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In vivo effects of vanadium on glucose transporter translocation in cardiac tissue Li, Shi Hong 2000

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IN VIVO E F F E C T S OF V A N A D I U M O N G L U C O S E T R A N S P O R T E R T R A N S L O C A T I O N I N C A R D I A C TISSUE By Shu Hong L i B.Sc , The JianXi Medical School, P.R. China, 1985 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E I N T H E F A C U L T Y OF G R A D U A T E STUDIES Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF BRITISH C O L U M B I A May 2000 ©Shu Hong L i , 2000 In p resen t ing this thesis in partial fu l f i lment of the requ i rements fo r an advanced deg ree at the Univers i ty of British C o l u m b i a , I agree that the Library shall make it f reely available fo r re ference and study. I further agree that pe rm i s s i on for extens ive c o p y i n g of this thesis f o r scholar ly p u r p o s e s may b e granted b y the h e a d of my depa r tmen t or by his o r her representat ives . It is u n d e r s t o o d that c o p y i n g or pub l i ca t i on of this thesis for f inancial gain shal l no t be a l l o w e d w i t hou t my wr i t ten pe rm iss ion . Depa r tmen t of The Univers i ty of British C o l u m b i a Vancouve r , C a n a d a DE-6 (2/88) ABSTRACT The effects of vanadium treatment on insulin-stimulated glucose transporter type 4 (GLUT4) translocation were studied in cardiac tissue of streptozotocin (STZ)-induced diabetic Wistar rats by determining the subcellular distribution of GLUT4. Four groups of rats were examined: control and diabetic, with or without bis(maltolato)oxovanadium(IV) (BMOV, an organic form of vanadium) treatment for 8 weeks. Plasma membrane and intracellular membrane fractions were purified from heart ventricles isolated from rats either in the basal state or injected with insulin. GLUT4 content in the membrane fractions was quantified with a competitive enzyme-linked immunosorbant assay (ELISA) and also with enhanced chemiluminescence (ECL) Western blot. A time-course study was first conducted in control rats to determine the points at which insulin stimulated an initial GLUT4 translocation and a maximal GLUT4 translocation. It was shown that after insulin injection plasma membrane GLUT4 level increased about 60% at 15 minutes and intracellular GLUT4 decreased about 40% at 5 minutes and remained at this level throughout the remaining 25 minutes. We therefore decided to study the effects of vanadium on insulin-induced GLUT4 translocation at 5 minutes as the initial insulin response and at 15 minutes after insulin injection as the maximal insulin response. Towards this goal, 50% of animals in each group at the time of termination received an intravenous (iv) dose of insulin (5 U/kg) and hearts were removed 5 minutes later. In another experiment using a second set of animals divided into groups as stated above, the same dose of insulin was administrated and the hearts were taken at 15 minutes after insulin injection. ii At 5 minutes after insulin injection, plasma membrane GLUT4 levels in the control group under basal and insulin-stimulated states were not significantly different from their respective control-treated groups. However, insulin stimulation caused a significant increase in plasma membrane GLUT4 level in the control-vanadium-treated group (-30%) but not in the control group (-12%). Basal plasma membrane GLUT4 level was significantly lower in the diabetic group (50%) when compared to the control and control-vanadium-treated groups. No significant difference in basal plasma membrane GLUT4 was detected between the diabetic and the diabetic-vanadium-treated groups. There was a significant increase in plasma membrane GLUT4 after insulin stimulation in these two groups. The magnitude of insulin-induced GLUT4 translocation was about 2.1-fold in the diabetic and 3.6-fold in the diabetic-vanadium-treated groups. On the one hand, plasma membrane GLUT4 levels following insulin administration in the diabetic group was still significantly lower than that of the corresponding control groups. On the other hand, plasma membrane GLUT4 level after insulin injection in the diabetic-vanadium-treated group was not different from the control groups and was significantly higher than that of the insulin-stimulated diabetic group, indicating an enhancement of insulin response on GLUT4 translocation brought about by vanadium treatment. At 15 minutes after insulin injection, all the groups had a significant increase in plasma membrane GLUT4 when compared to their corresponding non-insulin treated groups. The magnitude of insulin-mediated GLUT4 translocation was about 60% in the control and control-vanadium-treated groups, which was consistent with that from the time course study whereas the degree of insulin-induced GLUT4 mobilization was about 2.8-fold in the diabetic and 2.4-fold in the diabetic-vanadium-treated groups. Consistent with what we found at the 5-minute time point, basal plasma membrane GLUT4 levels were significantly lower in the diabetic and diabetic-vanadium-treated groups (50%) when compared to the control groups. In contrast to that at 5 i i i minutes after insulin injection, no significant difference in the plasma membrane GLUT4 level was observed between the diabetic and the diabetic-vanadium-treated groups at this time point. GLUT4 mobilization from the intracellular pool in response to insulin, as indicated by a decrease in GLUT4 level from the basal level, was also investigated at 15 minutes after insulin injection. A l l the groups had a significant decrease in intracellular membrane GLUT4 when compared to their corresponding non-insulin treated groups. Intracellular GLUT4 dropped by 44-57% after insulin stimulation in the control and control-vanadium-treated groups, which was again consistent with that from the time course study. In the diabetic and diabetic-vanadium-treated groups, insulin stimulation resulted in 60% and 45% decrease, respectively, in intracellular GLUT4 pool. Intracellular GLUT4 content under basal and insulin-stimulated conditions was significantly higher in the diabetic-vanadium-treated group when compared to the diabetic group under the same condition. Both basal and insulin-stimulated levels of intracellular GLUT4 in the diabetic-vanadium-treated group were comparable to their corresponding control groups. Taken together, these results show that firstly, insulin stimulation significantly increased plasma membrane GLUT4 content which correlated to the decrease in the intracellular GLUT4 pool at 15 minutes post-insulin injection in the control and control-vanadium-treated groups. Secondly, long-term insulin deficiency (9-week STZ diabetes) resulted in a decrease of total cellular GLUT4 content, but hearts from long term STZ-diabetic animals were still responsive to supraphysiological dose of insulin in terms of GLUT4 translocation. Thirdly, the observation that basal intracellular GLUT4 level was significantly higher in the diabetic-vanadium-treated group when compared to that of the diabetic group indicated that vanadium treatment may restore total cellular GLUT4 content in the diabetic group. However, increased basal intracellular GLUT4 in iv the diabetic-vanadium-treated group did not result in more insulin-mediated GLUT4 translocation at 15 minutes after insulin injection. Vanadium treatment did, however, enhance the insulin response in terms of GLUT4 translocation at 5 minutes in the cardiac tissue of diabetic rats. Finally, the finding that plasma membrane GLUT4 in the diabetic-treated group was significantly higher than that of the diabetic group at 5 minutes but not at 15 minutes post-insulin injection indicated that vanadium treatment enhances insulin-mediated GLUT4 translocation by enhancing its early response and by accelerating this process. This was further supported by data showing that insulin stimulation significantly increases plasma membrane GLUT4 in the control-vanadium-treated but not in the control group at 5 minutes after insulin administration. We also verified the ELISA method, which was developed in our laboratory. The data from the ELISA assay showed a pattern similar to that from the E C L Western blot, indicating that the ELISA assay was reliable. T A B L E OF C O N T E N T S A B S T R A C T ii T A B L E OF CONTENTS vi LIST OF T A B L E S viii LIST OF FIGURES ix LIST OF ABBREVIATIONS x_ A D C K N O W L E D G M E N T S xiii. DEDICATION xiv INTRODUCTION 1 I) Overview of Facilitative Glucose transporters: Isoform Distribution and Physiological Function: 1 II) Signaling Mechanisms that Regulate GLUT4 Translocation 3 III) Molecular Basis of Insulin-Stimulated GLUT4 Vesicle Trafficking 8 IV) GLUT4 and Insulin Resistance: Insights from Animals Models and Humans 13 V) Diabetic Cardiomyopathy 18 VI) Vanadium, an Insulin Mimetic/Insulin-Enhancing Agent 19 VII) Experimental Rationale and Objectives 20 MATERIALS A N D METHODS 23 I) Chemicals and Materials 23 II) Treatment Protocol 24 III) Blood Sample Analyses 26 IV) Subcellular Fractionation of Frozen Heart Ventricles 26 V) 5'Nucleotidase Marker Enzyme Assay 29 VI) ELISA 30 VII) E C L Western Blot 32 VIII) Statistical Analyses 33 RESULTS 34 I) General Features of STZ-Diabetic and Diabetic-Treated with Vanadium Rats 34 II) Plasma Parameters at Termination 41 III) 5'Nucleotidase Activity in Subcellular Fractions 44 IV) GLUT4 Levels in Various Fractions from the Fractionation Procedure 44 V) Temporal Effects of Insulin on Cardiac GLUT4 Mobilization 46 VI) The Effects of Vanadium on Insulin-Mediated GLUT4 Translocation in the Control and Diabetic Rats at 5 Minutes after Insulin Administration (Early Insulin Response) 48 VII) The Effects of Vanadium on Insulin-Regulated GLUT4 Translocation in the Control and Diabetic Rats at 15 Minutes after Insulin vi Injection (Maximal Insulin Response) 51 VIII) Results from the ELISA Assay 54 DISCUSSION 59 I) Evaluation of the Subcellular Fractionation Method 59 II) Insulin Resistance in the Long-Term STZ-Diabetic Rats 60 III) Lower Expression of Cardiac GLUT4 in the STZ-Diabetic Rat and the Restoration of Cardiac GLUT4 Level by Vanadium Treatment 61 IV) Cardiac GLUT4 Translocation in the Control and Diabetic Rats 64 V) The Effects of Vanadium on Insulin-Regulated GLUT4 Translocation in the Control and Diabetic Animals 68 VI) Evaluation of the ELISA Assay 70 FUTURE EXPERIMENTS 72 I) The Effects of Vanadium in vivo on GLUT4 Translocation in Skeletal Muscle 72 II) The Effects of Vanadium on PI 3-Kinase Activity and Expression in Insulin Sensitive Tissue 73 III) The Effects of Vanadium in vivo on GLUT4 Gene Expression in the Heart 74 IV) The Effects of Vanadium on Cardiac GLUT1 Expression, Functional Activity and Translocation 74 CONCLUSIONS 76 REFERENCES 78 LIST OF TABLES Table Pages I) Regulators and signaling pathways reported to stimulate glucose transport 7 II) Tissue-specific expression of GLUT4 15 III) Plasma parameters at termination following 8 weeks of B M O V treatment at 5 minutes after insulin injection 42 IV) Plasma parameters at termination following 8 weeks of B M O V treatment at 15 minutes after insulin injection 43 V) Na + /K + ATPase and 5'nucleotidase activity in subcellular fractions 45 viii LIST OF FIGURES Figures Pages 1. The overall experimental design of the present study 25 2. Subcellular fractionation of frozen heart ventricles 28 3. Body weight following B M O V treatment for 8 weeks 35 4. Food intake following B M O V treatment for 8 weeks 36 5. Fluid intake following B M O V treatment for 8 weeks 37 6. Plasma glucose levels following B M O V treatment for 8 weeks 38 7. Plasma insulin levels following B M O V treatment for 8 weeks 39 8. Plasma triglyceride levels following B M O V treatment for 8 weeks 40 9. GLUT4 content following the fractionation procedure in a control Wistar rat heart 47 10. GLUT4 content following the fractionation procedure in a control plus insulin stimulated Wistar rat heart 47 11. Plasma membrane GLUT4 levels at 0, 5, 15 and 30 minutes after insulin injection 49 12. Intracellular membrane GLUT4 levels at 0, 5, 15 and 30 minutes after insulin injection 50 13. Plasma membrane GLUT4 levels at 5 minutes after insulin injection 52 14. Plasma membrane GLUT4 levels at 15 minutes after insulin injection 53 15. Intracellular membrane GLUT4 levels at 15 minutes after insulin injection 55 16. Plasma membrane GLUT4 content at 0, 5, 15 and 30 minutes after insulin injection determined by the ELISA assay 56 17. Intracellular membrane GLUT4 content at 0, 5, 15 and 30 minutes after insulin injection determined by the ELISA assay 57 18. Plasma membrane GLUT4 levels at 5 minutes after insulin injection determined by the ELISA assay 58 ix LIST OF ABBREVIATIONS A M P adenosine monophosphate A M P K 5'-AMP-activated protein kinase A N O V A analysis of variance AOPCP a, pmethyleneadenosine 5'-diphosphate ARF ADP ribosylation exchange factor B A T brown adipose tissue Bis N , N'-methylene-bis-acrylamide B M O V bis(maltolato)oxovanadium (IV) BSA bovine serum albumin C control CV control-treated with vanadium D diabetic DV diabetic-treated with vanadium ECL enhanced chemiluminescence EDL extensor digitorum longus muscle ELISA enzyme-linked immunosorbant assay FFA free fatty acid G L M - A N O V A general linear model of A N O V A GLUT glucose transporter GRP-1 general receptor for phosphoinositides-1 HbAlc hemoglobin A l e IGF insulin-like growth factor ip intraperitoneal IRAP insulin-responsive aminopeptidase IRS insulin receptor substrate iv intravenous KBS Krebs buffered saline 5 'ND 5'nucleotidase PBS phosphate buffered saline PDGF platelet-derived growth factor PDK1 3 -phosphoinositide-dependent protein kinase-1 PDK2 3-phosphoinositide-dependent protein kinase-2 PH pleckstrin homology PI 3-kinase phosphatidylinositol 3-kinase P K A cAMP-dependent protein kinase PKB protein kinase B PKC protein kinase C PMSF phenylmethylsulfonylfluoride Ptdlns(3, 4)-P2 phosphatidylinositol 3, 4-bisphosphate Ptdlns(3, 4, 5)-P3 phosphatidylinositol 3, 4, 5-trisphosphate PtdIns-3-P phosphatidylinositol 3-monophosphate R A C related to A and C kinase SDS sodium dodecyl sulfate SH s rc homology SNARE soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor SR sarcoplasmic reticulum STZ streptozotocin TEMED N,N,N',N'-tetra-methyl-ethylenediamine TGN trans Golgi network Tris tris(hydroxymethyl)aminomethane T-tubules transverse tubules T-V elements tubulo-vesicular elements V A M P 2 vesicle associated membrane protein 2 X l l ACKNOWLEDGMENTS I would like to express my greatest appreciation to Dr. John McNeill for accepting me as his student at first place, and for his patience, constant encouragement and instructive advice during the course of the study. I would also like to thank my supervisory committee members, Dr. Brian Rodrigues, Dr. Steven Peleck, Dr. Ray Pederson and Dr. Roger Brownsey for their constructive criticism. Special thanks to Dr. Don Lyster for his skillful chairing of the committee meetings. Great • appreciation to Ms. Maggie L i for leading me into the research field and teaching me most of the techniques required for this study. I would also like to thank Ms. Mary Battell, our lab manager, for her help. Special thanks to Ms. Violet Yuen for excellent technical assistance and training. Special appreciation to Ms. Sylvia Chan for her excellent secretarial assistance and to Ms. Becky Dinesen and Ms. Erica Vera for helping with the treatment of the animals and for assistance with the insulin and triglyceride assays. Great appreciation to all other McNeillians for constant encouragement and for making my study an unforgettable experience. x i i i DEDICATION I would like to dedicate this work to my loving parents who have been a guiding light throughout my life. I would also like to dedicate this work to my daughter who is the most important person in my life. I would also like to dedicate this work to my husband for his fully support, valuable advice and discussion. It is impossible to accomplish this work without his help and support. xiv INTRODUCTION I) Overview of facilitative glucose transporters: isoform distribution and physiological function In mammalian cells, the transport of glucose across the cell membrane by facilitated diffusion is mediated by a family of glucose transporters. The mammalian glucose transporters belong to a superfamily of transport proteins including bacterial sugar-proton symporters, bacterial transporters of carboxylic acids, and yeast sugar transporters (Muckler et al., 1994). Molecular cloning studies over the last decade have led to the identification of seven genes for glucose transporters, which are named GLUT1 through GLUT7 in the order in which they were cloned (Bell et al., 1990; Muckler, 1990; Waddell et al., 1992). To date only four members of this gene family have been described and documented to function as authentic glucose transporters. For example, although GLUT5 was originally thought to be a glucose transporter, it was subsequently identified as a facilitative fructose transporter (Shepherd et al., 1992). GLUT5 is predominantly located on the luminal surface of the small intestine. This protein appears to be localized exclusively to the apical brush border on the luminal side of the epithelial cells (Shepherd et al., 1992). Thus, the primary role of GLUT5 would be the uptake of dietary fructose (Froehner et al., 1988). In addition, the GLUT5 protein has been identified in a range of tissues, including muscle and adipose tissue, which exhibit acute insulin-stimulated glucose transport. It appears that, unlike GLUT4, this transporter does not undergo insulin-stimulated translocation in adipocytes, consistent with an apparent lack of insulin-stimulated fructose transport in human adipocytes (Shepherd et al., 1992). GLUT6 is a pseudogene that is not expressed at the protein level (Froehner et al., 1988). GLUT7, the latest member of the glucose transporter family, is responsible for the transport of glucose produced from glucose-phosphate during gluconeogenesis and glycogenolysis across the endoplasmic reticulum of hepatocytes to be released into the circulation (Davidson et al., 1992). Of the four established glucose transporter isoforms, GLUT1, the best-studied glucose transporter, is present in many tissues and cells. It is expressed at the highest levels in brain but it is also enriched in the cells of the blood-tissue barriers such as the blood-brain/nerve barrier, the placenta and the retina (Gould et al., 1994). Although GLUT1 undergoes insulin-dependent translocation in adipocytes and myocytes, the magnitude of this response is small because a large fraction of GLUT1 is constitutively targeted to the cell surface, even in the absence of insulin and is responsible for transporting glucose in the basal state and across blood-tissue barriers (Shane et al., 1997; Merrall et al., 1993). Also, it appears that a variety of stimuli, including viral infection, hypoglycemia, stress, and numerous growth factors, can trigger GLUT1 but not GLUT4 translocation (Sviderskaya et al., 1996; Thoren et al., 1990). GLUT2 is expressed at the highest levels in the liver, pancreatic (3-cell, and on the basolateral surface of kidney and small-intenstine epithelia (Orci et al., 1989; Johnson et al., 1990). GLUT2 has a high capacity for glucose transport and is proposed to be involved in the net release of glucose during fasting (liver), glucose sensing (|3-cells) and transepithelial transport of glucose (kidney and small intestine). It appears that high GLUT3 protein expression levels are confined generally to the tissues which exhibit a high glucose demand (brain, nerve). Therefore, this isoform may be specialized to act in tandem with GLUT1 to meet the high energy demands of such tissues (Froehner et al., 1988). It is well established that the major glucose transporter expressed at the blood-nerve and blood-brain barrier is GLUT1 which has a higher equilibrium-exchange KD than GLUT3. However, under conditions of. either high glucose demand or hypoglycemia, the