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Studies on the mechanism of the insulin-mimetic effects of vanadium in streptozotocin-diabetic rats Cam, Margaret C. 1996

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S T U D I E S O N T H E M E C H A N I S M O F T H E INSUL IN-MIMETIC E F F E C T S O F V A N A D I U M IN S T R E P T O Z O T O C I N - D I A B E T I C R A T S by M A R G A R E T C . C A M B . S c , University of British Co lumb ia , 1988 A thesis submitted in partial fulfillment of the requirement for the degree of Doctor of Phi losophy in The Faculty of Graduate Studies Division of Pharmaco logy and Toxicology Faculty of Pharmaceut ica l S c i e n c e s W e accept this thesis as conforming to the required standard V The University of British Co lumbia July 1996 © Margaret C . C a m ,ii996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) II ) A B S T R A C T A ser ies of studies was conducted to examine the possib le mechanism(s) involved in the insulin-mimetic effects of vanadium in streptozotocin (STZ)-d iabet ic rats. It w a s hypothesized that vanadium treatment may induce a glucose- lower ing effect and an overal l amelioration of the diabetic state by 3 general mechan isms : i) by preventing the STZ- induced cytotoxic effects on (i-cells, resulting in the chronic reversal of the diabetic state, ii) by reducing food intake, or iii) by enhancing the peripheral effects of insulin. The goal was to examine the signif icance of the apparent multiple roles for vanad ium both at the level of the R-cell and in peripheral t issues. S ince de layed vanadium treatment up to 17 days after S T Z did not reduce the incidence of normoglycemia or correction of ad ipose t issue function, it is unlikely that vanad ium treatment shortly (3 days) after the induction of STZ-d iabe tes resulted from a direct inhibition of the acute STZ- induced R-cytotoxicity. However, the residual insulin secretory function in these animals may have contributed to an effective response to vanad ium. Indeed, the more severely diabetic animals which were previously unresponsive to vanad ium treatment developed normoglycemia with higher amounts of either nagl ivan (a more orally potent organic vanadyl compound) or vanadyl sulfate, suggest ing that vanad ium and insulin work in a complementary manner in vivo. Vanadium-treated diabetic animals were found to maintain long-term (20-30 weeks) normoglycemia after withdrawal from treatment, a phenomenon which was linked to an improved R-cell secretory function. The post-withdrawal normoglycemia could not be demonstrated in rats treated with vanadium prior to the administrat ion of S T Z alone, suggest ing the lack of a direct inhibitory effect of vanad ium on STZ- i nduced fi-cytotoxicity in vivo. However, continued treatment for 2 weeks after S T Z induced a partial preservat ion of f i-cells which was sufficient for a chronic reversal of the diabet ic state. It appeared that the smal l changes in pancreat ic insulin content had profound consequences on g lucose homeostas is in animals with a reduced ft-cell mass . Although the modest effects of a reduced food intake on improving glycemia and residual pancreatic insulin content suggest that it may contribute to the overall effects of vanadium treatment, the partial lowering of glycemia in pair-fed diabetic rats was linked to residual insulin levels in the plasma and pancreas, unlike vanadium-treated animals which maintained normoglycemia at relatively low levels of both circulating and pancreatic insulin. Thus, the mechanism(s) of glucose-lowering by food reduction is distinct from that of vanadium. However, the protection of residual insulin stores by vanadium or food restriction may be secondary to a higher threshold for insulin release. A possible mechanism for vanadium in improving glucose tolerance in diabetic animals is an enhanced