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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  degree  this  at the  thesis  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  freely available for copying  of  department publication  this or of  reference and study.  thesis by  this  for  his thesis  scholarly  or for  her  I further  purposes  gain shall  permission.  Department of  fxWi/>r>^c£xott<^fJo  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  <\%-0<i-0H  /4-CAM^X^&  requirements that  agree  may  representatives.  financial  the  be  It not  that  the  Library  permission  granted  is  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  T h e 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.  T h e integrity of t h e  M A P kinase p a t h w a y was evaluated by studying the activation of E R K l , ERK.2 and p90 r s . T h e activity of p70  6  was also evaluated as a possible downstream e n z y m e 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. H i n d limb muscle s u b j e c t e d to specific immunoprecipitation  extracts  experiments  were  prepared  for the kinases  and  mentioned  above. T h e first part of the study examined the effects of B M O V on p r o t e i n - s e r i n e kinases in Z u c k e r Diabetic Fatty (ZDF) rats. p r o v i d e d an ideal model to study  The Zucker  the activity  of protein  Diabetic Fatty kinases  (ZDF)  in noninsulin-  d e p e n d e n t diabetes mellitus (N1DDM), as the d e v e l o p m e n t of the disease in t h e s e rats closely parallels N I D D M in humans.  B M O V treatment for 8 weeks  significantly  (p < 0.05) d e c r e a s e d the basal activity of p70 S 6 K by 30% when c o m p a r e d to the untreated group.  F u t h e r m o r e , following B M O V t r e a t m e n t the basal kinase a c t i v i t y  of ERK-1 and ERK-2 significantly decreased by 4 0 % and 3 5 % , respectively, c o m p a r e d to the u n t r e a t e d group.  p90 rsk is postulated to be downstream of ERK-2  and any changes in ERK-2 could be reflected t r e a t m e n t d e c r e a s e d p90 r s untreated  group.  diabetes  pancreatic (3-cells.  in the activity of p90 r s .  BMOV  activity by approximately 75% when c o m p a r e d to t h e  T h e activity  of protein  S t r e p t o z o t o c i n (STZ) Diabetic Wistar rat. dependent  when  melliuts  kinases was also investigated  This model is r e p r e s e n t a t i v e  (IDDM), because  the chemical,  STZ  in t h e  of insulin-  destroys  the  p70 S 6 K activity was altered basally and following 5 U / k g 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 p90 rsk . 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 p90 rsk . In the diabetic state, ERK-2 activity was markedly active in response to insulin stimulation, whereas p90rsk showed no change. BMOV treatment decreased the activity of ERK-2, whereas p90 rs increased with BMOV treatment.  in  Finally,  activity  was  TABLE OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF FIGURES  viii  LIST OF ABBREVIATIONS  x  ACKNOWLEDGEMENTS  xiii  l.  INTRODUCTION 1.1  DIABETES MELLITUS  l  1.2  CLASSIFICATION OF DIABETES MELLITUS  I  1.3  1.4  1.2.1  Type I: Insulin-Dependent Diabetes Mellitus  2  1.2.2  Type II: Noninsulin-Dependent Diabetes Mellitus  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  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  iv  30  1.6.2 1.7  1.  3.  The Streptozotocin (STZ) Diabetic Wistar Rat  RATIONALE, RESEARCH OBJECTIVE AND HYPOTHESES  31 35  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  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  3.1.2  Basal MBP Phosphotransferase Activity and Effect of BMOV Treatment  47  47  3.1.3  Basal S6-10 Peptide Phosphotransferase Activity and Effect of BMOV Treatment  3.2  6  3.1.4  p70  3.1.5  ERK-1 and ERK-2 Immunoprecipitation Study  52  3.1.6  p90rs Immunoprecipitation Study  54  EFFECTS OF  Immunoprecipitation Study  49  BMOV ON PROTEIN-SERINE KINASES IN STZ-DIABETIC WISTAR  RATS  55  3.2.1  Plasma Glucose Levels in the STZ-Diabetic Wistar Rat  3.2.2  Basal and Insulin Stimulated MBP Phosphotransferase Activity and the Effect of BMOV Treatment  3.2.3  49  55  59  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  T H E ROLE OF P 7 0 S 6 K IN INSULIN A C T I O N  76  4.3  4.2.1  Basal p70S6K Activity and the Effect of BMOV in ZDF Rats  4.2.2  P 70  THE  S6K  Activity and the Effect of BMOV in STZ-Wistar Rats  MAP KINASE (MAPK) PATHWAY  78 79 80  4.3.1  Basal MAPK Pathway Activity and the Effect of BMOV in ZDF Rats  4.3.2  81  MAPK Pathway Activity and the Effect of BMOV in STZWistar 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  6.  