expression of GLUT3 in the brain with a low KD for hexoses may be required to successfully utilize low concentration of blood glucose (Froehner et al., 1988). GLUT4, which plays an important role in whole body glucose homeostasis, is mainly expressed in insulin sensitive tissues including skeletal and cardiac muscle and adipose tissue. Found almost 2 exclusively in intracellular storage vesicles in the absence of insulin, the GLUT4 isoform is translocated to the plasma membrane upon insulin stimulation, resulting in a substantial increase in glucose transport (Suzuki et al., 1980; Cushman et al., 1980). Glucose transport appears to be rate-limiting for glucose utilization at low physiological glucose and insulin levels and in diabetes (Ziel et al., 1988). The rate and extent of insulin-mediated glucose uptake is critically dependent on cellular GLUT4 levels and their ability to translocate to the cell surface. II) Signaling mechanisms that regulate GLUT4 translocation Insulin, the major hormonal regulator of glucose transport in humans, has served as a prototypic molecule for understanding cell signaling pathways. However, our understanding of how insulin and the related proteins, insulin-like growth factors I and II, stimulate glucose transport (via the translocation of GLUT4 from intracellular to plasma membranes) is fragmentary. It appears that insulin or insulin-like growth factors activate glucose transport via the activation of receptor-tyrosine kinases, phosphorylation of insulin receptor substrate (IRS.) proteins and activation of phosphatidylinositol 3-kinase (PI 3-kinase) (Zierath., 1995). PI 3-kinase, a dual protein and lipid kinase is a heterodimeric enzyme composed of a 110-kDa catalytic subunit (pllO) associated with an 85-kDa regulatory subunit (p85). Two isoforms of the catalytic subunit (pi 10a and pllOp) and several isoforms of the regulatory subunit (p55a, p55PIK, P85a, and p85P) have been cloned so far. The regulatory subunit contains several well known functional domains: one Src homology 3 (SH3) domain, homology to the breakpoint cluster region(bcr) gene, two proline-rich motifs, and two Src homology region 2 (SH2) domains (Dhand et al., 1994). Following insulin stimulation, the phosphorylated Y X X M motifs in IRSs binds to the SH2 domains of p85 activating the lipid kinase activity of the pllO subunit. The activation of PI 3-kinase leads to the phosphorylation of phosphatidylinositol, 3 phosphatidylinositol-4-monophosphate, and phosphatidylinositol-4,5-bisphosphate on the D-3 position of the inositol ring producing phosphatidylinositol 3-monophosphate [PtdIns-3-P], phosphatidylinositol 3,4-bisphosphate [Ptdlns(3, 4)-P2], and phosphatidylinositol 3,4,5-trisphosphate [Ptdlns(3, 4, 5)-P3], respectively. Insulin causes an acute increase in Ptdlns(3, 4, 5)-P3 and the metabolic effects of insulin are primarily activated by PI 3-kinase-dependent steps (Kaburagi et al., 1997; Krook et al., 1996). Several lines of evidence have indicated that PI 3-kinase activation is essential for insulin-stimulated GLUT4 translocation. First, PI 3-kinase inhibitors (e.g. wortmannin, LY294002) have been shown to block insulin-stimulated glucose transport and GLUT4 translocation in rat and 3T3-L1 adipocytes (Okada et al., 1994; Yeh et al., 1995). Second, inhibition of endogenous PI 3-kinase by microinjection of glutathione S-transferase-p85a subunit fusion protein (Haruta et al., 1995) or a dominant negative mutant of the p85a regulatory subunit of PI 3-kinase (Kotani et al., 1995; Katagiri et al., 1997) also inhibits GLUT4 translocation induced by insulin in 3T3-L1 adipocytes. Third, the over-expression of wild-type or constitutively active pi 10a induces the translocation of GLUT4 to the plasma membrane in 3T3-L1 cells, regardless of insulin stimulation, indicating that PI 3-kinase is also sufficient for glucose transporter translocation (Katagiri et al., 1996; Martin et al., 1996). Although a key role for PI 3-kinase in GLUT4 regulation by insulin seems almost certain, it is instructive to review the limitations related to the current supporting data. First, the inhibitors wortmannin and LY294002 are not fully specific for PI 3-kinase activity. The use of the truncated p85 subunit containing SH2 domains as a dominant inhibitory reagent to inhibit native p85/pl 10 PI 3-kinase function also has the potential limitation of inadequate specificity. When expressed at high concentrations, SH2 domains can be promiscuous in binding phosphotyrosines within various amino acid sequences and thus may not be restricted to binding sites specific for endogenous p85/pl 10-type PI 3-kinase. 4 Recent results call into question the hypothesis that PI 3-kinase activation by insulin is sufficient for GLUT4 translocation. For example, it is well estabished that recruitment of PI 3-kinase to phosphotyrosine on the platelet-derived growth factor (PDGF) receptor in response to PDGF, which occurs in 3T3-L1 adipocytes to the same extent as recruitment of PI 3-kinase to protein phosphotyrosines in response to insulin, has virtually no effect on GLUT4 translocation (Baldini et al., 1991). Recruitment of PI 3-kinase to IRS proteins in response to interleukin-4 (Elmendorf et al., 1998) or cell surface integrin cross-linking (Yel et al., 1995) also fails to enhance glucose transport in the absence or presence of submaximal concentrations of insulin. Conversely, severe inhibition of insulin-mediated IRS protein tyrosine phosphorylation and recruitment of PI 3-kinase in response to incubation of 3T3-L1 adipocytes with PDGF failed to diminish glucose transport stimulation by insulin. The lack of correlation between PI 3-kinase activation and GLUT4 translocation in the studies cited above may reflect an additional insulin-specific signaling pathway or pathways required to operate in conjunction with PI 3-kinase activation to effect this biological response. Recent surprising results support this hypothesis. In these experiments, a cell-permeable analog of the PI 3-kinase product Ptdlns(3, 4, 5)-P3 was unable to cause GLUT4 translocation when added alone to cells . However in the presence of insulin and wortmannin, a condition where no stimulation of glucose transport is observed, the Ptdlns(3, 4, 5)-P3 analog was able to enhance cellular uptake of glucose (Kishi et al., 1996). These results indicate that in the presence of wortmannin, insulin may uniquely act to initiate PI 3-kinase-independent signaling events that act in conjunction with the PI 3-kinase signaling pathway to effect GLUT4 translocation. Other potential mediators such as protein kinase B (PKB) have been suggested in the signaling process regulating glucose transport and GLUT4 translocation. P K B , the cellular 5 homologue of the viral oncogene v-Akt, is a protein-serine/threonine kinase. The catalytic domain of P K B is most similar to that of cAMP-dependent protein (PKA) and the protein kinase C (PKC) findings that gave rise to two of its names, PKB (i.e. between P K A and PKC) and R A C (related to A and C kinase) (Jones et al., 1991). The three isoforms of P K B - P K B a , PKBp and PKBy-display>80% sequence identity and contain a PH domain amino-terminal to the catalytic domain. Stimulation of cells with insulin or survival factors leads to a 10- to 100-fold increase in the concentration of Ptdlns(3, 4, 5)-P3 at the plasma membrane (Vanhaesbroeck et al., 1997). PKB then interacts with Ptdlns(3, 4, 5)-P3 and/or PtdIns(3,4-)-P2 the immediate breakdown product of Ptdlns(3, 4, 5)-P3 through its PH domain, and is thus recruited from the cytosol to the plasma membrane. The interaction of P K B with Ptdlns(3, 4, 5)-P3 alters its conformation and the subsequent phosphorylation of PKB at Thr-308 by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and Ser-473 by PDK2 activates PKB. Monica et al. (1998) suggested that PKBp co-localizes with GLUT4 in the basal state and insulin increases the association of PKBp with GLUT4-containing vesicles. It has also been suggested that PKB lies immediately downstream from PI 3-kinase and expression of a constitutively active form of P K B induced glucose uptake by stimulating GLUT4 translocation in fat and L6 skeletal muscle cells (Kohjiro et al., 1998). Moreover, recently it was shown that a " kinase-dead " PKB could act in a dominant negative fashion to suppress the acute translocation of GLUT4 when transfected into isolated rat adipocytes (Cong et al., 1997). Substantial recent information indicates that PI 3-kinase-independent mechanisms also regulate GLUT4 translocation. Recent findings indicate that exercise also stimulates GLUT4 translocation probably through the activation of the 5'-AMP-activated protein kinase (AMPK) (Hayashi et al., 1998). Another cell signaling pathway that appears to markedly stimulate glucose uptake in muscle, presumably through GLUT4 translocation, involves nitric oxide which 6 stimulate guanylate cyclase to produce cyclic GMP (Etgen et al., 1997). Phorbol ester-sensitive protein kinase C isoforms may also be significant regulators of muscle glucose uptake. The effect of phorbol ester on muscle glucose uptake is additive to the effect of insulin or hypoxia, indicating that protein kinase C acts through a separate pathway and is an unlikely candidate to mediate the responses to these stimulants. Trimeric G protein-linked receptor agonists have been reported to exert significant stimulatory effects on glucose transport, as exemplified by P-adrenergic agonists in skeletal muscle (Han et al., 1998) and brown fat (Shimizu et al., 1996), a-adrenergic agonists in heart muscle (Fischer et al., 1996), bradykinin in skeletal muscle (Kishi et al., 1998), and adenosine in white (Smith et al., 1984) and brown fat (Omatsu-Kanbe et al., 1996). These effects of catecholamines and bradykinin are not blocked by wortmannin, indicating independence of the actions of most known isoforms of PI 3-kinase. Table I summarizes many of the cellular regulators reported to stimulate glucose transport, and the isoform likely to be involved. TABLE I Regulators and signaling pathways reported to stimulate glucose transport Regulator Signaling pathway GLUT isoform Cell type Insulin IR, PI 3-kinase GLUT4 Muscle, fat IGF-I IGF-IR, PI 3-kinase GLUT4 Muscle, fat IGF-II IGF-IR, PI 3-kinase GLUT4 Muscle, fat Contraction 5'-AMP-activated protein GLUT4, Skeletal muscle kinase GLUT1? Hypoxia Unknown GLUT4 Skeletal muscle Nitric oxide cGMP and other? Presumed Skeletal muscle GLUT4 Phorbol ester Protein kinase C Presumed Skeletal muscle GLUT4 p-Adrenergic G s protein GLUT4 Brown fat, skeletal agonists muscle a-Adrenergic Gj protein Presumed Heart muscle agonists GLUT4 Bradykinin G q protein GLUT4 Skeletal muscle Adenosine G a protein GLUT4 White and brown fat Adapted from Czech MP and Corvera S (1999) 7 Ill) Molecular basis of insuiin-stimulated GLUT4 vesicle trafficking GLUT4 translocation in an insulin-responsive manner is evidently a complicated process. Numerous integral membrane proteins continually recycle between the plasma membrane and specific intracellular loci by way of a series of discontinuous tubular and vesicular structures, collectively referred to as the endosomal compartment. Using immunoelectron microscopy, GLUT4 has been localized to several elements of the endosomal recycling pathway including the trans Golgi network (TGN), clathrin-coated vesicles, and endosomes. However, the vast majority of GLUT4 (-60%) is found in tubulo-vesicular (T-V) elements clustered in the cytoplasm, often just beneath the cell surface (Slot et al., 1991a). The available data indicate that GLUT4 does populate the recycling endosomal system to some extent, but that a large proportion of the intracellular GLUT4 resides in a compartment that may have properties more akin to specialized secretory vesicles. The retention and synaptic models are two general models that can account for the insulin-mediated translocation of these intracellular GLUT4-containing vesicles to the plasma membrane. The retention model predicts that GLUT4-containing vesicles are sequestered away from the constitutive endosome recycling system by the specific association of certain sequences within GLUT4 or other co-localized vesicular proteins with a retention receptor. Insulin stimulation results in the release of these GLUT4-containing vesicles, which can then enter the recycling endosome system and thereby accumulate at the cell surface. This model is consistent with the recent observations that expression of the GLUT4 C-terminal domain, or introduction of the cytoplasmic N-terminal domain of the insulin-responsive aminopeptidase (IRAP) which displays some homology with the GLUT4 C-terminus, results in plasma membrane translocation 8 of GLUT4 (Lee et al., 1997; Waters et al., 1997). This model is also consistent with the predominant localization of GLUT4 to small vesicles and tubulovesicular structures adjacent to endosomes typically underlying the plasma membrane (Slot et al., 1991a). In addition, expression of GLUT4 in neuroendocrine cells and fibroblast cell lines results in the localization of GLUT4 to secretory granules and to endosomes, indicating the presence of specific GLUT4 intracellular sequestration sequences that can function in heterologous cell systems (Czech et al., 1991; Hudson etal., 1993). Although several aspects of the retention model are quite appealing, there are several lines of evidence inconsistent with this model. First, a substantial proportion of the GLUT4-containing vesicles appears to be segregated away from recycling endosome markers, having characteristics analogous to small synaptic vesicles. This includes the co-localization of GLUT4 with vesicle associated membrane protein 2 (VAMP2) and the fact that endosomal recycling proteins, such as the transferrin receptor and cellubrevin, can be specifically ablated, whereas a large fraction of GLUT4 cannot (Martin et al., 1996). Furthermore, ablation of the transferrin receptor-containing endosomes does not prevent insulin-stimulated GLUT4 translocation (Martin et al., 1998). These data demonstrate that insulin-regulated GLUT4 trafficking can occur independent of the recycling endosome system. The synaptic vesicle or the SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) hypothesis was subsequently proposed for the GLUT4 trafficking in response to insulin, and it described that, for all membrane trafficking events, a high-affinity match between a ligand in a transport vesicle (v-SNARE) and a receptor in the target membrane (t-SNARE) would be required to consummate a docking and fusion reaction (Shane et al., 1997). In the case of regulated exocytosis in neurons, the v- and t-SNAREs are V A M P 2 and SyntaxinlA/SNAP-25, respectively. According to this hypothesis, GLUT4 vesicles are localized to both small synaptic-like vesicles as well as larger 9 tubulovesicular compartments, both enriched for the v-SNARE protein V A M P 2 . Insulin stimulation results in the association of GLUT4-containing vesicles with the plasma membrane through the interaction of V A M P 2 with the t-SNARE complex composed of Syntaxin 4 and SNAP23. This model is supported by the finding that synthetic peptides that comprise unique V A M P 2 domains block GLUT4 exocytosis but not the constitutive trafficking of GLUT1 in permeabilized 3T3-L1 adipocytes (Shane et al., 1997). Moreover, it is shown that the introduction of either a recombinant fusion protein encoding the cytoplasmic tail of Syntaxin 4 or antibodies directed against Syntaxin 4 specifically blocked insulin-induced GLUT4 translocation in permeabilized 3T3-L1 adipocytes (Tellam et al., 1997, Volchuk et al., 1996). Two Syntaxin-binding proteins, namely, Munc-18c and Synip, have been identified in adipocytes. Although the precise role of these proteins has yet to be elucidated, it appears that these two proteins modulate vesicle docking efficiency by directly regulating the availability of Syntaxin where Munc-18c is a negative and Synip is a positive modulator (Shane et al., 1997; Pevsner et al., 1994). In either case, it is important to recognize that both the retention and synaptic vesicle models are not necessarily mutually exclusive. For example, insulin could induce the trafficking of multiple GLUT4 vesicle populations through different mechanisms. Alternatively, GLUT4 may be stored in synaptic-like vesicles that are sequestered because of GLUT4 and/or IRAP retention signals. Recent studies revealed that some proteins involved in vesicle transport are regulated by the phospholipids generated by PI 3-kinase. GRP-1 (general receptor for phosphoinositides-1) was discovered because of its ability to bind to Ptdlns(3, 4, 5)-P3 in an expression cloning study (Klarlund et al., 1997). GRP-1 is a member of a family of proteins with N-terminal Sec7 homology domains that function as ADP ribosylation exchange factor (ARF) and C-terminal 10 Pleckstrin Homology (PH) domains . The PH domain was shown to have high affinity and high selectivity for Ptdlns(3, 4, 5)-P3 (Jes et al., 1998). Further, the Sec7 domain of GRP-1 is found to catalyze guanine nucleotide exchange of ARF-1 and -5 and Ptdlns(3, 4, 5)-P3 markedly enhances the A R F exchange activity of GRP-1 (Ferro-Novick et al., 1993). The Rab protein family of Ras-related small GTP-binding proteins has been implicated in the regulation of intracellular vesicular traffic (Novick et al., 1993; Nuoffer et al., 1994; Balch et al., 1990; Ullrich et al., 1994). These proteins, with a molecular mass between 20-30 kDa, are highly homologous to the yeast YPT1 and SEC4 proteins, which play a crucial role in endocytosis and exocytosis. More that 30 members have been identified in mammalian cells, and individual Rab proteins are localized to distinct compartment of both the endocytotic and exocytotic pathways. A l l members contain highly conserved domains required for guanine nucleotide binding, GTP/GDP exchange, and GTP hydrolysis (Holman et al., 1994). Nucleotide binding and subsequent hydrolysis are essential for proper targeting and function of the Rab molecules (Holman et al., 1994). Rab proteins are proto-oncogene products highly involved in the growth factor and insulin signaling cascade (Nuoffer et al., 1994). Recent experimental findings indicate that Rab proteins are involved in GLUT4 vesicle trafficking (Robinson et al., 1992). It has been shown that a nonhydrolyzable analog of GTP, GTPyS, could induce GLUT4 translocation in permeabilized 3T3-L1 adipocytes (Robinson et al., 1992) and rat adipocytes (Baldini et al., 1991). A 24 kDa GTP-binding protein has been found to associate with the intracellular GLUT4 compartment in isolated rat cardiomyocytes (Uphues et al., 1994). Furthermore, GLUT4 translocation was found to be associated with the translocation of small G-proteins to the plasma membrane from intracellular vesicle enriched in GLUT4, although the particular isoform of Rab has not been identified (Etgen et al., 1993). More recently,.several lines of evidence support a specific role for Rab4 in the insulin-dependent translocation of GLUT4. First, Rab4 has been 11 found in GLUT4-containing vesicles in rat adipocytes (Cormont et al., 1993) and skeletal muscle (Sherman et al., 1996). Insulin stimulation induces a redistribution of Rab4 from the GLUT4-containing vesicles to the cytoplasm (Sherman et al., 1996). This effect is reversible after insulin withdrawal. Second, it is shown by Vollenweider et al. (1997) that microinjection of a GTP-binding defective mutant and Rab4 antibodies inhibits insulin-induced GLUT4 translocation by 50%. Third, 3T3-L1 adipocytes made insulin-resistant by chronic treatment with insulin show a reduced intracellular pool of GLUT4 and no recruitment of the glucose transporter to the plasma membrane upon insulin stimulation; under these conditions, a parallel alteration in Rab4 movement from intracellular membrane to the cytosol is observed in response to insulin (Ricort et al., 1994). Finally, a synthetic peptide corresponding to the hypervariable C-terminal domain of Rab4 inhibits insulin-induced GLUT4 translocation in rat adipocytes (Shibata et al., 1996). Moreover, Hiroshi et al. (1997) showed that insulin stimulates guanine nucleotide exchange on Rab4 via a wortmannin-sensitive signaling pathway in rat adiocytes. A l l these results indicate that Rab4 functions in directing GLUT4 to the cell surface in response to insulin. It is hypothesized that under normal circumstances, Rab4 might be passively carried into GLUT4 vesicles and there its hypervariable region is inactivated, perhaps through interaction with V A M P 2 . Among other effects, the addition of insulin would lead to the reactivation of Rab4, and GLUT4 vesicles would then be free to fuse with the cell surface via a Rab4 mediated trafficking event. Consistent with this model, it was recently shown that the activation of PI 3-kinase by insulin directly leads to an increase in the GTP loading of Rab4 (Shibata et al., 1997). Taken together, it is tempting to postulate that the activation of PI 3-kinase by insulin leads to the generation of Ptdlns(3, 4, 5)-P3. The binding of Ptdlns(3, 4, 5)-P3 to GRP-1 might lead to the GTP loading and the subsequent activation of Rab4 which in turn stimulates the translocation of GLUT4. 