glucose transport in insulin-sensitive tissues. The effect of vanadium treatment on the adipose tissue insulin-regulatable glucose transporter ( G L U T 4 ) translocation in vivo in response to an i.v. glucose load was examined. Vanadium treatment did not enhance the effects of insulin on G L U T 4 translocation at least in adipose tissue in control and diabetic animals. Alternately, the improved glucose tolerance and maintenance of adipose tissue intracellular G L U T 4 pool in diabetic rats treated with vanadium appear to be secondary to the preservation of pancreatic insulin stores. iv T A B L E OF CONTENTS Page A B S T R A C T ii T A B L E O F C O N T E N T S iv L IST O F T A B L E S xii L IST O F F I G U R E S xiii L IST O F A B B R E V I A T I O N S xvii A C K N O W L E D G M E N T S xxii P R E F A C E xxiii C H A P T E R 1 INTRODUCTION 1.1 D I A B E T E S 1 1.1.1. Classi f icat ion of d iabetes 2 1.1.2. Treatment of Type 1 d iabetes 4 1.2. S T R E P T O Z O T O C I N - D I A B E T E S 5 1.2.1. Mechan ism of the diabetogenic effect of S T Z 5 1.2.2. Character ist ics of STZ-d iabe tes in rats 6 1.3. V A N A D I U M IN B I O L O G Y 8 1.3.1. Introduction 8 1.3.2. Chemist ry 9 1.3.3. Absorpt ion, distribution and excretion 9 1.3.4. Intracellular forms and levels 10 1.3.5. Nutritional role 11 1.3.6. Effects on enzyme systems 12 1.3.6.1. A T P a s e s 12 1.3.6.2. Adeny la te cyc lase and phosphodiesterase 13 V 1.3.6.3. Misce l laneous 13 1.3.6.4. N A D ( P ) H Oxidat ion 14 1.3.7. Effects on physiological sys tems 15 1.4. INSUL IN-MIMETIC E F F E C T S O F V A N A D I U M IN V I T R O 16 1.4.1. Carbohydrate Metabol ism 16 1.4.2. Lipid metabol ism 20 1.4.3. Protein metabol ism 22 1.4.4. Mitogenic effects 22 1.4.5. Non-insul in mimetic effects of vanadium 24 1.5. INSUL IN-MIMETIC E F F E C T S O F V A N A D I U M IN V I V O 25 1.5.1. Contro l an imals 25 1.5.2. An ima l models of Type 1 d iabetes 26 1.5.2.1. Streptozotocin-diabetes 26 1.5.2.2. Spontaneous ly diabetic B iobreeding (BB) rats 29 1.5.2.3. Partially pancreatectomized rats 29 1.5.3. An ima l models of Type 2 diabetes 30 1.5.3.1. Neonata l STZ-d iabet ic rats 30 1.5.3.2. Genet ical ly obese fa/fa rats and ob/ob mice 30 1.6. T O X I C O L O G I C A L E F F E C T S O F V A N A D I U M C O M P O U N D S IN D I A B E T E S 31 1.7. M E C H A N I S M O F INSULIN-MIMETIC E F F E C T S O F V A N A D I U M 32 1.7.1. Insulin receptor 32 1.7.2. Post-receptor mechan isms 34 1.7.3. Other proposed second messengers 35 1.8. P E R O X O V A N A D I U M C O M P O U N D S 36 1.9. N E W O R G A N I C V A N A D I U M C O M P O U N D S 38 1.10. H U M A N T R I A L S 39 1.11 T H E S I S I N V E S T I G A T I O N 40 vi C H A P T E R 2 L O N G - T E R M E F F E C T I V E N E S S O F O R A L V A N A D Y L S U L F A T E IN S T R E P T O Z O T O C I N - D I A B E T I C R A T S 2.1. I N T R O D U C T I O N 43 2.2. M A T E R I A L S A N D M E T H O D S 44 2.2.1. Treatment and maintenance of animals 44 2.2.2. Oral g lucose tolerance test (OGTT) 45 2.2.3. Pancreat ic perfusion 45 2.2.4. Glycero l output from isolated ad ipose t issue 45 2.2.5. T i ssue histopathology 46 2.2.6. Vanad ium levels 46 2.2.7. Statistical analys is 46 2.3. R E S U L T S 47 2.3.1. Effects of vanadyl treatment on body weight, food and fluid intake, and p lasma g lucose 47 2.3.2. Effects of vanadyl on p lasma parameters at 5 months of treatment 53 2.3.3. Oral g lucose tolerance test 53 2.3.4. R e s p o n s e of isolated pancreas to 16.65 m M g lucose + 0-1 ng/ml G I P 56 2.3.5. Glycero l output in isolated ad ipose t issue 56 2.3.6. Histopathology results 59 2.3.7. Vanad ium levels 59 2.4. D I S C U S S I O N 61 V I I C H A P T E R 3 IN VIVO ANTIDIABETIC ACTIONS OF NAGLIVAN, A N ORGANIC V A N A D Y L COMPOUND IN STREPTOZOTOCIN- INDUCED DIABETES 3.1. I N T R O D U C T I O N 65 3.2. M A T E R I A L S A N D M E T H O D