APPENDIX  92 107  Vll  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  Figure 3.1  35  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  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  p90 rs immunoprecipitation study in the ZDF rats  58  Figure 3.7  Plasma glucose levels in the Wistar rats  60  Figure 3.8  M o n o Q profiles of MBP phosphotransferase activity in the Wistar  6K  rats Figure 3.9  63  M o n o Q 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  Figure 4.1  The regulation of glycogen synthase activity by the MAPK  study in the STZ-diabetic rats  pathway and RSK-3 in skeletal muscle by insulin in vivo  IX  74  86  LIST OF ABBREVIATIONS  BB  biobreeding  BMOV  bis(maltolato)oxovanadium(IV)  BSA  bovine serum albumin  cAMP  cyclic adenosine 3',5'-monophosphate  DNA  deoxyribonucleic acid  EGF  epidermal growth factor  ERK  extracellular signal-regulated protein kinase  GSK-3  glycogen synthase kinase-3  IDDM  insulin-dependent diabetes mellitus  IgG  immunoglobulin G  IGT  impaired glucose tolerance  IP  immunoprecipitate  IRS  insulin receptor substrate  kDa  kiloDalton  kg  kilogram  M  molar  MAPK  MAP kinase  MBP  myelin basic protein  MEK  MAP kinase kinase microgram microgram microlitre  \iM  micromolar  ml  millilitre  mM  millimolar  NIDDM  noninsulin-dependent diabetes mellitus  NOD  non-obese  p70 S6K  70 kDa ribosomal S6 kinase  P 90  rsk  90 kDa ribosomal S6 kinase  PDGF  platelet derived growth factor  PDKl  3-phosphoinositide-dependent protein kinase  PH  pleckstrin-homology  PI  phosphatidyl inositol  PI3K  phosphoinositide-3-OH kinase  PKB  protein kinase B  PPG-1  protein phosphatase-1  PTK  protein tyrosine kinase  SOS  son-of-sevenless  STZ  streptozotocin  U/kg  units/kilogram  ZDF  Zucker Diabetic Fatty  XI  Single and three letter codes for amino acids:  A  Ala  Alanine  R  Arg  Arginine  N  Asn  Asparagine  D  Asp  Aspartic Acid  C  Cys  Cysteine  Q  Gin  Glutamic Acid  G  Gly  Glycine  H  His  Histidine  I  He  Isoleucine  M  Met  Methionine  F  Phe  Phenylalanine  P  Pro  Proline  S  Ser  Serine  T  Thr  Threonine  W  Trp  Tryptophan  Y  Tyr  Tyrosine  V  Val  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. 1.1  INTRODUCTION  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,  abnormalities of glucose, lipid and protein metabolism (Horton, 1995). causes  blindness,  kidney  failure,  If  neuropathy,  and left  uncontrolled,  diabetes  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  yet  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.  1  (3) The third  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 b y 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 b y multiple  subdiabetogenic  doses  autoimmunity, which contributes provides  of  streptozotocin  to loss of (3-cells.  appears  to  Consequently,  evidence that a primary (3-cell insult can result  elicit  (3-cell  this model  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 N O D 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 T y p e 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. absence of carbohydrate  intolerance, compensatory  hyperinsulinemia  In the  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. has not been completely successful  Current available drug therapy  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,  thiazolidinediones.  the  biguanides,  the  (X-glucosidase  inhibitors  and  However, about 20-40% of the patients on these drugs  primary failures and about 10% of the patients are secondary failures. failures are patients that do not respond to oral hypoglycemics. patients are likely to be unrecognized Type I diabetes patients.  are  Primary  Some of these  In the majority of  primary failures, the precise mechanism for the drug failure is unknown. failure, refers to an initial successful  the  Secondary  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  5  associated  with  insulin  therapy,  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 (V v ), it predominantly exists in an anionic form that resembles phosphate, namely metavanadate ( V 0 3 ) or orthovanadate ( H 2 V 0 4 ) . oxidation state (V ), vanadium exists in a cationic form, vanadyl  In the +4 (V0 2 + ), and  resembles Mg 2+ . 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  T H E 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,  6  two  independent  groups  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 output from perfused liver (Bruck etal,  also inhibits glucose  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 synthase (Tamura  (Shechter and Ron, 1986), and stimulate  et al., 1983) in rat adipocytes.  