12 IV) GLUT4 and insulin resistance: insights from animal models and humans A n important pathologic feature of obesity, noninsulin-dependent diabetes mellitus (Type 2) and to a lesser extent insulin-dependent diabetes mellitus (Type 1) is impaired glucose utilization in insulin sensitive tissue (Kahn et al., 1996). Intensive research over the last decade has addressed how defects in GLUT4 gene expression or in function of the GLUT4 protein may contribute to diabetes and how understanding these defects might lead to new therapies to prevent or ameliorate diabetes. Injection of streptozotocin, a (3-cell toxin, in rodents results in insulinopenic diabetes that shares phenotypic characteristics with Type 1 diabetes in humans. Mechanisms for impaired insulin-stimulated glucose transport have been studied in the STZ-induced diabetic rat model. In adipocytes from diabetic rats, the recruitment of GLUT4 in response to insulin is markedly reduced. However, when rats are treated with insulin, their adipose cells regain the ability to translocate GLUT4 to the plasma membrane in response to acute insulin exposure. GLUT4 transporters in the intracellular pool show parallel changes, with a reduction of GLUT4 in the low-density microsomes of cells from diabetic rats and a restoration with insulin treatment. Therefore, the insulin resistance in adipose cells from diabetic rats appears to result from decreased GLUT4 in the intracellular membrane, with fewer GLUT4 available to be translocated to the plasma membrane in response to insulin (Kahn et al., 1996). In skeletal muscle, evidence from several studies indicates that skeletal muscle insulin resistance cannot be fully accounted for by the reduction in GLUT4 protein content (Richardson et al., 1991; Youn et al., 1994). This was shown by depressed insulin-stimulated skeletal muscle glycogen synthesis and glycolysis and glucose transport which preceded the decrease in GLUT4 protein content (Richardson et al., 1991; Youn et al., 1994). As well, the magnitude of the decrease in the response to insulin is 13 higher than the reduction in GLUT4 protein (Youn et al., 1994). Furthermore, many studies have now shown that in most insulin-resistant states in rodents, GLUT4 expression does not downregulate in skeletal muscle, indicating the additional defects in the glucose transport system including impaired glucose transporter translocation and a lower intrinsic activity of glucose transporters may be present (Abel et al., 1996; Zierath et al., 1996). In fact, It has been shown that insulin-mediated GLUT4 translocation to the transverse tubules (T-tubules) is significantly reduced in skeletal muscle of STZ-diabetic rats ( Dombrowski et al., 1998). The differences in the regulation of GLUT4 protein levels in muscle and fat indicate tissue-specific regulation. This phenomenon is seen in many different models of diabetes, obesity, and insulin resistance (Table II). In a new model of obesity and Type 2 diabetes created by transgenic ablation of brown adipose tissue, mice become progressively obese, markedly hyperinsulinemic, and eventually overtly diabetic (Lowell et al., 1993; Hamann et al., 1995). As insulin resistance progresses, GLUT4 downregulates in adipocytes but not in skeletal muscle (Frevert et al., 1995). In models of genetic or dietary-induced obesity without overt diabetes, tissue-specific regulation of GLUT4 is also seen. The Zucker fa/fa rat has endogenous hyperinsulinemia with insulin-resistant glucose uptake in vivo but normoglycemia, as in human obesity. In 5-week-old Zucker rats, GLUT4 is overexpressed in adipocytes, contributing to accelerated lipogenesis (Hainault et al., 1992; Pedersen et al., 1992). As obesity and hyperinsulinemia progress, there is progressive downregulation of GLUT4 in adipocytes (Pedersen et al., 1992). When diabetes is acutely superimposed in 20-week-old Zucker rats by injection of STZ, GLUT4 levels in adipocytes decrease further. However, GLUT4 expression is not altered in skeletal muscle from the same rats (Kahn et al., 1993). 14 TABLE II Tissue-specific expression of GLUT4 Fat Muscle Obesity 1 NIDDM I IDDM N D STZ diabetes I B A T deficiency I Zucker (young) t Zucker (old) I Adapted from Kahn BB (1996) Tissue-specific regulation of GLUT4 protein levels in adipose tissue and muscle from humans and rodents with insulin resistance. BAT, brown adipose tissue, ND, not determined, no change, T: increase, ^ : decrease. The mechanisms for insulin resistance in humans have also been investigated. GLUT4 expression was measured in vastus lateralis muscle of lean and obese nondiabetic and Type 2 diabetic subjects (Garvey et al., 1992). The level of GLUT4 mRNA was similar in all groups. Parallel results are seen with GLUT4 protein in muscle biopsies from these subjects (Garvey et al., 1992). Similarly, in a younger cohort of Type 1 diabetic subjects, GLUT4 levels in muscle are also unchanged from those of age-matched control subjects ( Kahn et al., 1992). In contrast, in adipose cells from subjects with obesity, Type 2 diabetes, or even impaired glucose tolerance and in some subjects with gestational diabetes, GLUT4 levels are decreased (Garvey et al., 1992, Sinha et al., 1991). Thus, in human obesity and diabetes mellitus, as in rodent models, there is a tissue-specific regulation of GLUT4. Changes in GLUT4 expression may explain insulin-resistant glucose transport in adipose cells but not in muscle. Importantly, changes in GLUT4 expression in skeletal muscle may not explain in vivo insulin-resistant glucose uptake. Knockout mice with one null allele of GLUT4 (GLUT4+/-) have been generated that exhibit reduced GLUT4 expression. Though all mice have the same mutation, GLUT4+/- mice 15 —> —> could be divided into three distinct groups: normal glycemia with normal insulin levels (N/N), or normal glycemia with high insulin levels (N/H), or hyperglycemia with high insulin levels (H/H). Beginning at 2 months of age all GLUT4+/- mice have reduced adipose and muscle GLUT4 expression (75 and 25-46%, respectively) (Stenbit et al., 1997). In vitro insulin-stimulated glucose uptake into soleus and extensor digitorum longus (EDL) muscle of prediabetic N / H GLUT4+/- mice is significantly diminished (Rossetti et al., 1997). Using euglycemic/ hyperinsulinemic clamps it was demonstrated that the in vivo peripheral insulin resistance of prediabetic (N/H) GLUT4+/- mice was as severe as in uncontrolled Type 2 diabetes in H/H mice (Rossetti et al., 1997). Additionally, the rate of insulin-stimulated glycogen synthesis was significantly impaired because of reduced muscle GLUT4-mediated glucose uptake. The effect was a direct effect on GLUT4 and not the result of defective activation of glycogen synthase. Myosin light chain 1-GLUT4 (MLC-GLUT4) transgenic mice that specifically overexpress GLUT4 in fast twitch muscle were mated into the genetic background of the GLUT4+/- mutation to assess the therapeutic merit of muscle GLUT4 gene therapy in Type 2 diabetes (Tsao et al., 1997). GLUT4 content and insulin-stimulated glucose uptake were normalized in fast twitch muscles of MLC-GLUT4+/- mice. Fed plasma glucose and insulin levels were normal throughout the lives of MLC-GLUT4+/- mice, and cardiac histopathologies were minimal. In vivo tracer studies demonstrated that whole body glucose utilization, glycolysis, and glycogen synthesis were normal in MLC-GLUT4. These data indicate that skeletal muscle GLUT4 plays a central role in peripheral insulin sensitivity and upregulation of muscle GLUT4 may attenuate or even correct insulin resistance in diabetes and other diseases although changes in GLUT4 expression in skeletal muscle does not fully explain in vivo insulin resistant glucose uptake. 16 GLUT4 null mice, which lack GLUT4, are also noteworthy. In comparison with GLUT4+/- mice it is both exciting and perplexing that GLUT4 null mice are not diabetic but do exhibit abnormalities in glucose and lipid metabolism (Charron et al., 1997). Surprisingly, blood glucose levels in GLUT4 null mice are normal under fasted and fed conditions. Although GLUT4 null mice have normal glucose tolerance, they do exhibit hyperinsulinemia in the fed state and impaired insulin tolerance, indicating insulin resistance. Northern and Western blot analyses verified that GLUT4 null mice could compensate for the lack of GLUT4 and maintain normal circulating glucose levels by a mechanism that did not involve overexpression of a known facilitative or Na+-dependent glucose transporter isoforms in skeletal muscle. The ability of two GLUT4 null muscle types to take up glucose in the presence of maximally stimulating concentrations of insulin was measured in vitro (Charron et al., 1997). Fast twitch E D L muscles failed to take up more glucose in response to insulin. Surprisingly, a 2-fold increase in insulin-stimulated glucose uptake was noted in female GLUT4 null slow twitch soleus muscle compared with a 3-fold increase in wild type controls. Soleus muscle of GLUT4 null males displayed a 2-fold increase in basal glucose uptake with no further increase following insulin stimulation. The molecular basis for the sexually dimorphic response to GLUT4 ablation in soleus muscle may be linked to the superior insulin sensitivity of female mice. This result, combined with the failure to detect increased expression of any known GLUT, led to the hypothesis that a novel glucose transport system is responsible for glucose uptake into the highly oxidative soleus muscle, which contributes to euglycemia in GLUT4 null mice. In contrast to GLUT4 null mice which never express GLUT4, GLUT4+/- mice express normal amounts of GLUT4 in their insulin-responsive tissue until 2 months of age. Therefore, the mechanisms that develop in GLUT4 null mice to help maintain euglycemia may not be apparent in GLUT4+/- mice because the function of the compensatory glucose transport system may be masked by the remaining functionally dominant GLUT4. 17 V) Diabetic cardiomyopathy Numerous clinical and epidemiological reports have confirmed that human diabetics appear particularly susceptible to heart failure, which is a leading cause of death in these patients (Kannel et al., 1979). Factors that appear largely to account for this increased incidence of cardiovascular dysfunction during diabetes include atherosclerosis of the coronary arteries, macroangiopathy, and autonomic neuropathy. However, it has also become apparent that these factors are not always responsible for the cardiac problems associated with diabetes. For example, a significant number of diabetic patients who do not develop atherosclerosis, macroangiopathy, and autonomic neuropathy still suffer from cardiomegaly, left ventricular dysfunction, and clinically overt congestive heart failure (D'elia et al., 1979; Regan et al., 1977). The existence of a specific cardiac muscle disease, referred to as diabetic cardiomyopathy was first suggested by Rubier et al. (1972). This was confirmed by Hamby et al. (1974) based on the fact that diabetic patients exhibited clinical hemodynamic evidence of cardiac dysfunction in the presence of normal coronary arteries. Diabetic cardiomyopathy may occur as a direct consequence of the insulin deficient state on myocardial cell function (Hamby et al., 1974; Rodrigues et al., 1992). The events causing the cardiomyopathy are not completely understood but have been extensively investigated. Increased stiffness of the ventricular wall accompanied by the accumulation of glycogen and collagen as well as perivascular thickening of the basement membranes has been documented as well as microangiopathy, autonomic dysfunction and altered vascular sensitivity (Rodrigues et al., 1992; McNeill, 1996a; McNeill et al., 1996b). A number of biochemical changes in the diabetic heart including altered myosin ATPase, depressed SR C a 2 + uptake and N a + / K + ATPase have also been identified (Rodrigues et al., 1992; McNeill et al., 1996b). The events that precede the structural and enzyme changes in diabetic hearts are not 18 well understood. However, In diabetic heart, there is an excessive oxidation of free fatty acid (FFA) and/or an impaired glucose oxidation, which occur concomitantly with a depressed cardiac function. Depressed cardiac glucose utilization in diabetes may be caused by impaired glucose transport. In fact, the insulin sensitive GLUT4 protein and mRNA are specifically reduced in cardiomyocytes in diabetic rats (Camps et al., 1992). Studies conducted by our laboratory also showed a lower GLUT4 content in both the plasma membrane and intracellular membrane in the fatty Zucker rat heart which may suggest an overall decrease in the cardiac expression of GLUT4 (Li et al., 1998). The reduction in cellular GLUT4 content restricts glucose transport across the sarcolemmal membrane into the myocardium, and hence impairs glucose utilization (Garvey et al., 1989). VI) Vanadium, an insulin-mimetic/insulin-enhancing agent Vanadium is a group V transition metal which exists in several valence states. An antidiabetic effect of vanadium was reported as early as 1899 when sodium vanadate was shown to decrease urinary glucose in two out of three diabetic patients (Lyonnet et al., 1899). The glucose-lowering effect of vanadium administrated as sodium orthovanadate was first demonstrated in vivo in diabetic rats in 1985 in our laboratory (Heyliger et al., 1985). In subsequent studies using both inorganic and organic compounds, the effectiveness of vanadium in the treatment of experimental diabetes has been substantiated (Meyerovitch et al., 1987; Ramanadham et al., 1989; Yuen et a l , 1993). Some studies have also been conducted on both Type 1 and 2 human diabetic subjects. In Type 1 diabetic patients, vanadium lowers insulin requirements without an effect on C-peptide levels indicating the absence of an influence on insulin release. In addition, two out of the five insulin-dependent subjects showed improved glucose utilization (Goldfine et al., 1995). In Type 2 diabetic subjects, recent studies have 19 demonstrated that 3 to 4 weeks of treatment with oral vanadyl sulfate 50 mg twice daily reduces fasting plasma glucose and HbAlc levels and improves insulin sensitivity. This improvement in insulin sensitivity is due to both augmented stimulation of glucose disposal and enhanced suppression of hepatic glucose output. Increased insulin-stimulated glycogen synthesis accounts for >80% of the increased glucose disposal with vanadyl sulfate. In Type 2 subjects, vanadyl sulfate is also associated with suppression of insulin-inhibited plasma free fatty acids and lipid oxidation. The reduction in hepatic glucose output and increase in glucose disposal are both highly correlated with the decline in plasma FFA concentrations. No such effect was observed in obese nondiabetic subjects. These data indicate that vanadium does not alter insulin sensitivity in nondiabetic subjects, but it does improve both hepatic and skeletal muscle insulin sensitivity in Type 2 subjects in part by enhancing insulin's inhibitory effect on lipolysis (Boden et al., 1996; Halberstamet al., 1996). The putative role of vanadium in the glucose transport system has also been indicated by the findings that include enhanced glucose transport activity in isolated rat adipocytes (Ramanadham et al., 1989), mouse brain (Amir et al., 1987), and rat skeletal muscle (Okumura et al., 1992); enhanced GLUT4 translocation in isolated adipocytes (Paquet et al., 1992); and enhanced expression of glucose transporter in NIH 3T3 mouse fibroblasts (Mountjoy et al., 1990). Furthermore, vanadium treatment restored skeletal muscle GLUT4 protein and mRNA content in STZ-diabetic rats (Strout et al., 1992). VII) Experimental rationale and objectives The present study aimed at studying the mechanisms of action of vanadium. As described above, the effectiveness of vanadium in the treatment of diabetes has been demonstrated in 20 animals as well as human studies. Vanadium treatment of diabetes in rats is also effective in correcting cardiac function. Several forms of vanadium, including vanadate, vanadyl sulphate, and bis(maltolato)oxovanadium(IV), have all been shown to significantly improve myocardial performance in diabetic rats (Meyerovitch et al., 1987; Ramanadham et al., 1989; Yuen et al., 1993). However, the exact mechanism of action of vanadium in treating diabetic cardiomyopathy is unknown. It was shown by a hyperinsulinemic euglycemic clamp experiment that the effect of vanadium was associated with improved glucose metabolism in various tissues with the most profound effect in the heart occurring without changing the expression of GLUT4 (Brichard et al., 1992), indicating the possible enhancement of cardiac GLUT4 translocation brought about by vanadium treatment. Furthermore, from the previous in vitro experiments, it was found that the insulin-sensitizing effect of vanadium on isolated cardiac myocytes is rapid, indicating that it is not a change in gene expression (Yao et al., 1996). It may be possible that the drug mediates at least some of its effects via stimulation of GLUT4 translocation. Therefore, we have hypothesized that vanadium treatment can enhance insulin-stimulated GLUT4 translocation in cardiac tissue of diabetic rats. We have developed a novel ELISA technique to quantitatively measure GLUT4 translocation in rat heart and fat tissue (Li et al., 1998; Cam et al., 1996). At present, there is solid evidence obtained from observations of immunoelectron microscopy, subcellular membrane fractionation, and photoaffinity labeling with A T B - B M P A , indicating insulin-induced GLUT4 translocation in heart muscle. The usual insulin responsiveness for GLUT4 translocation is around 100-300% or even higher when measured by the methods mentioned above (Watanabe et al., 1984; Slot et al., 1991b; Fischer et al., 1997). The magnitude of cardiac GLUT4 translocation when measured by the experimental competitive ELISA method is around 30% in lean Zucker rats. The discrepancy in the magnitude of GLUT4 translocation may be due to either 21 the difference in the experimental methods or the difference between an in vivo and in vitro study. The secondary purpose of this study was to verify the ELISA method by measuring GLUT4 content in the same membrane fractions with the ELISA assay and also with E C L Western blotting which is an universally used method for detection of GLUT4. The overall objective of this study was to examine the effects of vanadium on glucose transporter function in cardiac tissue of the STZ-diabetic rat. Specifically, the present study investigated the effect of vanadium on in vivo GLUT4 translocation in the heart of STZ-diabetic Wistar rat. Toward this goal, firstly, we determined the time course of insulin-mediated GLUT4 translocation in the control rat hearts. Secondly, we investigated the effects of vanadium on cardiac GLUT4 translocation by measuring insulin-induced GLUT4 translocation in vanadium-treated-STZ diabetic rats at the time points of initial and maximal insulin response. Lastly, we verified the ELISA method by measuring GLUT4 content in the same membrane fractions with the ELISA assay and also with E C L Western blotting. 