S 67 3.2.1. Formula and characterizat ion of the compound 67 3.2.2. Treatment and maintenance of animals 67 3.2.3. Isolated working heart study 68 3.2.4. P l a s m a Ana lys is 69 3.2.5. Statistical Ana lys is 69 3.3. R E S U L T S 70 3.3.1. Effects of nagl ivan and/or insulin treatment on body weight, food and fluid intake 70 3.3.2. Effects of nagl ivan and insulin treatment on p lasma g lucose 72 3.3.3. Effects of nagl ivan treatment on exogenous insulin requirement 72 3.3.4. Effects of nagl ivan and insulin treatment on p lasma lipids, G O T , creatinine, B U N , insulin and % glycosylated hemoglobin at eight weeks 75 3.3.5. Effects of nagl ivan and insulin treatment on h e a i t b o d y weight and heart function at eight weeks 78 3.4. D I S C U S S I O N 81 C H A P T E R 4 CONCENTRATION-DEPENDENT G L U C O S E LOWERING E F F E C T S OF O R A L V A N A D Y L A R E MAINTAINED FOLLOWING T R E A T M E N T WITHDRAWAL IN STZ-DIABETIC RATS 4.1 . I N T R O D U C T I O N 85 4.2. M A T E R I A L S A N D M E T H O D S 86 VIII 4.2 .1 . Treatment and maintenance of animals. 86 4.2.2. Analyt ical Methods 87 4.2.3. Statistical Ana lys is 87 4.3 . R E S U L T S 88 4 .3 .1 . Var ious parameters of diabetic animals treated with vanadyl sulfate 88 4.3.2. P l a s m a g lucose and insulin profile before and after 10 week treatment 93 4.3.3. P l a s m a g lucose and insulin after withdrawal from vanadium treatment 93 4.3.4. P l a s m a g lucose and insulin after withdrawal from treatment in [1.50] 98 4.3.5. O G T T s at 10 weeks of treatment and after treatment withdrawal 100 4.3.6. Integrated g lucose and insulin responses during O G T T s 102 4.3.7. Glucose- lower ing effects of an organic vanadyl compound, nagl ivan 106 4.4. D I S C U S S I O N 109 C H A P T E R 5 PARTIAL PRESERVATION OF PANCREATIC IJ-CELLS B Y VANADIUM: EVIDENCE FOR A MECHANISM OF CHRONIC AMELIORATION OF DIABETES 5.1. I N T R O D U C T I O N 113 5.2. M A T E R I A L S A N D M E T H O D S 114 5.2.1. Treatment and maintenance of animals 114 5.2.2. P l a s m a and Vanad ium Ana l yses 114 5.2.3. Oral g lucose tolerance test ( O G T T ) 115 5.2.4. Pancreat ic insulin extraction 115 ix 5.2.5. Histological analys is 115 5.2.6. Statist ical analys is 115 5.3. R E S U L T S 116 5.3.1. Genera l characterist ics of diabetic rats treated with vanad ium 116 5.3.2. Chron ic normoglycemia in diabetic rats after withdrawal from vanadium 118 5.3.3. Pancreat ic insulin content 120 5.3.4. Classi f icat ion of diabetic animals 120 5.3.5. Correlat ions between residual insulin content and g lycemic status 123 5.3.6. Histological examinat ion of f i-cells 126 5.4. D I S C U S S I O N 129 C H A P T E R 6 THE ANTIDIABETIC E F F E C T S OF VANADIUM TREATMENT IN STZ-DIABETIC RATS A R E INDEPENDENT OF ITS E F F E C T S ON REDUCING FOOD INTAKE 6.1. I N T R O D U C T I O N 133 6.2. M A T E R I A L S A N D M E T H O D S 134 6.2.1. Treatment and maintenance of animals 134 6.2.2. Oral g lucose tolerance test (OGTT) 135 6.2.3. Pancreat ic insulin extraction 135 6.2.4. Vanad ium levels 135 6.2.5. Statistical analys is 135 6.3. R E S U L T S 136 6.3.1. Genera l characterist ics of an imals 136 6.3.2. Effect of vanadium treatment and pair-feeding on g lycemia 138 X 6.3.3. Effects of vanadium treatment and pair-feeding on p lasma insulin 141 6.3.4. Oral g lucose tolerance test 141 6.3.5. P l a s m a parameters at termination 144 6.3.6. Effects of vanadium and pair-feeding on pancreat ic insulin content 144 6.4. D I S C U S S I O N 147 C H A P T E R 7 THE E F F E C T OF VANADIUM TREATMENT ON ADIPOSE TISSUE GLUT4 TRANSLOCATION IN VIVO IN STZ-DIABETIC RATS 7.1. I N T R O D U C T I O N 152 7.2 M A T E R I A L S A N D M E T H O D S 154 7.2.1. Treatment and maintenance of animals 154 7.2.2. Oral and intravenous g lucose tolerance tests ( O G T T / I V G T T ) 154 7.2.3. Subcel lu lar fractionation of ad ipose t