For example, does BMOV increase or decrease kinase activity. The STZ-Wistar rat model allowed the examination of protein-serine kinase activity in an insulin deficient state. In the present study, very high non-physiological insulin concentrations (5 and 10 U/kg) were used to elicit kinase activation. The main reason for using high concentrations of insulin was to ensure kinase activation would be achieved in the diabetic state. However, to get a be t ter understanding of the signalling pathways involved in glucose utilization, pharmacological doses of insulin (1-2 U/kg) need to be used. Furthermore, other models of insulin deficiency need to be used to examine kinase activities. Other models are needed because in the STZ-diabetic rats, the increased kinase activities could be due to streptozotocin's direct effect on the kinases and not secondary to insulin deficiency caused by P-cell destruction. This seems unlikely, since STZ's half-life is 15 min. One potential model is the spontaneously diabetic (BB Wistar) rat. Finally, the activity of upstream kinases need to be examined in the diabetic state and following BMOV treatment. Important upstream kinases that need to be studied are PI3K, PKB and GSK-3. Recent evidence from the literature implicate these kinases in the glucoregulatory effects of insulin. In addition, through the use of established kinase inhibitors such as wortmannin and LY 294002 (inhibit PI3K.) and quercetin (insulin-receptor tyrosine kinase inhibitor), kinase pathways affected by BMOV treatment can be elucidated. 90 4.6 CONCLUSIONS ZDF STUDY Chronic BMOV treatment is able to decrease the basal activities of several serine kinases, p70S6K, ERK-1, ERK-2, and p90rsk, in Zucker Diabetic Fatty rats. 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