glycogen  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 N I H 3T3 mouse fibroblasts (Mountjoy and Flier, 1990).  Finally, vanadate mimics the  secondary actions of insulin, such as an increase in calcium influx, inhibition of Ca 2+ /Mg 2+ -ATPases  in plasma membranes,  elevation of cytoplasmic pH (Shechter, 1990).  7  stimulation of potassium  uptake  and  1.3.2  T H E 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 STZdiabetic 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 related to diabetes.  a number of secondary  complications  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  (Domingo et al, 199lA ; Domingo et al, 199lB ; Domingo et al., 1992).  in tissues 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.  11  It was also  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  T H E 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 a 2 B 2 -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 highaffinity insulin binding (Yip, 1992). A chimeric IGF-1 receptor containing the Nterminal residues (1-68) from the insulin receptor resulted in a hybrid receptor that binds insulin with high affinity (Kjeldsen et al., 1991). terminal  have  also been  described  In addition, regions more C-  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  13  on tyrosine  residues  in the  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  •ss-  intracellular 1030 Lya  phosphorylated tyrosines  1162  ^  domain  -SS-  -ss-  beta subunit  Insulin Binding  ~7~  Juxtamembrane ATP Binding Regulat ory  1163  Tyrosine Kinase domain  1326  C-Terminal  1334"  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 r e c e p t o r 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  (pp60 IRS3 ) 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  T H E MAP  The  KINASE PATHWAY  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 p2Inactivity through a noncovalent interaction of its SH3 domain with the recently identified p21 ras 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 asm a  Others? (lRS-l/IRS-2  Glut4  18  )  <S>  m em b r a n e  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 p21 ras 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 p21 ras is found in the GDP bound inactive form. However, after stimulation by insulin, there is a rapid increase in the amount of GTP bound p21 ras . 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 p21 ras 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 p21 ras in the absence  of any detectable  Tyr  phosphorylation of IRS-1 (Cheatham and Kahn, 1995). The downstream target for p21 ras is Raf-1, a 74 kDa serine/threonine kinase. It has been shown that p21 ras , specifically in the GTP bound, form activates  19  Raf-1  (Avruch etal., 1994). Mutations in the effector domain of p21 ras that render it inactive in cells also abolish Raf-1 binding. The binding of Raf-1 directly to p21 ras 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 p21 ras 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 b y  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 p90 rs . 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  T H E 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 r e c e p t o r 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-4,5P 2 to PI3P, PI-3,4P 2 and PI-3,4,5P 3 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. central  PKB contains an amino terminal pleckstrin-homology (PH) domain, a  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-3,4P 2 and PI-3,4,5P 3 ) 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-3,4,5P 3 to the P H domain does not activate its kinase activity in vitro (Klippel et al., 1997), whereas, PI3,4P2 can directly stimulate PKB activity (Franke etal, 1997A). Furthermore, Franke et al. (1997A) also demonstrated that binding of PI-3,4P 2 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 wortmanninsensitive, 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, 3phosphoinositide-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  23  of lipid vesicles  containing PI-3,4P2 or PI-3,4,5P 3 . These results disagree with reports that claim that PKB is activated directly by PI-3,4P 2 .  It is possible that reports  showing PKB  activation directly by PI-3,4P 2 may have been the result of contamination of PKB preparations with trace PDKl activity. Although PKB is not activated by PI-3,4P 2 or PI-3,4,5P 3 , the interaction with these lipids may facilitate the activation of PKB b y 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). GSK3 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 p70 S6K . 