22 MATERIALS AND METHODS I) Chemicals and materials Subcellular fractionation of frozen heart ventricles Tris[hydroxymethyl]aminomethane (Tris) and phenylmethylsulfonylfluoride (PMSF) were obtained from Sigma Chemical Company ( St. Louis, MO). KC1 and NaCI were obtained from B D H Inc. (Toronto, ON). Sucrose was obtained from ICN Biomedicals, Inc. (Aurora, OH). 5' Nucleotidase (5f ND) marker enzyme assay Most of the chemicals for this assay including a,P-methyleneadenosine 5'-diphosphate (AOPCP), 5' nucleotidase control (5' N D control), adenosine monophosphate (AMP), ammonium salt, ZnS04, and Ba(OH)2 were obtained from Sigma Chemical Company (St. Louis, MO). ), [8-14C]adenosine 5'-monophosphate was obtained from Amersham Life Science (Oakville, ON). Scintiverse was obtained from Fisher Scientific Co. (Fair Lawn, NJ) The competitive enzyme-linked immunosorbant assay (ELISA) K H 2 P O 4 (monobasic), sodium carbonate (anhydrous), H2SO4, and hydrogen peroxide were purchased from B D H Inc. (Toronto, ON). Polyoxyethylenesorbitan monolaurate (Tween 20), citric acid (monohydrate), citric acid (trisodium dihydrate), bovine serum albumin (BSA, ELISA grade) and O-phenylenediamine dihydrochloride were obtained from Sigma Chemical Company (St. Louis, MO). Na2HP04 (dibasic, anhydrous) and sodium bicarbonate were obtained from Fisher Scientific (Fair Lawn, NJ). Skim milk powder (Carnation Inc., Toronto, ON) was purchased from a local supermarket. Anti-GLUT4 monoclonal antibody (1F8) was produced by Biogenesis Inc. (Sandown, NH). Synthetic GLUT4 C-terminus and goat serum were obtained 23 from East Acres Biologicals (Southbridge, M A , now, Biogenesis Inc.). Horseradish peroxidase-linked anti-mouse antibody was obtained from Amersham Life Science (Oakville, ON). The enhanced chemiluminescence Western blotting (ECL Western blot) Acrylamide (99.9%), N,N'-methylene-bis-acrylamide (Bis), N,N,N',N'-tetra-methyl-ethylenediamine (TEMED), ammonium persulfate, sodium dodecyl sulfate (SDS), bromophenol blue were obtained from Bio-Rad (Hercules, CA). Sodium azide and glycerol were obtained from Sigma Chemical Company (St. Louis, MO). Pure nitrocellulose membrane (0.45 pm) was also purchased from Bio-Rad. E C L Western blotting detection reagents and hyperfilm were obtained from Amersham Life Science (Oakville, ON). G B X developer and fixer stock solution was obtained from Kodak (Rochester, NY) . II) Treatment protocol The STZ-diabetic Wistar rat, a commonly used animal model of Type 1 diabetes, was chosen for the present study. Male Wistar rats (160-220g) were obtained from the Animal Care Centre at U B C . Diabetes was induced by a single iv injection of STZ (60 mg/kg, from Sigma Chemical Company, St. Louis, MO) via the tail vein. Diabetes was confirmed 3 days later by measuring plasma glucose level. Only animals with plasma glucose at 13 mmol/1 or ever higher were used. We were interested in using the organic forms of vanadium such as B M O V , to study the mechanism of vanadium since B M O V has been shown in our laboratory to be more effective and less toxic than inorganic forms of vanadium in ameliorating the symptoms of diabetes (Yuen et al., 1993). B M O V treatment was started one week after STZ injection. B M O V was given in drinking water beginning at a concentration of 0.25 mg/ml and adjusted to a maximal 24 concentration of 1 mg/ml for a period of 8 weeks. Treatment groups were control, control-treated, diabetic and diabetic-treated. Body weight, food and fluid intake, and five-hour-fasted plasma glucose were monitored weekly. Plasma insulin and triglycerides were determined at the beginning and the end of the treatment as well as at the time of termination. At termination, after an overnight fast, the animals were anesthetized using sodium pentobarbital (65 mg/kg, ip). Half of the animals in each group received an iv injection of regular beef/pork insulin (Iletin R) at 5 U/kg via the tail vein. Animals not injected with insulin were used as controls in the basal state. The time profile of cardiac GLUT4 translocation from our time course study showed that the initial response was detected at 5 minutes and the maximal response was reached at 15 minutes after insulin injection. Thus, the hearts were removed at 5 and 15 minutes post-insulin administration. For this purpose, after 5 and 15 minutes, the chest cavity was opened and 1 ml of blood was withdrawn from the heart using a syringe. The heart was immediately removed and washed in distilled water to remove blood. After the removal of the aorta and atrial tissue, the ventricles were cut open, blotted dry and freeze-clamped in liquid nitrogen and stored at -70°C until use for subcellular fractionation. Wistar Rats cv o% + nsulin) 50% 50% + (insulin) 50% STZ-D STZ-DV 50% + (insulin) 50% 50% + (insulin) 50% Figure 1. The overall experimental design of the present study (C: control, V : B M O V , D: diabetic). 25 Ill) Blood sample analyses Blood was centrifuged for 20 minutes at 17,500 x g using a Beckman centrifuge (Allegra™ 21R, rotor F3602) to separate plasma. Plasma samples were stored at -70°C until the time of analyses. Glucose was determined using a Beckman Glucose Analyzer 2. Triglycerides was determined using a triglyceride assay kit obtained from Boehringer Mannheim (Laval, PQ, currently Roche). Plasma insulin was measured using a radioimmunoassay kit specific for rat insulin (St. Charles, MO). IV) Subcellular fractionation of frozen heart ventricles The fractionation procedure was a modification of the method of Mansier et al. (1983) used to isolate highly purified sarcolemmal membrane using fresh heart ventricles. A schematic diagram of the fractionation procedure is illustrated in Figure 2. A l l fractionation procedures were performed at 4 °C. Frozen heart ventricles were powdered in a mortar cooled with liquid nitrogen and then transferred to centrifuge tubes with 8 ml of buffer I which contained 10 mM Tris, and 0.1 m M PMSF at pH 7.8. The mixture was then homogenized using a polytron with a PT 10-35 probe (Brinkman Instruments Ltd, Toronto, ON) at half maximum speed with 3 bursts. The heart homogenate was subjected to hypotonic lysis by stirring in the hypotonic buffer (buffer I) on ice for 20 minutes. Hypotonic lysis was stopped by adding 2 ml of a solution containing concentrated KC1 (600 mM), NaCI (150 mM) and sucrose (1250 mM) which brought the final concentration in the homogenate to 120 mM KC1, 30 m M NaCI, and 250 m M sucrose. The subsequent isolation of membrane fractions was done by differential centrifugation. A l l low-speed centrifugation (<31,000g) were done using the Beckman J2-21 centrifuge and JA-17 rotor. After the hypotonic lysis, the mixture was centrifuged for 20 minutes at 500 x g to remove 26 ventricular tissue which had not been broken down in the homogenization procedure. Supernatant (200-300 ml) was collected as homogenate in several aliquots and then quickly frozen in liquid nitrogen. The remaining supernatant was made up to 15 ml with buffer II which contained 30 mM Tris, 120 mM KCI, 30 mM NaCI, 250 mM sucrose and 0.1 mM PMSF (pH 6.8 at 4 °C). After a 20-minute centrifugation at 7,000 x g, the supernatant was centrifuged for another 40 minutes at 31,000 x g. The resultant pellet was collected for the further purification of sarcolemmal membrane whereas the supernatant was set aside for isolation of intracellular membrane. The pellet was resuspended in 1 ml of buffer II using the homogenizer (Dyna-mix, Fisher Scientific, Fair Lawn, NJ) and then layered on top of 4 ml of 33.3% sucrose solution containing 300 mM KCI, 100 mM Tris and 33.3% sucrose (w/v, pH 7.6). After ultracentrifugation at 90,000 x g for 90 minutes using the SW 41 rotor, sarcolemmal membrane, seen as an opaque band on top of the 33.3% sucrose cushion, was collected and 4-fold diluted with wash buffer (30 m M Tris, and 120 mM KCI, pH 6.8). After ultracentrifugation at 31,000 x g for 30 minutes using the Ti 70.1 rotor, sarcolemmal membrane was pelleted and then resuspended in 50 pi of buffer III (120 mM KCI, 250 mM sucrose, and 30 m M Tris, pH 6.8) by passing the suspension through a 1 ml syringe and 25G needle for several times. The supernatant, which was set aside for isolation of intracellular membrane, was ultracentrifuged at 150,000 x g for an hour using the Ti 50.2 rotor. The sediments were resuspended in 1 ml buffer II by the same homogenizer mentioned above and then layered on top of 8 ml of 50% (w/v) sucrose with the same ingredients as the 33.3% sucrose. After ultracentrifugation at 125,000 x g for 90 minutes using the SW 41 rotor, intracellular membrane was obtained from the top layer of the 50% sucrose first by washing using the wash buffer and then by pelleting down by centrifugation at 65,000 x g for 30 minutes using the Ti 70.1 rotor. The intracellular membrane fractions were also resuspended in 50 pi of buffer III using the 1 ml syringe and 25 G needle. A l l fractions collected were stored at -70°C until further analysis. 27 Powder Frozen Heart Ventricle V Homogenize using polytron (half speed, 3 bursts) Hypotonic lysis Add cone, salt and sucrose 500xg (20 min) V discard P * discard Aliquot S Dilute with buffer II • 7,000 x g'(20 min) I S * 31,000xg (40 min) Homogenate Plasma membrane p I 33% 90,000xg (90 min) 33% V 150,000xg (60 min) 50% * discard * 50% 125,000xg (90 min) • 65,000xg (30 min) \ Intracellular membrane Figure 2. Subcellular fractionation of frozen heart ventricles. P and S denote pellet and supernatant, respectively. 28 V) 5' Nucleotidase ( 5f ND) marker enzyme assay 5' N D is an enzyme generally found in the plasma membrane. Thus it is often used as a biochemical marker for the organelle. It catalyses the hydrolysis of A M P to adenosine and inorganic phosphate. In the enzyme assay, trace amount of l 4 C labeled A M P is used as a substrate in combination with non-radioactive A M P . The rate of adenosine production as an estimate of A M P hydrolysis is used to calculate 5' N D enzymatic activity. The assay is performed with two sets of tubes in duplicates. The first pair is used to measure total A M P hydrolysis. The second pair of tubes is used to measure A M P hydrolysis in the presence of AOPCP, a specific inhibitor of 5' ND. Thus 5' ND activity is determined by measuring the AOPCP-sensitive A M P hydrolysis: AOPCP-sensitive A M P hydrolysis = Total A M P hydrolysis - A M P hydrolysis in the presence of AOPCP 5' N D was assayed in a 1 ml volume containing 0.5 ml of Tris-HCl (0.1 M , pH 7.4), 20 pi of 20 uM AOPCP to the tubes with AOPCP, 10 u.1 of samples, and 0.1 ml of substrate (0.6 uM hot A M P and 100 p M cold AMP). The rest of volume was made up by distilled water. Blank samples contained no enzyme. The reaction was conducted at 37°C for 20 minutes. The reaction was stopped by the addition of 0.2 ml of 0.3 M zinc sulphate. Protein and unhydrolyzed A M P were precipitated by the addition of 0.2 ml of 0.3 M barium hydroxide. The tubes were centrifuged at 4,000 x g for 30 minutes using the Beckman J-6B centrifuge. A 0.9 ml aliquot of the supernatant was aspirated and counted in 10 ml of Scintiverse. 5' N D activity was expressed as umol adenosine produced per mg protein per hour. The following formula was used to convert results from cpm to umol/mg/hr: 29 (cpm*) x (1.4 ml) x (100 uM) x (0.0014 L) x (60 min) (total cpm*) x (0.9 ml) x (protein cone, in mg/ml) x (sample vol in ml ) x (20 min) Note: * cpm was obtained by: cpm without AOPCP - cpm with AOPCP. Total cpm was obtained by counting 0.1 ml substrate with 0.8 ml water in 10 ml of Scintiverse. VI) ELISA The competitive ELISA method involves competition between the GLUT4 protein and the GLUT4 C-terminal synthetic peptide to bind with the monoclonal antibody. This procedure is based on the fact that the epitope specifically recognized by the GLUT4 monoclonal antibody is thought to be the carboxy terminal end and that the relative affinity of antibody for native GLUT4 protein (in membrane fractions) is 103-fold higher than that for the synthetic peptide (James et al., 1988). The competitive ELISA method consists of five steps as outlined below: 1. Coating the plate: Lyophilized GLUT4 C-terminal peptide (CTELEYLGPEND) was initially made up to 0.125 mg/ml in deionized distilled water and stored in aliquots at -70°C. The coating antigen was prepared by diluting the peptide solution 1:10 in 0.1 M sodium carbonate-bicarbonate buffer (pH 9.3). and adding 50 pl/well of diluted peptide to high-binding Maxisorp 96-well ELISA plates (Nunc, Denmark). Coated plates were baked overnight at 50°C in a dry oven. A l l subsequent incubation steps of the coated ELISA plates were performed at 37°C in a shaking, covered water bath. Between each step, the reaction plate was washed 3 times and then dried by inverting the plate on paper towels. Each wash consisted of filling each well of the plate with wash buffer (0.1% Tween 20 in modified phosphate-buffered saline [KBS]: 0.138 M NaCI, 0.01 M KH2PO4, 0.0IM Na2HP04, 0.05% Tween 20) and then shaking the plate manually for 15 30 seconds. 2. Blocking: After the washing, the coated plates were blocked with 10% heat-inactivated goat serum for 2 hours at 37°C. Heat inactivation of goat serum was done in advance by incubating at 56 °C for 30 minutes. 3. Primary antibody incubation: Membrane fraction samples were first solubilized by adding TritonX-100 to a final concentration of 1% and then serial diluted (1:2) in dilution buffer with KBS containing 1% TritonX-100 in a low affinity binding plate (Evergreen Scientific, Los Angeles, CA). Just prior to the end of the blocking period, 35 pi of the monoclonal antibody (diluted 1:3000 in KBS containing 3% [w/v] skim milk, 1% [w/v] BSA [ELISA grade], and 0.05% [v/v] Tween 20) were added to 35 pi of the membrane samples in the low affinity plate. Subsequently, 50 pi of the mixture were transferred to the reaction plate. Incubation with the monoclonal antibody was conducted for 2 hours. 4. Secondary antibody incubation: One hundred pi of the secondary antibody (diluted 1:1000 in KBS containing 1.5% [w/v] skim milk, 0.5% [w/v] BSA, and 0.05% [v/v] Tween 20) was added to each well of the reaction plate and secondary antibody incubation was also conducted for 2 hours. 5. Substrate incubation: For substrate incubation, 0.15 ml of substrate solution (20 mg 0-phenylenediamine dihydrochloride in 50 ml of 0.1 M citrate buffer [pH 5.5]) were added to each well and the reaction was carried out for 20 minutes. Three minutes before the addition of the substrate, 20 pi of H2O2 were added to 50 ml of the substrate solution. The enzymatic reaction was stopped by adding 40 pi of 8 N sulphuric acid to each well. Color development was allowed 31 for 10 minutes before absorbance readings are taken at 490 nm using an automated microplate reader (model EL 309, Bio-Tek Instruments). GLUT4 content was determined as amount per mg membrane protein and the relative amount was obtained by comparing to an internal standard (heart homogenate of control Wistar rats) which was performed in every assay. Protein content of all fractions was quantitated using a commercial protein assay kit modified from the Lowry method (Bio-Rad, Hercules, CA). VII) ECL Western blot E C L Western blotting is a light emitting non-radioactive method for detection of immobilized specific antigens, conjugated directly or indirectly with horseradish peroxidase-labeled antibodies. Membranes (5 pg of proteins unless otherwise indicated) were subjected to a SDS-PAGE in 10% mini-gel slabs as described by Laemmli (1970). The separated GLUT4 protein was electrically transferred to a nitrocellulose membrane. The membrane was then blocked with 5% skim milk in PBS (2.6 mM KC1, 136 mM NaCI, 6.4 mM Na 2 HP0 4 , 1.46 mM KH2PO4) overnight at 4°C. Subsequently, the membrane was incubated with the anti-GLUT4 monoclonal antibody (diluted 1:400 in PBS containing 1% skim milk) for 1 hour at room temperature. After 3 x 5 minutes of washing with PBS containing 0.1% Tween 20, the membrane was incubated with the horseradish peroxidase-linked secondary antibody (diluted 1:3000 in PBS) for 30 minutes at room temperature. The membrane was again washed 3 x 5 minutes in the same PBS buffer mentioned above. Visualization of the immunolabeled bands was carried out by autoradiography after addition of a chemiluminescence reagent (ECL). After autoradiography, the density of the labeled 45-kDa bands was measured using a laser scanning densitometer (Quantity One, PDI Inc, Huntington Station, NY). 32 VIII) Statistical analyses Values are presented as means ± standard error of mean. Statistical analysis was done by analysis of variance (ANOVA) using one-way, two-way or general linear model ( G L M -A N O V A ) as appropriate. Significant difference was determined by a Neuman Keuls a posteriori test, p<0.05 taken as significant. 33 RESULTS I) General features of STZ-diabetic and vanadium-treated diabetic rats The STZ-diabetic rats exhibited progressive symptoms of Type 1 diabetes. On the one hand, when compared to controls, diabetic rats had significantly lower body weight during the whole experimental period (Figure 3). Diabetic rats also had dramatically elevated fluid (polydipsia) and food (hyperphagia) intake (Figure 4, 5). Five-hour fasted plasma glucose levels of diabetic animals were elevated within 24 hours after diabetes induction and remained elevated 3~4-fold for the rest of the study (Figure 6). On the other hand, plasma insulin levels measured at the beginning (one week after STZ-injection, 0.96±0.13 ng/ml) and at the end of the study (week 8, 0.37±0.05 ng/ml) were significantly lower than their corresponding control groups (1.73±0.25 vs 1.44±0.19 ng/ml, Figure 7). Figure 8 shows plasma triglyceride levels in the experimental rats. Plasma triglyceride levels were elevated in diabetic rats at week 8 (2.84±0.48 vs 1.19±0.08 mM of controls) although the basal levels (at one week after diabetes induction and before the treatment of vanadium) were not different from controls. Vanadium treatment failed to correct the reduced body weight gain in the diabetic rats (Figure 3). However, food and fluid intake were gradually normalized by vanadium in the diabetic-treated group (Figure 4, 5). Five-hour fasted plasma glucose was restored to control level after 3 weeks of vanadium treatment and remained at the same level for the rest of the study (Figure 6). Like the diabetic rats, vanadium-treated diabetic rats were hypoinsulinemie during the experimental period even though they maintained euglycemie, demonstrating the insulin-enhancing effect of vanadium (Figure 7). However, in contrast to the diabetic group, plasma 34 TIME (Weeks) Figure 3. Body weight following bis(maltolato)oxovanadium (IV) treatment for 8 weeks. The four treatment groups were: control (n=10), control-BMOV (n=l 1), diabetic (n=l 1) and diabetic-BMOV (n=l 1). Statistical analysis was done by G L M - A N O V A followed by Newman-Keuls, p < 0.05. * different from control groups. 35 60 55 50 45 40 35 30 25 20 Control Control-BMOV Diabetic Diabetic-BMOV TIME (Weeks) Figure 4. Food intake following bis(maltolato)oxovanadium (IV) treatment for 8 weeks. The four treatment groups were: control (n=10), control-BMOV (n=l 1), diabetic (n=l 1) and diabetic-BMOV (n=l 1). Statistical analysis was done by G L M -A N O V A followed by Newman-Keuls, p < 0.05. * different from control groups, @ different from all other groups. 36 300 Control 0 J , 1 1 1 , 1 0 2 4 6 8 TIME (Weeks) Figure 5. Fluid intake following bis(maltolato)oxovanadium (IV) treatment for 8 weeks. The four treatment groups were: control (n=10), control-BMOV (n=l 1), diabetic (n=l 1) and diabetic-BMOV (n=l 1). Statistical analysis was done by G L M - A N O V A followed by Newman-Keuls, p < 0.05. * different from control groups, @ different from all other groups. 