issue 155 7.2.4. Membrane marker enzyme a s s a y s 156 7.2.5. G L U T 4 analys is 158 7.2.6. Protein assay 159 7.2.7. Statistical analys is 159 7.3. R E S U L T S 160 7.3.1. Genera l characterist ics of diabetic animals prior to vanadium treatment 160 7.3.2. Effects of vanadium on body weight, food and fluid intake 160 7.3.3. Effects of vanadium on g lycemia and g lucose to lerance 163 7.3.4. Character izat ion of ad ipose t issue subcel lu lar fractionation 167 xi 7.3.5. Quantitation of G L U T 4 in subcel lular fractions by competit ive E L I S A 169 7.3.6. Effect of vanadium treatment on maximal G L U T 4 translocation in vivo in response to an i.v. g lucose load in control and STZ-d iabet ic rats 171 7.3.7 Relat ionship between residual insulin store, g lycemia A U . C g and G L U T 4 171 7.4. D I S C U S S I O N 175 C O N C L U S I O N S 180 R E F E R E N C E S 184 APPENDIX 1 A SENSITIVE RADIOIMMUNOASSAY OPTIMIZED FOR R E P R O D U C I B L E M E A S U R E M E N T OF RAT P L A S M A INSULIN A 1 . 1 . I N T R O D U C T I O N 214 A 1 . 2 . M A T E R I A L S A N D M E T H O D S 216 A1 .2 .1 . Materials 216 A1 .2 .2 . RIA Procedure 217 A 1 . 3 . R E S U L T S 218 A1 .3 .1 . Effects of C E P on the standard curve 218 A1 .3 .2 . Correct ing for C E P (%Bp/Bo) binding 218 A1 .3 .3 . Effect of increased charcoal-binding 223 A1.3 .4 . Recovery Exper iment 223 A1 .3 .5 . Reproducibi l i ty of the a s s a y 225 A1 .3 .6 . Effects of site of p lasma sampl ing 228 A1 .4 . D I S C U S S I O N 230 xii LIST OF T A B L E S Table P a g e 1. Character ist ic features of d iabetes subtypes 3 2. In vitro effects of vanadium on carbohydrate metabol ism 17 3. In vitro vanad ium effects on enzymes involved in g lucose metabol ism 19 4. In vitro effects of vanadium on lipid metabol ism 21 5. In vitro effects of vanadium on protein metabol ism and mi togenesis 23 6. In vitro effects of vanadium on the insulin signal ing pathway 33 7. In vitro effects of peroxides(s) of vanadium 37 8. P l a s m a parameters of var ious groups at 5 months 54 9. Effects of nagl ivan and insulin treatment on var ious p lasma parameters at 8 weeks after the S T Z injection 76 10. Heart and body weight of the var ious groups at 8 weeks after the S T Z injection 79 11. Number of euglycemic animals at the end of 10-week treatment period according to vanadyl concentrat ion in the drinking water 89 12. Var ious characterist ics of animals at the end of a 10-week treatment period accord ing to vanadyl concentrat ion in the drinking water 91 13. Parameters of various groups at weeks 4-5 117 14. Classi f icat ion of STZ-d iabet ic animals according to g lycemic status 124 15. P l a s m a parameters at termination 145 16. Insulin levels calculated for p lasma samp les before and after correction of % B / B o 221 Xlll LIST OF FIGURES Figure P a g e Chapter 2 2.1 . Body weight and food intake over 15 weeks 48 2.2. Fluid intake and vanadyl dose over 15 weeks 49 2.3. Effect of vanad ium treatment on g lycemia in diabet ic rats 51 2.4. Diabetic subgroups according to hypoglycemic response to vanadyl (0.75 mg/ml) treatment over 15 weeks 52 2.5. Oral g lucose tolerance test at 5 months of study 55 2.6. In situ pancreat ic perfusion at 5 months of study 57 2.7. Effects of vanadium on ad ipose t issue lipolytic rates in diabet ic rats 58 2.8. Prevent ion of morphological changes in diabetic kidney by vanad ium 60 Chapter 3 3.1. Bis(cysteine, amide N-octyl)oxovanadium IV (naglivan) 66 3.2. Body weight, food and fluid intake of various groups for 8 weeks 71 3.3. Effect of nagl ivan and/or short-term insulin treatment on g lycemia 73 3.4. R e d u c e d exogenous insulin requirement with nagl ivan treatment 74 3.5. P l a s m a insulin and % glycosylated hemoglobin at termination 77 3.6. Preservat ion of card iac function in diabetic rats by nagl ivan treatment 80 Chapter 4 4 .1 . Ca lcu