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 and its regulation.  6  activation  Rapamycin blocks activation of p70S6K by all known mitogens b y  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  mTOR/FRAP upstream of P 70 S6K .  complex  mTor/FRAP  actually  interacts  with  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 p70 S6K 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 p70 S6K . 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). p70 S6K , overexpression  Whilst PKB has not been shown to directly of a ^^-fusion  (Burgering and Coffer, 1995).  of PKB constitutively  phosphorylate  activates  p70 S6K  It is postulated that PKB is either upstream of, or acts  in parallel to, the rapamycin-sensitive step in the activation of p70  25  6K  . The latter is  likely, as wortmannin and rapamycin block p70  6K  by inhibiting different  inputs  (Chung eta!., 1994).  1.5  T H E 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 cellpermeable  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).  26  There are two major classes of  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 r e c e p t o r 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 w e r e  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 has been used to define the different roles of the two PTKs.  27  staurosporine  It is thought that  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 r e c e p t o r activation and IRS-1 phosphorylation. activate Pl3K ; however,  Vanadate and insulin have been shown to  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 polyacrylamide  gels  in PKB mobility was seen on sodium dodecyl  (Wijkander  et al., 1997). This is indicative  phosphorylation, which correlates with increased kinase activity.  of  sulfateincreased  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, p90 rs and p70S6K in C H O cells overexpressing a normal human insulin r e c e p t o r (Pandey et al,  1995).  Among the three vanadium salts tested, vanadyl  28  sulfate  appeared to be slightly more potent than the others in stimulating MAP kinases and p70 S6K 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  modulation of adenylyl cyclase signalling through G-protein Srivastava et al., 1995).  involved  regulation  in the (Anand-  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  29  correlation  between  animal and human disease.  Animal models will never  characteristics of diabetes identical to those in humans.  reproduce  the  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 crossbreeding 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 (Considine et al., 1996).  body fat  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  hyperlipidemia, similar to those observed in the Zucker rat (Yuen et al,  30  and 1997).  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 Z D F 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 CH3_N—NO Figure 1.4 The structure of a-streptozotocin.  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. growth  Insulinopenia appears to be responsible for the metabolic and  disturbances,  because  hormone replacement,  transplantation rapidly reverses the disease. seen  include  high circulating  levels  of  33  islet cell, or  pancreatic  Some of the endocrine abnormalities glucagon,  somatostatin,  vasopressin,  corticosterone, atrial natriuretic peptide, and reduced levels of renin, angiotensin II, aldosterone, and thyroid hormones T 4 andT 3 (Tomlinson etal., 1992) A wealth of knowledge on the mechanism of STZ diabetogenicity accumulated.  has  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 (CH 3 + ), formed during the decomposition of STZ. The CH 3 + 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. (3cells 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  z  BREAKS  ^ I activation  . poly(ADP-ribose) synthetase  chromatin Streptozotocin (STZ)  I  CH3+ (?)  (ADP-ribose) n 4 *  fc  nucleus  NAD y  ^ ^ ^ ^ ^ 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 B M O V treatment completely normalized the kinase activity. activity was decreased  Likewise, basal S6 kinase  (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. stimulation of both kinases was observed by selenium.  Likewise,  significant  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 b y 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. hypotheses proposed are outlined accordingly.  