37 Control Control-BMOV Diabetic Diabetic-BMOV 4 8 TIME (Weeks) Figure 6. Plasma glucose levels following bis(maltolato)oxovanadium (IV) treatment for 8 weeks. The four treatment groups were: control (n=10), control-BMOV (n=l 1), diabetic (n=l 1) and diabetic-BMOV (n=l 1). Statistical analysis was done by G L M - A N O V A followed by Newman-Keuls, p < 0.05. * different from control groups, @ different from all other groups. 38 E CO 1 CO < • BASAL 3 WEEK 8 I # X Con Con-BMOV Dia Dia-BMOV TREATMENT GROUPS Figure 7. Plasma insulin levels following bis(maltolato)oxovanadium (IV) treatment for 8 weeks. The four treatment groups were: control (n=10), control-BMOV (n=l 1), diabetic (n=l 1) and diabetic-BMOV (n=l 1). Statistical analysis was done by G L M - A N O V A followed by Newman-Keuls, p < 0.05. * different from control basal, @ different from control at week 8, # different from respective basal. Basal value was determined one week after STZ injection and before the treatment of B M O V . 39 BASAL WEEK 8 T Con Con-BMOV Dia Dia-BMOV TREATMENT GROUPS Figure 8. Plasma triglyceride levels following bis(maltolato)oxovanadium (IV) treatment for 8 weeks. The four treatment groups were: control (n=10), control-BMOV (n=l 1), diabetic (n=l 1) and diabetic-BMOV (n=l 1). Statistical analysis was done by G L M - A N O V A followed by Newman-Keuls, p < 0.05. * different from other three groups at week 8. 40 I insulin levels at week 8 in the vanadium-treated diabetic group were not different from their basal levels, indicating the preservation of pancreatic function by vanadium treatment. Vanadium-treated diabetic rats had normal plasma triglyceride level during the whole study period (Figure 8). No significant difference was observed between the control and vanadium-treated control groups for all the parameters mentioned above except the lower basal insulin level in the control-treated group when compared to the controls (1.17±0.14 vs 1.73±0.25 ng/ml, Figure 7). The possible explanation for this result is currently unknown. However, at week 8, plasma insulin levels in the control and vanadium-treated control groups were not different from each other. II) Plasma parameters at termination Table III shows plasma glucose and insulin levels at termination following 8 weeks of vanadium treatment. Half of the animals in each group received an iv dose of insulin at 5 U/kg and blood samples were collected at 5 minutes post-insulin injection. Plasma insulin levels were elevated over 400-fold in all of the insulin-injected groups compared to the non-insulin-treated groups. However, plasma glucose levels were unchanged in all of the insulin-injected groups compared to their corresponding non-insulin groups. In the second experiment, which had the same treatment protocol and received the same dose of insulin at termination, blood samples were collected at 15 minutes post insulin injection. Plasma insulin levels were over 200-fold higher in the insulin-treated animals as compared to their respective non-insulin injected groups (Table IV). However, plasma insulin levels at 15 minutes post-injection were about 2- to 3-times lower than that at 5 minutes post-injection (Table 41 T A B L E III P L A S M A P A R A M E T E R T R E A T M E N T G R O U P S G L U C O S E (mM) C O N C . INSULIN (ng/ml) C O N C . Control (-) (n=4) 7.4+0.4 5.4±0.6 Control-BMOV (-) (n=5) 8.8±0.6 4.111.2 Diabetic (-) (n=3) 22.510.2* 0.410.1* Diabetic-BMOV (-) (n=4) 9.1±0.3 0.410.1* Control (+) (n=6) 8.2±0.6 1324156.7 Control-BMOV(+) (n=5) 7.2±0.3 10521219 Diabetic (+) (n=7) 23.210.9* 1267174 Diabetic-BMOV (+) (n=6) 8.4±0.7 10631118 Plasma parameters at termination following 8 weeks of BMOV treatment in the drinking water in control and diabetic rats in the presence (+) or absence (-) of intravenous insulin administration (5 U/kg, 5 minutes). Statistical analysis was done by two-way ANOVA followed by Newman-Keuls, p<0.05. * different from control. 42 T A B L E IV P L A S M A P A R A M E T E R T R E A T M E N T GROUPS Glucose Cone. (mM) Insulin Cone, (ng/ml) Triglyceride Cone. (mM) Control (-) 7.2±0.16 1.4010.32 0.2510.05 Control-BMOV (-) 5.911.03 3.211.48 0.4210.2 Diabetic (-) 14.2±1.03 * 0.53+0.05 * 0.3910.1 Diabetic-BMOV (-) 7.7+1.1 0.6610.19 * 0.2410.08 Control (+) 3.110.07 # 284110.3 0.1310.04 Control-BMOV (+) 3.410.28 # 432173.5 0.2410.08 Diabetic (+) 16.313.62* 207114.9 1.1310.35* Diabetic-BMOV(+) 3.510.21# 396134 0.1910.1 Plasma parameters at termination following 8 weeks of BMOV in the control and diabetic rats in the presence (+) or absence (-) of intravenous insulin injection (5 U/kg, 15 minutes). Statistical analysis was done by two-way ANOVA followed by Newman-Keuls, p<0.05. * different from control group, # different from respective non-insulin group. 43 III+IV). Plasma glucose levels were significantly lowered by insulin in the control, vanadium-treated control and vanadium-treated diabetic groups. However, insulin administration did not r change plasma glucose levels in the diabetic group, indicating the existence of insulin-resistance in this group of animals. Plasma triglyceride levels were not affected by insulin administration. However, the plasma triglyceride level in the diabetic plus insulin group was significantly higher than that of all other groups. III) 5' nucleotidase activity in subcellular fractions of cardiac tissue The subcellular fractionation method used in the current study had been evaluated in a previous study by the use of a plasma membrane marker enzyme, N a + / K + ATPase (106). From the previous study, we found that the plasma membrane was 88 times more enriched in N a + / K + ATPase activity when compared to the homogenate. There was little plasma membrane contamination in the intracellular membrane. Only 15% of the N a + / K + ATPase activity in the plasma membrane fraction was present in the intracellular membrane fraction (Table V). To further verify this method, the activity of another plasma membrane marker enzyme, 5' nucleotidase, was measured in the various fractions obtained in the present study. We found that the plasma membrane was 107-fold more enriched in 5' nucleotidase activity when compared to the homogenate, again indicating the membrane fractions obtained from this fractionation procedure were highly purified (Table V). IV) GLUT4 levels in various fractions from the fractionation procedure We also investigated GLUT4 levels following the fractionation steps by E C L Western blot. GLUT4 was highly enriched in the plasma and intracellular membrane fractions (Figure 9) 44 T A B L E V 5 Nucleotidase N a / K + ATPase Activity Activity H o m o g e n a t e 1 . 2 2 ± 0 . 4 5 1 . 3 ± 0 . 5 ( n = l l ) ( n = 6 ) P l a s m a m e m b r a n e 1 3 0 ± 2 5 1 1 4 ± 9 . 4 ( n = l l ) ( n = l l ) I n t r a c e l l u l a r 1 6 . 8 ± 2 . 6 m e m b r a n e ( n = 1 0 ) N a + / K + ATPase and 5' Nucleotidase activity (pmole/mg/hr) in subcellular fractions obtained from Wistar rat hearts. Activity values represent mean ± S E M . "n" denotes the number of animals in each group (the data for N a + / K + ATPase was adopted from L i et al., 1998). 45 whereas it was barely detected in 6 other fractions from this fractionation procedure (data not shown, see Figure 2 for fractionation procedure). As expected, the GLUT4 level increased in plasma and decreased in intracellular membrane fractions in response to insulin stimulation (Figure 10). Interestingly, we found an unidentified intracellular fraction which was also highly enriched in GLUT4 (Figure 9, 10). Like the intracellular GLUT4 pool, this unidentified portion also declined substantially upon insulin stimulation (Figure 10). Later we found that this unidentified intracellular fraction also had a similar enzymatic profile as that of the intracellular membrane fraction. Therefore, the unidentified intracellular portion was pooled together with the intracellular membrane fraction as the insulin-responsive intracellular GLUT4 pool. The yield of GLUT4 from the plasma and intracellular membrane fractions was also calculated in a control Wistar rat heart (total yield of GLUT4 from each fraction = value from the densitometer/pg of protein X total amount of protein in pg from each fraction), it was found that the intracellular membrane fraction had about 9 times more GLUT4 than that of the plasma membrane fraction under basal condition (21.7arbitrary units in the plasma membrane vs 193.4 arbitrary units in the intracellular membrane). V) Temporal effects of insulin on cardiac GLUT4 mobilization The time course of GLUT4 translocation in the heart of Wistar control rats was studied at 0, 5, 15, and 30 minutes after insulin injection. As shown in Figure 11, the plasma membrane GLUT4 level reached its maximum at 15 minutes after insulin injection. At the 30-minute time point, plasma membrane GLUT4 declined almost back to the basal level. GLUT4 mobilization to the plasma membrane was shown by a 60% increase in plasma membrane GLUT4 level at 15 minutes after insulin administration. The elevation in plasma membrane GLUT4 level was statistically significant when compared to the basal level at this time point. 46 Figure 9. GLUT4 content following the fractionation procedure in a control Wistar rat heart determined by E C L Western blot. P M : plasma membrane, IM?: unidentified intracellular GLUT4 pool. IM: intracellular membrane. Figure 10. GLUT4 content following the fractionation procedure in a control and a control plus insulin stimulated (iv, 5U/kg, 15 minutes) Wistar rat hearts determined by E C L Western blot. +: in the presence of insulin, -'. in the absence of insulin. The time-dependent effect of insulin on GLUT4 mobilization from the intracellular membrane fraction was also examined and is shown in Figure 12. GLUT4 levels in the intracellular membrane fraction decreased about 40% at 5 minutes after insulin injection. There was no further GLUT4 mobilization from the intracellular membrane fraction beyond 5 minutes after insulin injection as shown by no significant difference among 5, 15, and 30 minutes after insulin injection. From the results of the time course study, we decided to study the effects of vanadium on insulin-induced GLUT4 translocation at 5 minutes as the initial insulin response and at 15 minutes after insulin injection as the maximal insulin response. VI) The effects of vanadium on insulin-mediated GLUT4 translocation in the control and diabetic rats at 5 minutes after insulin administration (early insulin response) Figure 13 summarized the effects of vanadium and insulin on GLUT4 mobilization in the control and diabetic groups. Plasma membrane GLUT4 levels under basal and insulin-stimulated states in the vanadium-treated control group were not significantly different from their respective controls. However, insulin stimulation caused a significant increase in plasma membrane GLUT4 level in the vanadium-treated control group (-30%) but not in the control group (-12%) when compared to their corresponding basal state. Basal plasma membrane GLUT4 level was significantly lower in the diabetic group (50%) when compared to the control and vanadium-treated control groups. No significant difference in basal plasma membrane GLUT4 was detected between the diabetic and the vanadium-treated diabetic groups. On the one hand, there was a significant increase in plasma 48 Figure 11. Plasma membrane GLUT4 levels at 0, 5, 15 and 30 minutes after insulin injection (5U/kg, iv) in the control Wistar rat heart determined by E C L Western blot. P M denotes plasma membrane. Values are the mean ± S E M from 5 to 6 determinations. Statistical analysis was performed using one-way A N O V A followed by Neuman-Keuls (p<0.05). * different from the basal level (PMO). 49 Figure 12. Intracellular membrane GLUT4 levels at 0, 5, 15 and 30 minutes after insulin injection (5U/kg, iv) in the control Wistar rat heart determined by E C L Western blot. IM denotes intracellular membrane. Values are the mean ± S E M from 5 to 6 determinations. Statistical analysis was performed using one-way A N O V A followed by Neuman-Keuls (p<0.05). * different from the basal level (IMO). 50 membrane GLUT4 after insulin stimulation in both diabetic groups. The magnitude of insulin-induced GLUT4 translocation was 2.1-fold in the diabetic and 3.6-fold in the diabetic-vanadium-treated groups. However, the plasma membrane GLUT4 level in the insulin-stimulated state in the diabetic group was still significantly lower than that of the insulin-stimulated control groups. On the other hand, the plasma membrane GLUT4 level after insulin injection in the vanadium-treated diabetic group was not different from its respective control and was significantly higher than that of its corresponding diabetic group. VII) The effects of vanadium on insulin-regulated GLUT4 translocation in the control and diabetic rats at 15 minutes after insulin injection (maximal insulin response) At 15 minutes after insulin injection, all the groups had a significant increase in plasma membrane GLUT4 when compared to their corresponding non-insulin treated groups (Figure 14). The magnitude of insulin-mediated GLUT4 translocation was about 60% in the control and control-vanadium-treated groups, which was consistent with that from the time course study, whereas the degree of insulin-induced GLUT4 mobilization was about 2.8-fold in the diabetic and 2.4-fold in the diabetic-vanadium-treated groups. Consistent with what we found at the 5-minute time point, basal plasma membrane GLUT4 was significantly lower in the diabetic and vanadium-treated diaetic groups when compared to the basal level of the controls. However, in contrast to 5 minutes post-insulin injection, plasma membrane insulin-stimulated GLUT4 level in the vanadium-treated diabetic group was not significantly different from its respective diabetic group. GLUT4 mobilization from the intracellular pool in response to insulin, as indicated by a decrease in GLUT4 level from the basal level, was also investigated at 15 minute after insulin 51 Figure 13. Plasma membrane GLUT4 levels at 5 minutes after insulin injection (5U/kg, iv) determined by E C L Western blot. Values are the mean ± SEM from 5 to 6 determinations. Statistical analysis was performed using G L M A N O V A (p<0.05). C: control, CV: control treated with vanadium; D: diabetic; DV: diabetic treated with vanadium; I: insulin. * different from corresponding non-insulin treated group, # D and DV were significantly lower than C and C V , @ D+I was significantly lower than C+I, CV+I, and DV+I. 52 GLU ntrol) 200 CD c o o 150 CD t _Q E CJ 100 CD ~~—' E CO " c CD •i—» 50 E C CO CO o o 0 C L @ C C+l CV CV+I D + o + + < + Figure 14. Plasma membrane G L U T 4 levels at 15 minutes after insulin injection (5U/kg, iv) determined by E C L Western blot. Values are the mean ± S E M from 5 to 6 determinations. Statistical analysis was performed using G L M A N O V A (p<0.05). C: control, C V : control treated with vanadium; D: diabetic; D V : diabetic treated with vanadium; I: insulin. * different from corresponding non-insulin treated group, # D and D V were significantly lower than C and C V . @ D+I and DV+I were significantly lower than C+I and CV+I. 53 injection (Figure 15). A l l the groups had a significant decrease in intracellular membrane GLUT4 when compared to their corresponding non-insulin treated groups. Intracellular GLUT4 dropped 44-57% after insulin stimulation in the control and vanadium-treated control groups, which was again consistent with data from the time course study. In the diabetic and vanadium-treated diabetic groups, insulin stimulation resulted in a 60% and 45% decrease, respectively, in the intracellular GLUT4 pool. The intracellular GLUT4 level in both the basal and insulin-stimulated states in the diabetic group was significantly lower than that of the corresponding controls. However, the intracellular GLUT4 level in the basal and the insulin-stimulated states in the vanadium-treated diabetic group was significantly higher than that of its corresponding diabetic group and was comparable to its respective control group. VIII) Results from the ELISA assay To verify the ELISA assay, we examined the time course of cardiac GLUT4 translocation and, in addition, the effects of vanadium on insulin-regulated GLUT4 translocation in the control and diabetic rats at the 5-minute time point by the ELISA method. Figure 16 and 17 indicated the changes in plasma and intracellular membrane GLUT4 levels at 0, 5, 15 and 30 minutes after insulin injection in the control hearts. As was found from the E C L Western blot, plasma membrane GLUT4 was increased significantly (about 60%) at 15 minutes after insulin injection. Intracellular membrane GLUT4 was also found decreased about 56% at 5 minutes and no further GLUT4 mobilization from the intracellular fraction beyond 5 minutes after insulin injection was observed. Figure 18 shows plasma membrane GLUT4 levels in the control, control-treated, diabetic and diabetic-treated as well as their corresponding insulin-stimulated groups. Because the "n" number was relatively small (2 to 3 determinations), it was not appropriate to do a statistical analysis on these data. However, the data from the ELISA assay did show a patternof results similar to that obtained with the ECL Western blot. 54 Figure 15. Intracellular membrane G L U T 4 content at 15 minutes post insulin injection (5U/kg, iv) determined by E C L Western blot. Values are the mean ± S E M from 5 to 6 determinations. Statistical analysis was performed using G L M A N O V A (p<0.05). C: control, C V : control treated with vanadium; D : diabetic; D V : diabetic treated with vanadium; I: insulin. * different from corresponding non-insulin treated group, # D was significantly lower than C, C V and D V . @ D+I was significantly lower than C+I, CV+I, and DV+I. 55 PMO PM5 PM15 PM30 Figure 16. Plasma membrane GLUT4 content at 0, 5, 15, and 30 minutes after insulin injection (5 U/kg, iv) in the control Wistar rat heart determined by the ELISA assay. P M denotes plasma membrane. Values are the mean ± S E M from 3 determinations. Statistical analysis was performed using one-way A N O V A followed by Neuman-Keuls (p<0.05). * different from the basal level (PMO). Figure 17. Intracellular membrane GLUT4 levels at 0, 5, 15 and 30 minutes after insulin injection (5 U/kg, iv) in the control Wistar rat heart determined by the ELISA assay. IM denotes intracellular membrane. Values are the mean ± S E M from 3 determinations. Statistical analysis was performed using one-way A N O V A followed by Neuman-Keuls (p<0.05). * different from the basal level (IMP). 57 Figure 18. Plasma membrane GLUT4 levels at 5 minutes after insulin injection (5 U/kg, iv) determined by the ELISA assay. Values are the mean ± S E M from 3 determinations. Where there were 2 determinations, no S E M was calculated (C+I, CV+I and D). C: control, CV: control treated with vanadium; D: diabetic; DV: diabetic treated with vanadium; I: insulin. 