36  Thus, the research  objective  and  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 STZdiabetic 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  orthovanadate, phenylmethylsulfonyl  fluoride,  acid, sodium  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). anti-Rsk2-PCT antibody was purchased  The  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 antiErk 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 phosphataseconjugated goat anti-rabbit and goat anti-mouse IgGs and horse radish peroxidaseconjugated  goat  anti-mouse  (Mississauga, Canada).  IgG  were  obtained  from  Bio-Rad  Laboratories  Protein A-Sepharose CL-4B and the HR5/5 Mono Q column  38  were purchased from Pharmacia Biotech (Baie d'Urfe, phosphocellulose Canada).  filter paper was purchased  Canada).  from VWR  Whatman P-81  Canlab  (Mississauga,  All other chemicals and reagents were of the highest grade commercially  available.  2.2  EXPERIMENTAL P R O T O C O L  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 B M O V 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 w e r e 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 b y 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 ( D T I ) 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 520 |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  chromatography system was used for all the chromatographic  liquid  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- 3 2 P]ATP (specific activity -2000 cpm/pmol) and assay dilution buffer, pH 7.2 (20 mM MOPS, 25 mM Bglycerophosphate, 20 mM M g C l ^ 5 mM EGTA, 2 mM EDTA, 1 mM DTT, 5 mM Bmethylaspartic 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 mM glycine, 20% methanol (v/v), pH 8.6). hydrated in transfer buffer  (20mM Tris, 120  The nitrocellulose membrane  for at least 1 min before the transfer.  was  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 b y  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-bromo4-chloro-3-indolyl  phosphate  (BCIP)/nitro blue  43  tetrazolium  (NBT)  development  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 MgCl 2 )). 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 ASepharose 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 M g C h , 5 mM EGTA, 0.25 mM DTT, 50 mM sodium fluoride), following which kinase assays were performed.  44  For the  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  appropriate. Results were considered significantly different if p < 0.05.  46  test  as  CHAPTER 3.  3.1  RESULTS  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  phosphotransferase  column  and  the  resultant  fractions  were  activity toward myelin basic protein (MBP).  examined  for  To produce a  column profile that was more representative of each group (untreated and B M O V 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. comparison of the untreated and the BMOV treated column profiles  On  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  P70 S6K 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) MBP phosphotransferase activity (pmol/min per ml)  o o  3 O  o  o  U\  o 3 3  o  o o  0\  o  o  taken to represent 100 percent activation of p70 S6K . 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 antip70 S6K 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 percent  activation  phosphotransferase ERK-1.  and  the  activity.  activity  of  ERK-1  was  assessed  Figure 3.4B shows a representative  The antibody, ERK-l-CT was used  to blot for  by  100 MBP  immunoblot of  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 ZD  -  A  >  ro  00 -  S 2 •K O  *  C  T 1  75 -  c  u  O CL,  a  50 -  vo t/1  25 -  0 B  . m  _..  . 69 kDa p70  -I  ^  ^  .  ^ ^  —_,  ^ -  S6K  •Antibody Band (49 kDa)  c E ra —  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 antip70S6K 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 conducted.  of the ERK-2 immunoprecipitation  experiments  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 treatment decreased kinase activity.  immunoprecipitated.  Once again B M O V  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 B M O V treatment decreases kinase activity. The band shift present in the untreated group is probably due to increased basal phosphorylation of ERK-2. phosphorylated,  they  polyacrylamide gels.  become  heavier  Following BMOV  and  thus  migrate  When proteins slower  treatment the band shift  on  are SDS-  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 p90 rsk . 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 p90 rsk . The antibody anti-RSK2-PCT was used to blot for p90 rsk . As before, the activity of p90rsk was adjusted for based upon the amount of protein present for each group. Therefore, the decrease in p90 rsk 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 w e r e 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 STZdiabetic rats when compared to the control animals. The plasma glucose levels w e r e 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  ERK-1 Immunoprecipitation 125  A 100  > y oj o ra * ; t. c  75  -  *  Ji o  T 1  C •*. 