58 DISCUSSION I) Evaluation of the subcellular fractionation method The current fractionation method was evaluated previously in our lab by measuring the activity of N a + / K + ATPase, a commonly used plasma membrane marker enzyme. From our previous study, we found the plasma membrane was highly enriched in Na / K ATPase (88-fold) when compared to homogenate and the plasma membrane contamination in the intracellular membrane was low (<15%) in both Wistar and Zucker rats (Li et al., 1998). In the present study, the activity of another plasma membrane marker enzyme, 5' nucleotidase, was measured in the various fractions obtained from this method. We found that plasma membrane was 107-fold more enriched in 5' nucleotidase activity when compared to the homogenate. Thus the plasma membrane fraction isolated from this procedure appeared to be relatively pure. As there is no currently available marker for the intracellular GLUT4 which is mostly located in T-V elements, the relative enrichment of the intracellular membrane fraction could not be determined. However as shown in the present study, a 40% decrease in the GLUT4 content in response to insulin stimulation in the control heart confirmed that the intracellular membrane obtained closely represents the insulin-regulated pool as shown by immunogold labeling experiments and other biochemical studies (Watanabe et al., 1984; Slot et al., 1991b; Fischer et al., 1997). Furthermore, results from ECL Western blot also showed that GLUT4 was mostly enriched in the plasma and intracellular membrane fractions but barely detectable in any other fractions using this method. Insulin stimulation caused an increase in plasma membrane GLUT4 that correlated with the decrease in the intracellular GLUT4 pool. 59 In the present study, the possibility of cross-contamination cannot be ruled out due to the non-availability of a marker specific for the intracellular GLUT4 compartment. Alternatively, the use of "negative marker enzymes" (see below) to determine the relative contamination in the plasma membrane is another indirect method for determining the relative purity of the membrane fractions. Mitochondrial and sarcoplasmic reticular membrane are the most probable contaminating membranes in the plasma membrane fraction. Thus sodium azide-sensitive N a + / K + ATPase, a marker for mitochondrial membrane, and C a 2 + ATPase, a marker for sarcoplasmic reticulum, are possible negative marker enzymes that could be measured to evaluate relative contamination in the plasma membrane fractions. The relative purity of the membrane fractions has been established previously in our laboratory ( L i et al., 1998). II) Insulin resistance in the long-term STZ-diabetic rats In the present study, exogenous insulin was shown to significantly lower plasma glucose in the control, control-treated and diabetic-treated animals but not in the diabetic animals at 15 minutes after insulin injection. This was not due to the lack of an increase in plasma insulin levels in vivo in the diabetic group since the insulin level in the diabetic group was comparable to the other three groups at an iv dose of 5 U/kg. Elevated plasma glucose level in the STZ-diabetic rats is generally believed to be due to insulin deficiency. However, long-term STZ-diabetes could result in insulin-resistance (Napoli et al., 1995). The inability of exogenous insulin to reduce plasma glucose in the STZ-diabetic rats observed in the present study may be due to both hepatic and peripheral insulin resistance. Results from the present study indicate that insulin resistance in the long-term STZ-diabetic rats appears to be associated with a decreased GLUT4 level in the intracellular membranes, with less GLUT4 available to be translocated to the plasma 60 membrane in response to insulin. However, the possibility that exogenous insulin may lower plasma glucose levels at a later time point in the diabetic group cannot be ruled out since the present study did not measure plasma glucose levels at times beyond 15 minutes after insulin injection. It was shown that plasma triglyceride level in the diabetic plus insulin group was significantly higher than that of all other groups whereas it was not different between diabetic and controls. One possible explanation for the difference in plasma triglycerides may be attributed to the range of levels observed in the diabetic group (1 - 5.31 mM) and that during the random division of the group into with or without insulin, animals with higher plasma triglycerides were grouped together to create an artifact in these data (week 8: 3.87±0.6 in the diabetic+insulin group vs 1.67±0.17 mM in the diabetic group). Ill) Lower expression of cardiac GLUT4 in the STZ-diabetic rat and the restoration of cardiac GLUT4 level by vanadium treatment The finding of a lower basal GLUT4 level in both plasma and intracellular membrane of the STZ-diabetic heart in the present study indicates that there may be an overall decrease in the cardiac expression of GLUT4. This is consistent with the in vitro study of Eckel et al. (1990) in which the mRNA level of GLUT4 as measured by Northern blot analysis of total R N A isolated from the cardiocytes of STZ-diabetic rats was found to decrease by 87%. A lower cardiac expression of GLUT4 in the STZ-diabetic rats was also demonstrated in several in vivo studies. Camps et al. (1992) assessed GLUT4 protein and mRNA levels in heart, red and white muscle, as well as in brown and white adipose tissue from 7-day STZ-induced diabetic and 48-h-fasted 61 rats. They found that GLUT4 mRNA decreased to a greater extent than GLUT4 protein in the heart in response to diabetes. They suggested that the maintenance of GLUT4 protein content may be due to the triggering of adaptive mechanisms at translational or post-translational steps. However, studies from other investigators demonstrated that these adaptive mechanisms are attenuated or lost as diabetes proceeds. Stanley et al. (1994) found that one month of STZ-diabetes resulted in a modest decrease in myocardial GLUT4 protein level (11-23%) when measured in left ventricular biopsies. However, in 10 to 12 week STZ-diabetic Sprague-Dawley rats, myocardial GLUT4 was decreased by 70% (Hall et al., 1995). A similar reduction in myocardial GLUT4 protein content was observed in 12 to 16 week STZ-diabetic Sprague-Dawley rats (Kainulainen et al., 1994). In the present study, basal plasma and intracellular membrane GLUT4 levels decreased about 50% and 40%, respectively, in 9 week STZ-diabetic rats which was a greater increase than that of the one month (Stanley et al., 1994) but lower than that of the 12 to 16 weeks (Kainulainen et al., 1994) of STZ-diabetes. A l l of these results indicate that there is a correlation between the length of diabetes and the level of myocardial GLUT4 protein. Myocardial GLUT4 protein content declines in conjunction with the progress of diabetes. In the present study, basal plasma membrane GLUT4 content was shown to be reduced by 50% in the STZ-diabetic rat heart. The contribution of cell-surface GLUT4 to basal glucose transport is not known since GLUT4 is mostly located intracellularly in the basal state. As mentioned previously, the evidence that cardiac GLUT1 is localized to the plasma membrane together with the demonstration of an absence of GLUT4 at the cardiomyocyte cell surface under basal conditions indicates that GLUT1 may be a major determinant of basal glucose transport in cardiac muscle (Slot et al., 1991b). Whereas the GLUT1 transporter is thought to be predominantly involved in basal glucose uptake, evidence indicates that GLUT4 plays a major 62 role in mediating insulin-stimulated glucose uptake in cardiac muscle (Camps et al., 1992; Slot et al., 1991b). However, a quantitative assessment of the precise contribution of each transporter to glucose uptake would be necessary to confirm this speculation and is currently lacking. Cardiac GLUT4 depletion may have implications for heart function in the STZ-diabetic rat but this is not clear at present. In the diabetic state, myocardial glucose transport was shown to be depressed as a result of glucose transporter depletion (Garvey et al., 1989). Recent evidence indicates that glucose transport/utilization by cardiac muscle cells is critical for the maintenance of normal function. Thus ablation of the GLUT4 in mice leads to cardiac hypertrophy and major morphologic alterations in this organ (Katz et al., 1995). Moreover, a high rate of cardiac glucose metabolism may be crucial in pathophysiologic conditions such as ischemia, as indicated by the beneficial effects of a selective increase in glucose utilization in animal and clinical studies (Mallet et al., 1990; Taegtmeyer et al., 1995). The impairment of heart glucose metabolism in diabetes mellitus may contribute to the mechanical dysfunction and cardiomyopathy observed in this disease. The observation that basal intracellular GLUT4 level was significantly higher in the diabetic-treated group when compared to the diabetic group in the same state indicates that vanadium treatment may restore total cellular GLUT4 content in the diabetic* group. The restoration of the intracellular GLUT4 pool in STZ-diabetic rats thus makes more GLUT4 available to be translocated to cell surface in response to insulin, therefore, improving insulin sensitivity. Vanadium treatment has been shown to restore GLUT4 expression in other tissues such as the skeletal muscle of STZ-diabetic rats (Strout et a l , 1990). In the heart, the effect of vanadium treatment on diabetes-induced alterations in GLUT4 transporter was also investigated by Kopp et al. (1997). In their study, vanadyl sulfate was administered orally during a 10-week 63 trial period to STZ-diabetic and control rats. They found that total cardiac myocyte and sarcolemmal GLUT4 protein levels were significantly lower in the diabetic group relative to control. Vanadium treatment of diabetic rats produced a normalization of both sarcolemmal GLUT4 and total cardiac myocyte levels towards control levels. Although most of their results were in agreement with those of the present study, the finding that sarcolemmal GLUT4 was normalized by vanadium treatment in the diabetic rats was contrary to our study in which we did not see any change in basal plasma membrane GLUT4 level in the diabetic animal after vanadium treatment. On the one hand, as described earlier, GLUT4 is mainly responsible for insulin and/or some other stimulus-regulated glucose uptake and the contribution of cell-surface GLUT4 to basal glucose transport is currently unknown. On the other hand, the fact that cardiac GLUT1 is localized to the plasma membrane under basal condition indicates that GLUT1 may be a major determinant of basal glucose transport in cardiac muscle (Slot et al., 1991b). It is possible that vanadium may mediate at least some of its effects by increasing GLUT1 content and/or functional activity. In fact, vanadium has been shown to enhance the expression of GLUT1 in NIH 3T3 mouse fibroblasts (Mountjou et al., 1990). IV) Cardiac GLUT4 translocation in the control and diabetic rats. In the present study, it was shown that plasma membrane GLUT4 level increased about 60% at 15 minutes after insulin injection (5 U/kg) in the control heart. Concomitant with the increase in plasma membrane GLUT4, the intracellular GLUT4 level decreased about 40-50%. The magnitude of insulin-stimulated GLUT4 translocation observed in the present study was in excellent agreement with the findings of Rattigan et al. (1991) and Fischer et al. (1995). In Rattigan's study, the heart from Wistar control rats was perfused with insulin and a 60% increase in plasma membrane GLUT4 level was found when determined by subcellular fractionation and 64 Western blot. In Fischer's study, isolated cardiomyocytes were incubated with insulin and a 60% increase in plasma membrane GLUT4 was also found when determined by the same methods. The magnitude of cardiac GLUT4 recruitment to the plasma membrane is also consistent with that reported by other biochemical studies in which a 1.4- to 2-fold increase occurred in skeletal muscle in response to insulin in vivo. However, several other studies showed a much higher degree of cardiac GLUT4 recruitment in response to insulin. A 2-fold increase in plasma membrane GLUT4 level was reported by Rett et al. (1996) while a 3-fold increase in plasma membrane GLUT4 was found by Sun et al. (1994) and Zaninetti et al. (1988) after cardiac perfusion with insulin. The fact that the data from our in vivo study is consistent with some of the in vitro studies indicates that the discrepancy in the magnitude of cardiac GLUT4 translocation may mainly be due to the limitations of the subcellular fractionation technique in estimating glucose transporter translocation. It has been shown by immunogold labeling studies that insulin caused a 40-fold increase in the amount of GLUT4 in the plasma membrane in both the heart and adipose tissue (Slot et al., 1991a; 1991b). In addition, a higher magnitude of GLUT4 translocation (5.7-fold) could be directly estimated by photolabeling glucose transporters on the cell surface whereas a much lower magnitude of cardiac GLUT4 translocation was found in the same study when measured by the subcellular fractionation method (Fischer et al., 1997). Neither of these alternative techniques involve the isolation of membrane fractions and therefore avoid cross contamination of subcellular membrane fractions. In spite of the limitations in fractionation techniques for the estimation of subcellular distribution of GLUT4, it remains the most commonly used method since it is relatively easy to perform and can be used for whole tissue whereas cell-surface photolabeling can be done only with isolated cells. The intracellular GLUT4 pool was decreased about 60% at 5 minutes and sustained up to 30 minutes but plasma membrane GLUT4 content was increased around 60% at 15 minutes post-65 insulin injection. The reason for the time difference between the increase in plasma membrane GLUT4 level and the decrease in the intracellular GLUT4 pool is currently unknown. Discrepant changes in plasma and intracellular membrane GLUT4 levels have also been reported in some other studies (Dombrowski et al., 1998; Rouru et al., 1995). This finding may be best explained by a model postulating two intracellular GLUT4 pools. On the basis of the proteins associated with the two intracellular GLUT4 pools, one might represent an endosomal compartment whereas the other one, referred to as intracellular GLUT4 storage vesicles, is a separate entity from the endosomal compartment. A growing body of evidence indicates that GLUT4 is constitutively recycled via the endosomal recycling pathway in which GLUT4 is internalized into early endosomes and then consucutively sorted back into the storage pool before reappearing on the cell surface. This model was supported by studies using photolabels (Holman et al., 1990; Jhun et al., 1992). The sorting of endocytosed GLUT4 into a storage pool was further supported by the findings from immunocytochemical studies (Slot et al., 1991a and 1991b). It was observed that GLUT4 recycles constantly between the endosomal compartment and the plasma membrane and the stimulation of the exocytotic rate constant is likely the major mechanism for GLUT4 translocation (Slot et al., 1991a and 1991b). In the insulin-stimulated state, endosomes stained positive for GLUT4 were also stained positive for albumin, an indicator of early endosomes. Some endosomes were connected with elongated tubular structures of T-V elements. These observations suggest that GLUT4 is recycled by endocytosis and then sorted back into T-V elements, the largest intracellar pool. The subcellular fractionation method employed in this study may selectively favor the isolation of the intracellular GLUT4 storage pool. Therefore, the decline in the plasma membrane GLUT4 occurred prior to the increase in the intracellular GLUT4 storage pool because of the endosomal GLUT4 comparment. It is possible that at the time when the decrease in plasma membrane GLUT4 was observed , GLUT4 66 was recycled back to the endosomal comportment before it can be sorted back into the storage compartment. In the current study, the magnitude of insulin-induced GLUT4 translocation in heart was about 2.8-fold in the diabetic group 15 minutes after insulin injection. However, only a 60% decrease in intracellular GLUT4 pool was found in the diabetic group at this time point. The discrepancy in these changes in the plasma and intracellular membrane GLUT4 levels may be explained by the fact that GLUT4 is mostly localized in the intracellular site in the absence of stimuli. The increase in plasma membrane GLUT4 could be prominent because of the lower basal GLUT4 level whereas the decrease in intracellular membrane GLUT4 could be less noticeable because of the relative larger intracellular pool. This phenomenon was also found in our control groups (60% increase in plasma membrane versus 40% decrease in intracellular membrane GLUT4) and in several other studies (Vogt et al., 1990; Pulido et al., 1996). The magnitude of insulin-induced GLUT4 recruitment was much higher in the diabetic group compared to the control group, indicating that long term insulin deficiency (9-week STZ diabetes) was still responsive to a supraphysiological dose of insulin in terms of GLUT4 translocation. This finding was consistent with that reported by Kahn et al. (1996) in which they found that after 7 days of insulin treatment of the diabetic rat, the glucose transport response to insulin was not only restored, but actually increased to 3-fold control levels. Although the magnitude of cardiac GLUT4 translocation was higher in the diabetic group when compared to the control group, the absolute levels of both plasma and intracellular membrane GLUT4 under basal and insulin-stimulated conditions were lower than that of their respective control groups. This may account for the ineffectiveness of insulin injection at a dose of 5 U/kg to lower plasma glucose in the diabetic group. These data also indicate that cardiac insulin resistance in long term STZ-diabetes (9-week STZ) may not result totally from impairment of insulin-regulated GLUT4 translocation but from the reduction of total cellular GLUT4 content. 67 V) The effects of vanadium on insulin-regulated GLUT4 translocation in the control and diabetic animals In the present study, basal plasma membrane GLUT4 level was not altered by vanadium treatment in either the control or diabetic groups. At 5 minutes after insulin administration, which was considered as the initial insulin response, insulin stimulation caused a significant increase in plasma membrane GLUT4 level in the control-treated group (-30%) but not in the control group. In the diabetic-treated group, plasma membrane GLUT4 levels were significantly higher than that of the diabetic group at this time point. However, at 15 minutes post-insulin injection, which was considered as the maximal insulin response, no significant differences in plasma membrane GLUT4 levels were observed between the control and control-treated groups and between the diabetic and the diabetic-treated groups. Consistent with the changes in plasma membrane GLUT4, the elevated basal intracellular GLUT4 level in the diabetic-treated group did not result in more translocation of GLUT4 to the plasma membrane at the 15-minute time point. The finding that the magnitude of insulin-mediated GLUT4 recruitment in the diabetic-treated group (3.6-fold) is significantly higher than that of the diabetic group (2.1-fold) at 5 minutes but not at 15 minutes post insulin injection (2.4 vs 2.8-fold) indicates that vanadium treatment enhances insulin response in the early time point and causes a shift of insulin response. Once the insulin response is maximal, the effects of vanadium are masked by insulin. This is also supported by the data showing that insulin stimulation significantly increases plasma membrane GLUT4 in the control-treated group but not in the control group at 5 minutes after insulin administration. At the time point of maximal insulin response (15 minutes after insulin), however, both the control and the control-treated group showed a similar degree of translocation. 68 Our results indicate that vanadium has a more insulin-enhancing effect than an insulin-mimetic effect in terms of cardiac GLUT4 translocation. Several in vitro studies have been conducted to examine the effects of vanadium on glucose uptake and GLUT4 translocation. Okumura et al. (1992) reported that incubation of vanadium with sarcolemmal vesicles prepared from rat hind limb skeletal muscles resulted in the stimulation of glucose uptake. The activation of D-glucose uptake was due to a large increase in the Vmax value. Therefore, they suggest that vanadium increase the intrinsic activity (turnover number) of skeletal muscle glucose transporters. However, they did not identify the type of glucose transporter in this study. In rat adipocytes, Paquet et al. (1992) found that under conditions where vanadium stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT4 in the plasma membrane was increased similarly by both insulin and vanadium, and its concentration was decreased in the low density microsomal fraction. The effects of vanadium and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT4 were not additive. They suggest that vanadium induced the recruitment of GLUT4 to the plasma membrane. The discrepancy between our study and Paquet's study may be due to the difference in experimental setting. In our study, vanadium was administrated orally and had to be absorbed from the gastrointestinal tract to reach its action site. In the in vitro study, cells were exposed to a much higher concentration of vanadium directly (3 mM in vitro [Paquet et al., 1992] vs 4 to 36 uM in vivo [Thompson et al., 1998]). It is possible that at such a high concentration, vanadium may act more like an insulin-mimetic than an insulin-enhancing agent. Several in vivo studies have been carried out recently on both Type 1 and 2 human diabetic subjects. Consistent with the present study, it was found that in Type 1 diabetic patients, vanadium lowers insulin requirements and improves glucose utilization (Goldfine et al., 1995). In Type 2 diabetes, it was also shown that vanadium enhances insulin sensitivity by both augmenting stimulation of glucose disposal and 69 enhancing suppression of hepatic glucose output (Boden et al., 1996; Halberstam et al., 1996). These studies did not investigate the glucose transporter system. To our knowledge, the present study is the first one to examine the in vivo effects of vanadium on the glucose transporter system and the interaction of vanadium with insulin in an animal model. We demonstrated that vanadium treatment could enhance insulin sensitivity in STZ-diabetic animals possibly by increasing insulin-mediated GLUT4 translocation early in the insulin response and/or by increasing GLUT4 expression with more GLUT4 available to be translocated to plasma membrane in response to insulin. VI) Evaluation of the ELISA assay We have developed a novel ELISA technique to quantitatively measure GLUT4 in various subcellular fractions. The ELISA was verified in our previous studies by running two or three internal standards (e. g. crude membranes) each time the assay was conducted (Cam et al., 1996). We found that the ELISA method is highly reproducible, having inter- and intra-assay coefficients of variation of <10%. However, the magnitude of cardiac GLUT4 translocation when measuring by the competitive ELISA method is around 30% in lean Zucker rats (Li et al., 1998) which is relatively low when compared to that being reported in the literature. Therefore, the ELISA assay was further verified in the present study. As we showed in the results, the data from the ELISA assay have a similar pattern to that from the E C L Western blot. The magnitude of cardiac GLUT4 translocation when measured by the ELISA method was around 60% in Wistar control rats which is in agreement with that from the E C L Western blot. Thus, the reliability of the ELISA technique was further confirmed in the present study. The discrepancy in the magnitude of cardiac GLUT4 translocation between the two rat strains, as explained earlier, 70 m a y m a i n l y b e d u e t o t h e l i m i t a t i o n s o f s u b c e l l u l a r f r a c t i o n a t i o n t e c h n i q u e i n e s t i m a t i n g g l u c o s e t r a n s p o r t e r t r a n s l o c a t i o n a n d m a y a l s o b e d u e t o d i f f e r e n c e s i n t h e a n i m a l s t r a i n ( Z u c k e r v s W i s t a r ) . 71 FUTURE EXPERIMENTS I) The effects of vanadium in vivo on GLUT4 translocation in skeletal muscle In the present study, we have investigated the in vivo effects of vanadium on cardiac G L U T 4 translocation. However, skeletal muscle is quantitatively the most important tissue involved in maintaining glucose homeostasis, and accounts for approximately 80% of the total body glucose disposal after a glucose infusion or ingestion (DeFronzo et al., 1981). Insulin-resistance commonly occurs in Type 1 and 2 diabetic patients. Desensitization of the glucose transport system in skeletal muscle has been proposed to be a major cause of insulin resistance (Pederson et al., 1982; Ciaraldi et al., 1982). A s mentioned earlier, it has been shown that insulin-mediated G L U T 4 translocation to T-tubules is significantly reduced in skeletal muscle of STZ-diabetic rats (Dombrowski et al., 1998). In vivo experiments have indicated that vanadium treatment in STZ-diabetes can restore glucose transporter expression in skeletal muscle in the STZ-diabetic rats (Strout et al., 1990). However, since altered expression of G L U T 4 cannot fully explain insulin resistance in skeletal muscle in the STZ-diabetic rat, vanadium may have additional effects in the regulation of glucose transport including enhanced translocation. It is of interest to examine the effects of vanadium in vivo on glucose transporter ( G L U T 4 ) translocation in skeletal muscle which is the major site of glucose disposal (DeFronzo et al., 1981). Using the STZ-diabetic rat, a model of Type 1 diabetes, and the insulin resistant fa/fa Zucker rat, a model of Type 2 diabetes, we can assess the effects of vanadium on insulin-stimulated G L U T 4 translocation in skeletal muscle of Type 1 and 2 diabetes. 72 II) The effects of vanadium on PI 3-kinase activity and expression in insulin sensitive tissue As described in the introduction, in the insulin signaling cascade, PI 3-kinase is activated when the SH2 domain of p85 subunit of PI 3-kinase binds to the tyrosine phosphorylated IRS-1. Considerable evidence indicates that activation of PI 3-kinase in insulin signaling leads to GLUT4 translocation (Kanai et al., 1993; Clarke et al., 1994; Kotani et al., 1995), although some data indicate that PI 3-kinase failed to stimulate GLUT4 translocation when it was activated by non-insulin stimuli (Isokiff et al., 1995). The difference may be due to the different distribution between PI 3-kinase activated by insulin and that activated by non-insulin stimuli. Indeed, it has recently been reported that insulin is able to target activated PI 3-kinase to specific GLUT4 containing vesicles (Robin et al., 1996). In the present study, vanadium has been shown to have insulin-enhancing activities. Therefore, it is of interest to examine whether vanadium is able to activate PI 3-kinase and, thereby, enhance GLUT4 translocation as well as its ability to target activated PI 3-kinase to GLUT4 containing vesicles in diabetic rats. It has been shown in in vitro studies that wortmannin, an inhibitor of PI 3-kinase, blocks vanadium-stimulated glucose transport (Berger et al., 1994), indicating that similar to insulin, vanadium stimulates glucose transport by activating PI 3-kinase. The activity of PI 3-kinase has been reported to be associated with insulin resistance. It has been shown that its activity is depressed in the insulin resistant ob/ob mice and its expression is reduced in the diabetic K K A y mice (Folli et al., 1993; Bonini et al., 1995). Furthermore, treatment of K K A y mice with pioglitazone, an insulin sensitizing agent, was able to restore the expression of PI 3-kinase. Since vanadium can sensitize the insulin effect in vivo, it 73 is also of interest to investigate the effects of vanadium on the expression and activity of PI 3-kinase in ob/ob mice or K K A y mice. Ill) The effects of vanadium in vivo on GLUT4 gene expression in the heart As shown in the results, the cellular protein content of cardiac GLUT4 is decreased in STZ-diabetic rats and vanadium treatment resulted in a modest increase in intracellular GLUT4 content in the diabetic rats. Furthermore, the restoration of GLUT4 content by vanadium treatment in the diabetic animals has also been reported in other studies (Kopp et al., 1997). Therefore, it is of interest to investigate whether vanadium treatment affects GLUT4 gene expression in the diabetic heart. Using the STZ-diabetic rat and the fa/fa Zucker rat as models of Type 1 and 2 diabetes, we can assess the mRNA content of GLUT4 in heart as well as the effect of treatment with vanadium. IV) The effects of vanadium on cardiac GLUT1 expression, functional activity and translocation Vanadium treatment has been shown to effectively lower plasma glucose levels in STZ-diabetic rats even in the absence of exogenous insulin. However, the lack of increase in basal plasma membrane GLUT4 in the vanadium-treated STZ-diabetic rats observed in the present study indicates that vanadium may exhibit its glucose lowering effects under basal condition by a mechanism other than an increase in basal plasma membrane GLUT4. As mentioned earlier, GLUT1 is mostly localized to the plasma membrane under basal conditions and is a major determinant of basal glucose uptake. It is possible that vanadium may mediate at least some of its effects by increasing GLUT1 content and functional activity, as well as 74 translocation in the cardiac muscle of diabetes. Therefore, it is of interest to extend our study to investigate the possible role of vanadium on G L U T 1 expression, functional activity and translocation. 75 CONCLUSIONS 1. Insulin stimulation significantly increases cardiac plasma membrane GLUT4 content which correlates to the decrease in the intracellular GLUT4 pool at 15 minutes post insulin injection in the control and vanadium-treated control groups. 2. Long term insulin deficiency (9-week STZ diabetes) results in a decrease of total cellular GLUT4 content but long term STZ-diabetic rats are still responsive to a supraphysiological dose of insulin in terms of cardiac GLUT4 translocation. 3. The observation that basal intracellular GLUT4 level is significantly higher in the diabetic-treated group when compared to that of the diabetic group indicates that vanadium treatment may restore total cellular GLUT4 content in the diabetic group. However, increased basal intracellular GLUT4 in the vanadium-treated diabetic group does not result in more insulin-mediated GLUT4 translocation at 15 minutes after insulin injection. Vanadium treatment dose, however, enhance the insulin response in terms of GLUT4 translocation at 5 minutes in the cardiac tissue of diabetic rats. 4. The finding that plasma membrane GLUT4 in the vanadium-treated diabetic group is significantly higher than that of the diabetic group at 5 minutes but not at 15 minutes after insulin injection indicates that vanadium treatment enhances insulin-mediated GLUT4 translocation by enhancing its early response and by accelerating this process. This is further supported by the data showing that insulin stimulation significantly increases plasma membrane GLUT4 in the vanadium-treated control group but not in the control group at 5 minutes after insulin administration. 76 The ELISA method for measuring GLUT4, which was developed in our laboratory was verified. The data from the ELISA assay showed a pattern similar to that from the Western blot. 77 R E F E R E N C E S Abel ED, Shepherd PR, Kahn BB. Glucose transporters and pathophysiologic states. In Diabetes Mellitus: A Fundamental and Clinacal Text. Dr Leroith, J M Olefsky, SI Taylor, Eds. Philadelphia, Lippincott, p530-543, 1996. Amir S, Meyerovitch J, Shechter Y . Vanadate ions: central nervous system action on glucoregulation. Brain Res. 419: 392-396, 1987. Balch WE. Small GTP-binding proteins in vesicular transport. Trends Biochem Sci. 15: 473-477, 1990. Baldini G, Holman R, Charron MJ, Lodish HF. Insulin and nonhydrolyzable GTP analogs induce translocation of GLUT4 to the plasma membrane in alpha-toxin-permeabilized rat adipose cells. J Biol Chem. 270: 4037-4040, 1991. Bell G l , Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Fukumoto H, Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care 13: 198-208, 1990. Berger J, Hayes N , Szalkowski D M , Bei Zhang. PI 3-kinase activation is required for insulin stimulation of glucose transport into L6 myotubes. Biochem Biophy Res Comm. 205: 570-576, 1994. 78 Boden G, Chen S, Ruiz J, George D V , Rossum V, Turco S. Effects of vanadyl sulfate on carbohydrate and lipid metabolism inpatients with non insulin dependent diabetes mellitus. Metabolism 45: 1130-1135, 1996. Bonini JA, Colca JR, Dailey C, White M , Hofmann C. Compensatory alterations for insulin signal transduction and glucose transport in insulin-resistant diabetes. A m J Physiol. 269: 759-765, 1995. Brichard SM, Assimacopoulos-Jeanner F, Jeanrenaud B. Vanadate treatment markedly increased glucose utilization in muscle of insulin-resistant fa/fa rats without modifying glucose transporter expression. Endocrinology 131: 311-317,1992. Cam M C . Studies on the mechanism of the insulin-mimetic effects of vanadium in streptozotocin-diabetic rats. Ph.D. Thesis, Faculty of Pharmaceutical Sciences, U B C , 1996. Camps M , Castello A , Munoz P, Mongar M , Testar X , Palacin M , Zorzano A. Effect of diabetes and fasting on GLUT4 (muscle, fat) glucose-transporter expression in insulin-sensitive tissue. Heterogeneous response in heart, red and white muscle. Biochem J. 282: 765-772, 1992. Charron MJ , Katz EB, Zierath JR. Metabolic and molecular consequences of modifying GLUT4 expression in skeletal muscle. Biochem Soc Trans. 25: 963-968, 1997. Charron MJ , Katz EB. Metabolic and therapeutic lessons from genetic manipulation of GLUT4. Mol Cell Biochem. 182: 143-152, 1998. 79 Ciaraldi TP, Kolterman OG, Scarlett JA, Kao M , Olefsky JM. Role of glucose transport in the postrecepter defect of non-insulin-dependent mellitus. Diabetes 31: 1016-1022,1982. Clarke JP, Young PW, Yonezawa K, Kasuga M , Holman GD. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem J. 300: 631-635, 1994. Cong L, Chen H, L i Y , Zhou L, McGibbon M A , Taylor SI, Quon M . Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose tissue. Mol Endo. 11: 1881-1890, 1997. Cormont M , Tanti JF, Zahraoui A , Van Obberghen E, Tavitian A , Le Marghand-Brostel Y . Insulin and okadaic acid induce Rab4 redistribution in adipocytes. J Biol Chem. 268: 19491-19497,1993. Cushman SW, Wardzala LJ. Potential mechanism of insulin action on glucose transport in the isolate rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem. 255: 4758-5762, 1980. Czech MP, Chawla A , Wook CW, Buxton J, Armoni M , Tang W, Joly M , Corvera S. Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization. J Cell Chem. 123: 127-135, 1993. Czech MP, Corvera S. Signaling mechanisms that regulate glucose transport. J Biol Chem. 274(4): 1865-1868, 1999. 80 Davidson NO, Hausman A M L , Ifkovits CA, Buse JB, Gould GW, Burant CF, Bell GL Human intestinal glucose transporter expression and localization of GLUT5. Am J Physiol. 262: C795-C800, 1992. DeFronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Pelber JP. The effect of insulin on the disposal of intravenous glucose: Results from calorimetry and hepatic and femoral venous catheterization. Diabetes 30: 1000-1007, 1981. D'Elia JA, Weinrauch L A , Healy RW, Libertino RW, Bradley RF, Leland OS. Myocardial dysfunction without coronary artery disease in diabetic renal failure. Am J Cardiol. 43: 193-199, 1979. Dhand R, Hara K, Hiles I, Bax B, Gout I, Panayotou G, Fry MJ , Yonezawa K, Kasuga M , Waterfield M D . PI 3-kinase: structural and functional analysis of intersubunit interactions. E M B O J. 13: 511-521, 1994. Dombrowski L, Roy D, Marette A. Selective Impairment in GLUT4 translocation to transverse tubules in skeletal muscle of STZ-induced diabetic rats. Diabetes 47: 5-12, 1998. Eckel J, Reinauer H. Insulin action on glucose transport in isolated cardiac myocytes: signalling pathways and diabetes-induced alteration. Biochem Soc Trans. 18:1125-1127, 1990. 81 Elmendorf JS, Chen D, Pessin JE. Guanosine 5'-0-(3-thiotriphosphate) (GTPgammaS) stimulation of GLUT4 translocation is tyrosine kinase-dependent. J Biol Chem. 273: 13289-13296, 1998. Etgen GJ, Fryburg DA, Gibbs E M . Nitric oxide stimulates skeletal muscle glucose transport through a calcium/contraction- and phosphatidylinositol-3-kinase-independent pathway. Diabetes 46: 1915-1919, 1997. Etgen Jr GJ, Memon AR, Thompson Jr GA, Ivy JL. Insulin and contraction stimulated translocation of GTP-binding proteins and GLUT4 protein in skeletal muscle. J Biol Chem. 268: 20164-20169, 1993. Ferro-Novick S, Novick P. The role of GTP-binding proteins in transport along the exocytic pathway. Annu Rev Cell Biol. 9: 575-599, 1993. Fischer Y , Thomas J, Rosen P, Kammermeier H. Action of metformin on glucose transport and glucose transporter GLUT1 and GLUT4 in heart muscle cells from healthy and diabetic rats. Endocrinology 136 (2): 412-420, 1995. Fischer Y , Kamp J, Thomas J, Popping S, Rose H, Carpene C, Kammermeier H. Signals mediating stimulation of cardiomyocyte glucose transport by the alpha-adrenergic agonist phenylephrine. Am J Physiol. 270: C1211-1220, 1996. Fischer Y , Thomas J, Sevilla L, Munoz P, Becker C, Holman GD, Kozka IJ, Palacin M , Testar X , Kammermeier H, Zorzano A. Insulin-induced recruitment of glucose transporters GLUT4 and 82 GLUT1 in isolated rat cardiac myocytes. Evidence for the existence of different intracellular GLUT4 vesicle populations. J Biol Chem. 272: 7085-7092, 1997. Folli F, Saad MJA, Backer JM, Kahn R. Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus. J Clin Invest. 92: 1787-1794, 1993. Frevert E U , Hamann A, Lowell BB, Flier JS, Kahn BB. Decreased insulin action precedes down regulation of Glut4 in adipocytes in a novel transgenic model of obesity and N I D D M (Abstract). Diabetes 44 (1): 38A, 1995. Froehner SC, Davies A , Baldwin SA, Lienhard GE. The blood-nerve barrier is rich in glucose transporter. J Neurocytol. 17: 173-178, 1988. Garvey WT, Huecksteadt TP, Birnbaum MJ. Pretranslational suppression of an insulin-responsive glucose transporter in rats with diabetes mellitus. Science 245: 60-63, 1989. Garvey WT, Maianu L, Hancock JA, Golichowski A M , Baron A. Gene expression of GLUT4 in skeletal muscle from insulin-resistant patients with obesity, IGT, G E M , and N I D D M . Diabetes 41: 465-475, 1992. Garvey WT. Glucose transport and NIDDM. Diabetes Care 15: 396-417, 1992. 83 Goldfine A B , Simonson DC, Folli F, Patti M E , Khan CR. Metabolic effects of sodium metavanadium in humans with insulin dependent and non insulin dependent diabetes mellitus in vivo and in vitro studies. Clin Endocrinol Metab. 80: 3311-3320, 1995. Gould GW, Merrall NW, Martin S, Jess TJ, Campbell IW, Calderhead D M , Gibbs E M , Holman GD, Plevin RJ. Growth factor-induced stimulation of hexose transport in 3T3-L1 adipocytes: evidence that insulin-induced translocation of GLUT4 is independent of activation of M A P kinase. Cell Signalling 6: 313-320,1994. Hainault I, Guerre-Millo M , Guichard C, Lavau M . Differential regulation of adipose tissue glucose transporters in genetic obesity (fatty rat). J Clin Invest. 87: 1127-1131, 1992. Halberstam M , Cohen N , Shlimovich P, Rossetti L, Shamoon H. Oral vanadyl sulfate improves insulin sentivity in N I D D M but not in obese non diabetic subjects. Diabetes 45: 695-666, 1996. Hall JL, Sexton WL, Stanley WC. Exercise training attenuates the reduction in myocardial GLUT-4 in diabetic rats. J Appl Physiol. 78 (1): 76-81, 1995. Hamann A, Benecke H, LeMarchand-Brustel Y , Susulic VS, Lowell B B , Flier JS. Characterization of insulin resistance and N I D D M in transgenic mice with reduced brown fat. Diabetes 44: 1266-1273, 1995. Hamby Rl , Zoneraich S, Sherman L. Diabetic cardiomyopathy. J A M A 229: 1749-1754, 1974. 84 Han X X , Bonen A. Epinephrine translocates GLUT-4 but inhibits insulin-stimulated glucose transport in rat muscle. Am J Physiol. 274: E700-E704, 1998. Haruta T, Morris AJ , Rose DW, Nelson JG, Mueckler M , Olefsky JM. Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway. J Biol Chem. 270:27991-27994,1995. Hayashi T, Hirshman MF, Kurth EJ, Winder WW, Goodyear LJ. Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes 47: 1369-1373, 1998. Heyliger CE, Tahiliani A G , McNeill JH. Effect of vanadate on elevated blood glucose and depressed cardiac performance of diabetic rats. Science 227: 1474-1477, 1985. Hiroshi S, Waka O, Itaru K. Insulin stimulated guanine nucleotide exchange on Rab4 via a wortmannin-sensitive signaling pathway in rat adipocytes. J Biol Chem. 272 (23): 14542-14546, 1997. Holman GD, Kozka IJ, Clark A E , Flower CJ, Saltis CJ, Habberfield A D , Simpson IA, Cushman SW. Cell-surface labeling of glucose transporter isoform Glut4 by bio-mannose photolabel. Correclation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J Biol Chem 265: 18172-18179, 1990. Holman GD, Cushman SW. Subcellular localization and trafficking of the GLUT4 glucose transporter isoform in insulin-responsive cells. Bioassays 16: 735-759, 1994. 85 Hudson A W , Fingar DC, Seidner GA, Griffiths G, Burke B, Birnbaum MJ . Targeting of the "insulin-responsive" glucose transporter (GLUT4) to the regulated secretory pathway in PC 12 cells. J Cell Biol. 122: 579-588, 1993. Isokoff SJ, Taha C, Rose E, Marcusohn J, Klip A , Skolnik EY. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake. Proc Natl Acad Sci USA 92: 10247-10251, 1995. James DE, Brown R, Navarro J, Pilch PF. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature 333: 183-185,1988. Jes K K , Lucia ER, Lewis CC, Joanne M B , John JH, Christoper S, Varsha P, Silvia C, Michael PC. Regulation of GRP-l-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol 3, 4, 5-trisphosphate. J Biol Chem. 273: 1859-1862, 1998. Jhun BH, Rampal A L , Liu H, Lachaal M , Jung C Y . Effects of insulin on steady state kinetics of Glut4 subcellular distribution in rat adipocytes. Evidence of constitutive Glut4 recycling. J Biol Chem 267: 1710-1715, 1992. Johnson JH, Ogawa A, Chem LO, Newgard CB, Alam T, Unger RH. Underexpression of beta cell high Km glucose transporters in noninsulin-dependent diabetes. Science 250: 546-549, 1990. 86 Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings B A . Molecular cloning and identification of a serine threonine protein-kinase of the 2nd-messenger subfamily. Proc Natl Acad Sci USA 88: 4171-4175 1991. Kaburagi Y , Satoh S, Tamemoto H, Yamamoto-Honda R, Tobe K, Veki K, Yamauchi T, Kono-Sugita E, Sekihara H, Aizawa S, Cushman SW, Akanuma Y , Yazaki Y , Kadowaki TJ. Role of insulin receptor substrate-1 and pp60 in the regulation of insulin-induced glucose transport and GLUT4 translocation in primary adipocytes. J Biol Chem. 272: 25839-25844, 1997. Kahn BB, Charron MJ , Lodish HF, Cushman SW, Flier JS. Different regulation of two glucose transporter in adipose cells from diabetic and insulin treated diabetic rats. J Clin Invest 84: 404-411,1989. Kahn BB, Rosen AS, Bak JF, Andersen PH, Lund S, Pedersen O. Expression of GLUT1 and GLUT4 glucose transporter in skeletal muscle of humans with insulin-dependent diabetes mellitus: regulatory effects of metabolic factors. J Clin Endocrinol Metab. 74: 1101-1109, 1992. Kahn BB, Pedersen O. Suppression of GLUT4 expression in skeletal muscle of rats that are obese from high fat feeding but not from high carbohydrate feeding or genetic obesity. Endocrinology 132: 13-22, 1993. Kahn B B . Glucose transport: Pivotal step in insulin action. Diabetes 45: 1644-1645, 1996. 87 Kainulainen H, Breiner M , Schurmann A, Marttinen A, Virjo A , Joost HG. In vivo glucose uptake and glucose transporter proteins GLUT1 and GLUT4 in heart and various types of skeletal muscle from streptozotocin-diabetic rats. Biochim Biophys Acta 1225: 275-282, 1994. Kanai F, Ito K, Todaka M , Hayashi H, Kamohara S, Ishii K, Okada T, Hazeki O, Ui M , Ebina Y . Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI 3-kinase. Biochem Biophy Res Comm. 195: 762-768, 1993. Kannel WB, McGee DL. Diabetes and cardiovascular disease: the Framingham study. J A M A 241: 2035-2038,1979. Katagiri H , Asano T, Ishihara H, Inukai K, Shibasake Y , Kikuchi M , Yazaki Y , Oka Y . Overexpression of catalytic subunit pi 10 alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 271: 16987-16990, 1996. Katagiri H , Asano T, Ishihara H, Inukai K, Shibasaki Y , Murata T, Terasaki J, Kikuchi M , Yazaki Y , Oka Y. Roles of PI 3-kinase and Ras on insulin-stimulated glucose transport in 3T3-L l adipocytes. Am J Physiol. 272: E326-E331, 1997. Katz EB, Stenbit A E , Hatton K, DePinho R, Charron MJ. Cardiac and adipose tissue abnormalities but not diabetes in mice deficient in GLUT4. Nature 377: 151-155, 1995. 88 Kishi K, Hayashi H, Wang L, Kamohara S, Tamaoka K, Shimizu T, Ushikubi F, Narumiya S, Ebina Y . Gq-coupled receptors transmit the signal for GLUT4 translocation via an insulin-independent pathway. J Biol Chem. 271: 26561-26568, 1996. Kishi K, Muromoto N , Kakaya Y , Miyata I, Hagi A , Hayashi H, Ebina Y . Bradykinin directly triggers GLUT4 translocation via an insulin-independent pathway. Diabetes 47: 550-558, 1998. Klarlund JK, Guilherme A, Holik JJ, Virbasius JV, Chawla A , Czech MP. Signaling by phosphoinositide-3, 4, 5-trisphosphate through proteins containing pleckstrin and Sec7 homology domains. Science 275: 1927-1930, 1997. Kohjiro U , Ritsuko Y H , Yasushi K, Toshimasa Y , Kazuyuki T, Boudewijn M T B , Paul JC, Issei K, Yasuo A, Yoshio Y , Takashi K. Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem. 273 (9): 5315-5322, 1998. Kopp SJ, Daar J, Paulson J, Romano FD, Laddaga R. Effects of oral vanadyl treatment on diabetes-induced alterations in the heart GLUT-4 transporter. J Mol Cell Cardiol. 29: 2355-2362, 1997. Kotani K, Carozzi AJ , Sakaue H, Hara K, Robinson LJ , Clark SF, Yonezawa K, James DE, Kasuga M . Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Biochem Biophy Res Comm. 209:343-348, 1995. Krook A , Moller DE, Dib K, O'Rahilly S. Two naturally occurring mutant insulin receptors phosphorylate insulin receptor substrate-1 (IRS-1) but fail to mediate the biological effects of 89 insulin. Evidence that IRS-1 phosphorylation is not sufficient for normal insulin action. J Biol Chem. 271: 7134-7140, 1996. Laemmli U K . Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970. Lee W, Jung C Y . A synthetic peptide corresponding to the GLUT4 C-terminal cytoplasmic domain causes insulin-like glucose transport stimulation and GLUT4 recruitment in rat adipocytes. J Biol Chem. 272: 21427-21431, 1997. LI W M , Cam M C , Poucheret P, McNeill JH. Insulin-induced Glut4 recruitment in the fatty Zucker rat heart is not associated with changes in Glut4 content in the intracellular membrane. Mol Cell Biochem. 183: 193-200, 1998. Lowell B B , Susulic V , Hamann A, Lawitts JA, Himms-Hagen J, Boyer B B , Kozak LP, Flier JS. Development of obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature 366: 740-742, 1993. Lyonnet B, Martz and Martin E. L'emploi therapeutique des derives du vanadium. La Presse Medicale 1: 191-192, 1899. Mallet RT, Hartman DA, Bunger R. Glucose requirement for postischemic recovery of perfused working heart. Eur J Biochem. 188: 481-493, 1990. Mansier P, Charlemagne D, Rossi B, Preteseille M , Swynghedauw B, Lelievre L. Isolation of impermeable inside-out vesicles from an enriched sarcolemma fraction of rat heart. J Biol Chem. 258: 6628-6635, 1983. Martin L B , Shewan A, Millar CA, Gould GW, James DE. Vesicle-associated membrane protein 2 plays a specific role in the insulin-dependent trafficking of the facilitative glucose transporter GLUT4 in 3T3-L1 adipocytes. J Biol Chem. 273: 1444-1452, 1998. Martin S, Tellam J, Livingstone C, Slot JW, Gould GW, James DE. The glucose transporter (GLUT4) and vesicle-associated membrane protein-2 (VAMP-2) are segregated from recycling endosomes in insulin-sensitive cells. J Cell Biol. 134: 625-635, 1996. Martin SS, Haruta T, Morris AJ , Klippel A , Williams Lt, Olefsky JM. Activated phosphatidylinositol 3-kinase is sufficient to mediate actin rearrangement and GLUT4 translocation in 3T3-L1 adipocytes. J Biol Chem. 271: 17605-17608, 1996. McNeill JH. Role of elevated lipids in diabetic cardiomyopathy. Diabetes Res Clinic Practice. 31: SupplS67-S71, 1996a. McNeill, JH, Cam M C , Sambandam N , Rodrigues B. Interventions in experimentally-induced diabetic cardiomyopathy. In: N.S. Dhalla, G.N. Pierce and V. Panagia (Eds), Pathophysiology of heart failure, Kluwer Academic Publishers, Boston, p32-45, 1996b. Merrall NW, Plevin R, Gould GW. Growth factors, mitogens, oncogenes and the regulation of glucose transport. Cell Signalling 5: 667-675, 1993. 91 Meyerovitch J, Fargel Z, Sack J, Schechter Y . Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. J Biol Chem. 262: 6658-6662, 1987. Monica RC, Carmen M , Hongzhi L, Amr KEJ , Morris JB, Paul FP. Insulin increases the association of Akt-2 with GLUT4-containing vesicles. J Biol Chem. 273 (13): 7201-7204, 1998. Mountjoy K G , Flier JS. Vanadate regulates glucose transporter (GLUT1) expression in NIH 3T3 mouse fibroblasts. Endocrinology 126: 2778-2787, 1990. Muckler M . Facilitative glucose transporters. Eur J Biochem. 219: 713-725, 1994. Muckler M . Family of Glucose-transporter genes: implications for glucose homeostasis and diabetes. Diabetes 39: 6-11, 1990. Napoli R , Hirshman MF, Horton ES. Mechanisms and time course of impaired skeletal muscle glucose transport activity in streptozocin diabetic rats. J Clin Invest. 96: 427-437, 1995. Novick P, Brennwald P. Friends and family: the role of the Rab GTPases in vesicular traffic. Cell 75: 597-599, 1993. Nuoffer C, Balch WE. GTPases: multifunctional molecular switches regulating vesicular traffic. Annu Rev Biochem. 63: 949-990, 1994. Okada T, Kawano Y , Sakakibara T, Hazeki O, Ui U . Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem. 269: 3568-3573, 1994. Okumura N , Shimazu T. Vanadate stimulates D-glucose transport into sarcolemmal vesicles from rat skeletal muscles. J Biochem. 112: 107-111, 1992. Omatsu-Kanbe M , Zarnowski MJ , Cushman SW. Hormonal regulation of glucose transport in a brown adipose cell preparation isolated from rats that shows a large response to insulin. Biochem J. 315: 25-31, 1996. Orci L, Thoren B, Ravazzola M , Lodish HF. Localization of the pancreatic beta cell glucose transporter to specific plasma membrane domains. Science 245: 295-297, 1989. Paquet MR, Romanek RJ, Sargeant RJ. Vanadate induces the recruitment of GLUT4 glucose transporter to the plasma membrane of rat adipocytes. Mol Cell Biochem. 109: 149-155, 1992. Pedersen O, Kahn CR, Kahn BB. Divergent regulation of the GLUT1 and GLUT4 glucose transporters in isolated adipocytes from Zucker rats. J Clin Invest. 89: 1964-1973, 1992. Pederson O, Hjolland E, Sorensen NS. Insulin receptor binding and insulin action in human and fat cells: effects of obesity and fasting. Metab Clin Exp. 31: 884-895, 1982. Pevsner J, Hsu S-C, Braun JEA, Calakos N , Ting A E , Bennett M K , Scheller RH. Specificity and regulation of a synaptic vesicle docking complex. Neuron 13: 353-361,1994. 93 Pulido N , Romero R, Suarez AI, Rodriguez E, Casanova B, Rovira A . Sulfonylureas stimulate glucose uptake through GLUT4 transporter translocation in rat skeletal muscle. Biochem Biophys Res Commun. 228 (2): 499-504, 1996. Ramanadham S, Mongold JJ, Brownsey RW, Cros FH, McNeill JH. Oral vanadyl sulfate in treatment of diabetes mellitus in rats. Am J Physiol. 257: H904-911, 1989. Rattigan S, Appleby GJ, Clark M G . Insulin-like action of catecholamines and C a 2 + to stimulate glucose transport and GLUT4 translocatioon in perfused rat heart. Biochim Biophys Acta 1094: 217-223, 1991. Regan TJ, Lyons M M , Ahmed SS. Evidence for cardiomyopathy in familial diabetes mellitus. J Clin Invest. 60: 885-889, 1977. Rett K, Wicklmayr M . Dietze GJ. Haring HU. Insulin-induced glucose transporter (GLUT1 and GLUT4) translocation in cardiac muscle tissue is mimicked by bradykinin. Diabetes 45: S66-69, 1996. Richardson JM, Balon TW, Treadway JL, Pessin JE. Differential regulation of glucose transporter activity and expression in red and white skeletal muscle. J Biol Chem. 266: 12690-12694, 1991. Ricort JM. Tanti JF. Cormont M . Van Obberghen E. Le Marchand-Brustel Y . Parallel changes in Glut 4 and Rab4 movements in two insulin-resistant states. FEBS Letters. 347: 42-44, 1994 94 Robin A , Harrion H, Morin M , Guilherme A, Czech MP. Insulin-mediated targeting of phosphatidylinositol 3-kinase to GLUT4-containing vesicles. J Biol Chem. 271: 10200-10204, 1996. Robinson LJ, Pang S, Hairns DA, Heuser J, James DE. Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes. Effects of ATP, insulin and GTPrS and localization of GLUT4 to clathrin lattices. J Cell Biol. 117: 1181-1196, 1992. Rodrigues B, Cam M C , McNeill JH. Myocardial substrate metabolism: implications for diabtic cardiomyopathy. J Mol Cell Cardiol 27: 169-179, 1995. Rodrigues B, McNeill JH. The diabetic heart: metabolic causes for the development of a cardiomyopathy. Cardiovasc Res 26: 913-922, 1992. Rossetti L, Stenbit A E , Chen W, Hu M , Barzilai N , Katz EB, Charron MJ. Peripheral but not hepatic insulin resistance in mice with one disrupted allele of the glucose transporter type 4 (GLUT4) gene. J Clin Invest. 100: 1831-1839, 1997. Rouru J, Koulu M , Peltonen J, Santti E, Hanninen V , Pesonen U , Huupponen R. Effects of metformin treatment on glucose transporter proteins in subcellular fractions of skeletal muscle in (fa/fa) Zucker rats. British J Pharmaco 115: 1182-1187, 1995. Rubier S, Dlugash J, Yeceoglu Y Z , Kumral T, Branwood A W , Grishman A . New type of cardiomyopathy associated with diabetic glomerulosclerosis. A m J Cardiol. 30: 595-602, 1972. 95 Shane Rea, David EJ. Moving GLUT4: the biogenesis and tranfficking of GLUT4 storage vesicles. Diabetes 46: 1667-1677, 1997. Shepherd PR., Gibbs E M , Weslau C, Gould GW, Kahn BB. Human small intestine facilitative fructose/glucose transporter (GLUT5) is also present in insulin-responsive tissue and brain: Investigation of biochemical characteristics and translocation. Diabetes 41: 1360-1365, 1992. Sherman L A , Hirshman MF, Cormont M , Le Marchand-Brustel Y , Goodyear LJ . Differential effects of insulin and exercise on Rab4 distribution in rat skeletal muscle. Endocrinology 137: 266-273, 1996. Shibata H , Omata W, Suzuki Y , Tanaka S, Kojima I. A synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain inhibits insulin action on glucose transport in rat adipocytes. J Biol Chem 271: 9704-9709, 1996. Shibata H, Omata W, Kojima I. Insulin stimulates guanine nucleotide exchange on Rab4 via a Wortmannin-sensitive signalling pathway in rat adipocytes. J Biol Chem 272: 14542-14546, 1997. Shimizu Y , Kielar D, Minokoshi Y , Shimazu T. Noradrenaline increases glucose transport into brown adipocytes in culture by a mechanism different from that of insulin. Biochem J. 314: 485-490, 1996. Sinha M K , Raineri-Maldonado C, Buchanan C, Pories WJ, Carter-Su C, Pilch PF, Caro JO. Adipose tissue glucose transporters in NIDDM. Diabetes 40: 472-477, 1991. 96 Slot JW, Geuze HJ, Gigengack S, Liehard GE, James DE. Immunolocalization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 113: 123-135, 1991a. Slot JW, Geuze HJ, Gigengack S, James DE, Lienhard GE. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci USA 88: 7815-7819, 1991b. Smith U , Kuroda M , Simpson IA. Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell. J Biol Chem. 259: 8758-8763, 1984. Stanley WC, Hall JL, Smith KR, Cartee GD, Hacker TA, Wisneski JA. Myocardial glucose transporters and glycolytic metabolism during ischemia in hyperglycemic diabetic swine. Metabolism 43: 61-69, 1994. Stenbit A E , Tsao T-S, L i J, Burcelin R, Geenen DL, Factor SM, Houseknecht K L , Katz EB, Charron MJ . GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes . Nature Med. 3: 1096-1101, 1997. Strout HV, Vicario PP, Biswis C, Saperstein R, Brady EJ, Pilch PF, Berger J. Vanadate treatment of streptozotocin-diabetic rats restores expression of the insulin-responsive glucose transporter in skeletal muscle. Endocrinology 126: 2728-2732, 1990. 97 Sun DQ, Nguyen N , DeGrado TR, Schwaiger M , Brosius FC. Ischemia induces translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane of cardiac myocytes. Circulation 89 (2): 793-798, 1994. Suzuki K, Kono T. Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc Natl Acad Sci USA 77: 2542-2545, 1980. Sviderskaya EV, Jazrawi E, Baldwin SA, Widnell CC, Pasternak CA. Cellular stress causes accumulation of the glucose transporter at the surface of cells independently of their insulin sensitivity. J Membrane Biol. 149: 133-140, 1996. Taegtmeyer H. Metabolic support for the postischemic heart. Lancet 345: 1552-1555, 1995. Tellam JT, Macaulay SL, Mcintosh S, Hewish DR, Ward CW, James DE. Characterization of Munc-18C and Syntaxin 4 in 3T3-L1 adipocytes. J Biol Chem. 272: 6179-6186, 1997. Thompson K H , Battell M , McNeill JH. Vanadium in the environment (volume 31, chapter 2 of part 2: toxicology of vanadium): 30, the Wiley Series in Advances in Environmental Science and Technology, Editor: Nriagu JO, 1998. Thorens B, Cheng Z-Q, Brown D, Lodish HF. Live glucose transporter: a basolateral protein in hepatocytes and intestine and kidney cells. Am J Physiol. 260: C279-C285, 1990. Tsao TS, Burcelin R, Katz EB, Huang L, Charron MJ. Enhanced insulin action due to targeted GLUT4 overexpression exclusively in muscle. Diabetes 45: 28-36, 1996. 98 Ullrich O, Horiuchi H , Bucci C, Zerial M . Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange. Nature 368: 157-160, 1994. Uphues I, Kolter T, Goud B, Eckel J. Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle studies of the role of small-molecular-mass GTP-binding proteins. Biochem J. 301: 177-182, 1994. Vanhaesbroeck B, Leevers SJ, Panayotoy G, Waterfield M D . Phosphoinositide 3 kinase: a conserved family of signal transducers. Trends Biochem Sci. 22: 276-272, 1997. Vogt B, Mushack J, Seffer E, Haring HU. The phorbol ester TPA induces a translocation of the insulin sensitive glucose carrier (GLUT4) in fat cells. Biochem Biophy Res Comm. 168: 1089-1094,1990. Vollenweider P. Martin SS. Haruta T. Morris AJ . Nelson JG. Cormont M . Le Marchand-Brustel Y. Rose DW. Olefsky JM. The small guanosine triphosphate-binding protein Rab4 is involved in insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-L1 cells. Endocrinology. 138(11):4941-9, 1997 Nov. 98006448 Volchuk A , Wang Q, Ewart S, Lin Z, He L, Bennett M K , Klip A . Syntaxin 4 in 3T3-Li adipocytes: regulation by insulin and participation in insulin-dependent glucose transport. Mol Biol Cell 7: 1075-1082, 1996. Waddell ID, Zomerschoe A G , Voice M W , Burchell A. Cloning and expression of a hepatic microsomal glucosetrnaporter protein. Biochem J. 286: 173-177, 1992. 99 Watanabe T, Smith M M , Robinson FW, Kono T. Insulin action on glucose transporter in cardiac muscle. J Biol Chem. 259: 13117-13122, 1984. Waters SB, D'Auria M , Martin SS, Nguyen C, Kozma L M , Luskey K L . The amino terminus of insulin-responsive aminopeptidase causes Glut4 translocation in 3T3-L1 adipocytes. J Biol Chem. 272: 23323-23327, 1997. Yao J. Effects of vanadium compounds on diabetes induced changes in STZ-diabetic rats. M.Sc. Thesis, Faculty of Pharmaceutical Sciences, UBC, 1996. Yeh JI, Gulve E A , Rameh L i , Birnbaum MJ . The effcets of wortmannin on rat skeletal muscle. Dissociation of signaling pathways for insulin-and contraction-activated hexose transport. J Biol Chem. 270:2107-2111, 1995. Youn JH, K i m JK, Buchanan TA. Time courses of changes in hepatic and skeletal muscle insulin action and GLUT4 protein in skeletal muscle after STZ injection. Diabetes 43: 564-571,1994. Yuen V G , Orvig C, McNeill JH. Glucose-lowering effects of a new organic vanadium complex, bis(maltolato)oxovanadium(lV). Can J Physiol Pharmaco. 71: 263-269,1993. Zaninetti D, Greco-Perotto R, Assimocopoulos-Jeannet F, Jeanrenaud B. Effects of insulin on glucose transport and glucose transporters in rat heart. Biochem J. 250: 277-282, 1988. 100 Ziel FH, Venkatesan N , Davidson M B . Glucose transport is rate limiting for skeletal muscle glucose metabolism in normal and STZ-induced diabetic rats. Diabetes 37: 885-890, 1988. Zierath JR. In vitro studies of humal skeletal muscle hormonal and metabolic regulation of glucose transport. Acta Physiol Scand. 155: 1-96, 1995. Zierath JR, Houseknecht K L , Kahn B B . Glucose transporters and diabetes. In Seminar in Cell and Developmental Biology 7: 295-307, 1996. 1 0 1 

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