10 o C 4-i  o  c  Q. fcf n  u  50  -  25  -  Q Q_  &  n  - 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  DC1/  p90 °  immunoprecipitation  A &  100  -  > Eg  75 -  g° •&y  50 -  O Qw J=  a *o  25  *- r  -  o-l  B «*«.«»  DIM*  <»••«(§  «•<•(»  «M—»  mmm>  • 94 kDa •p90RSK  h  £j rs o 00  3  Pi E  i E cra '  *w& ^m  Untreated  aa .-^^ ML,  -•-  - Antibody Band (49 kDa)  BMOV treated  Figure 3.6 p90 rs 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-p90 rsk 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 B M O V  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  59  the  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 subdivided 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 b y 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 w e r e 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) 01  O  _l_  _l_  Plasma Glucose (mmol/l)  o  _l_  _L_  +  as  o  c > era  13  +  ON  H*  kfej—i OQ  W  + O  CfQ  ^  ^!  o i*  ^ H o  o  n  n  muscle extracts and the fractions were analyzed for phosphotransferase towards myelin basic protein  (MBP).  To produce  a kinase profile  activity 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  Diabetic Group 40- insulin + 5 U/Kg insulin 30-  0 | • •• • | i • i • | ii i i | i i i i | n 0  10  20  30  40  •• | n 50  i i | • •• • ! 60  0  70  10  20  30  40  50  60  70  Mono Q fraction i  Mono Q traction no.  Control Treated Group  Diabetic Treated Group •2 40 -0 ••#• 3  • insulin + 5 U/Kg insulin  30-  III 20-  10  i iii  0  111 i i  10  111 i i  20  11 i i i  30  1 1 1 1 1 1 i*0i 11 i i i  40  50  60  i  0 •  70  Mono Q fraction no.  •^•^•^•^•^r^^^^^^^^^^^^^^^^  10  20  30  ^ ^ ^ ^ ^ ^ H ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^  40  Mono Q fraction no.  Figure 3.8 M o n o Q 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  50  60  70  Control Group A  t  Diabetic Group • insulin + 5 U/Kg insulin  200-  100-  II  c S ^ l 1L  I  ryOOOQO i 11 i  020  10  30  40  50  60  70  20  Mono Q fraction no.  — O — t  70  40  Mono Q fraction no.  Control Treated Group B  30  Diabetic Treated Group  -insulin + 5 U/Kg insulin  O •  • insulin = 5 U/Kg insulin  40  50  n  200-  100-  1 v" X «b  1  lv  o0  10  20  30  40  50  60  70  10  20  30  Mono Q fraction no.  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  P70 S6K IMMUNOPRECIPITATION STUDY  Figure  3.10A shows the results  conducted to assess p70  6  of the immunoprecipitation  experiments  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 p70 S6lc 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. maximum stimulation was achieved with 5 U/kg, the activity of p70  6  Because  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. diabetic group, p70  6K  With the  was increased in the basal state, although this increase in  activity was not statistically significant. the basal control group.  Its activity was about 3-4 folds higher than  Insulin stimulation did not activate p70S6K any further.  BMOV treatment did not seem to correct this defective activity of p70 diabetic state in the basal or insulin stimulated states.  Therefore,  6  the  in the major  conclusion that can be drawn about p70S6K activity is that in the diabetic state, p70 S6K activity is altered and BMOV treatment is unable to restore p70S6K activity to normal levels.  65  p 7 0 S6K Immunoprecipitation 1000  >  >JJ C IS  750  O u O u  _  T  1 500  -  J= c O  T  U  T  a Jj a  250  T  1 rr  -  _ 69 kDa 'P70^6K •Antibody Band (49 kDa)  Control  Diabetic  Control Treated  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-p70 S6K 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 S6KN T antibody.  Following quantitative analysis of the immunoblots, the activity of  p70 S6K 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 p70 S6K , 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 ERK1 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 B M O V  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  ERK-1 Immunoprecipitation 500  u o U  I  400  >  1  o  O  2i  300  JJ o C vi_ ra O is *••  T  o c  200  1*  JL  1  T  1 T  100  -Antibody Band (49 kDa) .44.5 kDa •ERK)  * 8  <*  UJ  3 £  s E so  c — ra  00  DC  ra  ra  Control  Control Treated  OB  j2  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  experiments. kinase.  3.12A  shows  the  results  of  the  As before, basal control represented  ERK-2  immunoprecipitation  100 percent activation of the  Following 5 U/kg insulin stimulation, there was approximately  stimulation of ERK-2 activity. above 5 U/kg were seen.  Following 10 U/kg insulin, no further  2 fold  stimulation  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  69  to the  control  group.  Its  MBP  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 p90 rs 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 p90 rs 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. difference between the basal and insulin stimulated groups. no difference  was seen between  compared to the control group.  There was no  In the diabetic animals,  the basal and insulin stimulated groups  when  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 p90 rsk . After BMOV treatment, p90 rsk activity increased by 2-2.5 fold above control. Therefore, the major conclusion that can be drawn from the p90 rs 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 p90 rs 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 p90 rsk . 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  experiments  are the (l) beads only and (2) antibody only (data not  immunoprecipitates. non-specifically antibody  conducted  in parallel  with  the  phosphotransferase  shown)  The beads only represents the phosphotransferase activity of  bound proteins to the protein A-Sepharose  being  immunoprecipitation  present.  Whereas,  the  antibody  beads without  only  represents  activity of the antibody without the presence  the the  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 600  T 500  1  u v O i/>  400  I-  2 c  ju o c «ra O o c n  300  u  O Q_  * *  200  a.  T  T  Q_  J. X  100  o ^ h  J.  us  B  • Antibody Band (49 kDa) •44.5kDa  * g  ; ; ; ;;  UJ g B E c — "ra ra  3C  bo  3  3  to  o  + Control  +  3 ra  5c  ra  • ERK-2  «M»  5£  "ra ra  3  3  >n +  (n +  in  Diabetic  Diabetic Treated  Control Treated  3 +  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  p90  Immunoprecipitation  400  # > u ra  T  ^ "o U u ui  re  *-•  c  300  -  200  -  100  -  1  Js s° IO  -w OJ  JS y  T 1  o o(X  T  -94 kDa •p90RSK  o  + Control  Ifl  a  SO  u  m  3 m +  •1-  Contro Treatec  3  U/Kg  3  LO  ra  basal  SB  U/Kg  basal  •Antibody Band (49 kDa)  ITl  m  t  +  Diabetic  Diabetic Treated  Figure 3.13 p90rsk immunoprecipitation study in the STZ-diabetic rats. (A) The effect of BMOV on basal and insulin stimulated p90 rsk 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-p90 rsk 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 ttest). The error bar for the group "basal control" was too small and did not appear on the graph  74  CHAPTER 4. 4.1  DISCUSSION  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 p90 rsk . 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  phosphotransferase activity appears in peak II and III.  of the MBP  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 m T O R 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 p70 S6K .  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 p70 S6K 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  phosphorylate and inactivate GSK-3 in vitro (Sutherland et al., 1993).  shown  to  However,  because p70 S6K 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 p70 S6K . If BMOV decreased the activity of an upstream regulator of p70 S6K , 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 BMOV treatment. p70  6  by  Thus, if BMOV treatment can decrease PDKl activity, then  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 activity.  p70 S6K on specific  residues  to decrease  its  phosphotransferase  Downregulation of activity has also been seen with other proteins.  For  example, serine and threonine phosphorylation of IRS-1 may downregulate IRSdependent 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  deoxyglucose uptake decreased.  phosphorylation,  while  PI3K  activation  This suggests that hyper-phosphorylated  and 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 p70 S6K 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  (Chang et al, 1995; M o d e et al, 1995,- Cross et al, 1995).  in glucose utilization 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. role of the MAPK pathway in glycogen synthesis is questionable.  80  The  It has been  proposed that p90rsk (downstream target of MAPK) phosphorylates the glycogenbinding 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. p90 rsk is postulated to be downstream of ERK-2 and any changes in ERK-2 could be  81  reflected in the activity of p90 rsk . BMOV treatment decreased p90rsk activity b y approximately 75% when compared to the untreated group. A role for the MAPK pathway in the activation of glycogen synthesis b y insulin has been excluded by several findings, as stated above. Briefly, p90 rsk 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 B M O V  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 p90 rsk , 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 p90 rs  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. insulin  was  sufficient  to achieve  This suggests that 5 U/kg  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. p90 rs , however, was surprisingly  not hyper-responsive  to insulin stimulation,- its  activity was very similar to control. The activity of p90rsk was expected to be hyperresponsive 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. treatment the activity of ERK-2 was completely However, stimulation.  BMOV  had no effect  normalized to control levels.  on ERK-1 hyper-responsiveness  Therefore, BMOV appears to have differential  isoforms of ERK.  Following B M O V  effects  to  insulin  on the two  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 B M O V  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 p90 rsk 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 p90 rsk . 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 p90 rsk . 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 p90 rsk . Secondly, ERK-2 activity was hyper-responsive to insulin stimulation in the diabetic state, whereas p90rsk showed no change.  Lastly, B M O V  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 p90 rsk , 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 b y 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 p90 rs , 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  Plasma Membrane  tt (lRS-l/IRS-2 )  (ERKjJ)  RSK3 /  \ \  y  /  PPi P; Glycogen ^ Synthase (less active)  Glycogen Synthase > (more active)  87  4.4  LIMITATIONS OF THE ZDF  AND S T Z - W I S T A R 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 Z D F 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 p r e v e n t 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 ((3 2 -adrenoceptor) actions on the liver and muscle, and through  88  indirect actions that include particularly  (0C2-adrenoceptor)  limitation of insulin  secretion, but also ((3 2 -adrenoceptor) stimulation of glucagon secretion and ((3,-,p 2 -,p 3 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  activities that we chose to measure. that  affect  the  kinase  Previous work from our laboratory has shown  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. hyperglycemic.  The ZDF rat is obese, hyperinsulinemic and also  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 b e t t e r 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 kinases and not secondary to insulin deficiency caused by P-cell destruction. seems unlikely,  since STZ's half-life  is 15 min.  on the This  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 b y 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, p70 S6K , ERK-1, ERK-2, and p90rsk, in Zucker Diabetic Fatty rats.  STZ-DlABETIC WlSTAR STUDY  1) p70 S6K activity is altered basally and following insulin stimulation in STZ-diabetic rats and BMOV treatment is unable to restore the activity to control. 2) In the diabetic state ERK-1 and ERK-2 are markedly activated following stimulation with 5 U/kg insulin when compared to control. 3) 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. 4) There appears to be a dissociation between the ERK-2 and p90 rs . i) 5 U/kg was required to activate ERK-2 to its maximal level, whereas 10 U/kg was required to achieve maximal activity of p90 rsk . ii) In the diabetic state, ERK-2 activity was markedly active in response to insulin stimulation, whereas p90rsk showed no change. iii) BMOV treatment decreased the activity of ERK-2, whereas p90rsk activity was increased with BMOV treatment  91  CHAPTER 5. REFERENCES Agarwal, M.K. (1980) Streptozotocin: Mechanisms of Action. 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APPENDIX  Sample Calculation: ZDF Study p70S6K  raw CPMs  Untreatd BMOV treated 5996.00 4441.00 4809.00 2634.00 6266.00 4043.00  AVG  5690.33  3706.00  Normalized Data  5696.20 5674.62 5702.06  4218.95 3108.12 3679.13  Corection factor (Densitometry)  1.00 1.00 1.00  1.23 0.94 0.95  Activity based upon protein levels  5696.20 5674.62 5702.06  5201.97 3303.00 3506.21  AVG SEM  5690.96 8.34  4003.73 601.99  100.00 0.15  70.35 10.58  % Control % SEM  107  

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