la ted vanadium dose in STZ-d iabet ic rats on var ious V O S 0 4 concentrat ions 90 4.2. P l a s m a vanadium levels of various diabetic groups at 10 weeks 92 xiv 4 .3 . Var iance in severity of diabetic state induced by 55 mg/kg S T Z 94 4.4. Effect of pretreatment p lasma g lucose and insulin levels on subsequent vanadyl requirement 95 4.5 . P l a s m a g lucose and insulin profile before and after vanadyl treatment 96 4.6. P l a s m a g lucose and insulin levels in control and diabetic rats over 5 months following withdrawal from vanadyl treatment 97 4.7. Assoc ia t ion between insulin and maintenance of normoglycemia in rats reverting to hyperglycemia after withdrawal from vanad ium treatment 99 4.8. Oral g lucose tolerance tests prior to and at 3 and 20 weeks after treatment withdrawal 101 4.9. Integrated g lucose and insulin responses during O G T T s 103 4.10. Relat ionship between glucose-to lerance and fed p lasma g lucose and between insulin secretory function and fed p lasma insulin levels at 20 weeks 104 4 .11 . Relat ionship between glucose-to lerance level and insulin secretory response during O G T T at 3 and 20 weeks after withdrawal from treatment 105 4.12. Effects of chronic nagl ivan treatment in STZ-d iabet ic rats 107 4 .13. O G T T in naglivan-treated rats at 30 weeks after treatment withdrawal 108 Chapter 5 5.1. Effect of vanad ium treatment fol lowed by withdrawal period on fed p lasma g lucose levels in control and diabetic rats 119 5.2. Effect of vanad ium treatment on pancreat ic insulin content 121 5.3. Oral g lucose tolerance test demonstrat ing the var iances in g lucose-tolerance and insulin response in the pooled diabetic animals 122 XV 5.4. Relat ionship between glycemic status and residual insulin content in pooled diabetic animals at 5 weeks pos t -STZ 125 5.5. Relat ionships between glucose-to lerance and insulin secretory function at 4 weeks pos t -STZ with residual insulin content in pooled diabetic rats 127 5.6. Photomicrographs of islets, stained for R>-cells 128 Chapter 6 6.1. Effects of vanadium treatment and pair-feeding on body weight and fluid intake in STZ-d iabet ic rats over 6 weeks 137 6.2. Effects of vanadium treatment and pair-feeding on food intake and p lasma g lucose levels in STZ-d iabet ic rats over 6 weeks 139 6.3. P l a s m a g lucose in vanadium-treated and pair-fed diabetic rats 140 6.4. Effect of vanadium treatment and pair-feeding on p lasma insulin levels 142 6.5. Effect of vanadium treatment and pair-feeding on oral g lucose tolerance at 5 weeks 143 6.6. Relat ionships between pancreat ic insulin store and g lycemia and insulin response in diabetic rats 146 Chapter 7 7.1. Modif ied protocol for subcel lular fractionation of whole epididymal rat fat pad 157 7.2. Oral g lucose tolerance test at week 1 following S T Z , prior to vanad ium treatment 161 7.3. Effects of vanad ium treatment on body weight and food and fluid intake in STZ-d iabet ic rats over 10 weeks pos t -STZ 162 xvi 7.4. Effects of vanadium treatment on fed p lasma g lucose and insulin levels and on oral g lucose tolerance in STZ-d iabet ic rats 164 7.5. Spon taneous reversion in STZ-d iabet ic rats: chronic fed g lycemia, p lasma insulin and oral g lucose tolerance levels 165 7.6. Intravenous g lucose tolerance test ( IVGTT) at week 9 166 7.7. Relat ionship between g lucose d isappearance and integrated insulin re lease over 30 minutes 168 7.8. Compet i t ive E L I S A method for measurement of subcel lu lar G L U T 4 content: Determination of maximal p lasma membrane G L U T 4 content in ad ipose t issue in response to i.v. g lucose 170 7.9. G L U T 4 content in ad ipose t issue subcel lular fractions at 7.5 minutes following an i.v. g lucose dose 172 7.10. Relat ionship between residual pancreat ic insulin content, fed p lasma g lucose levels and A U C g 173 7.11. Relat ionships between residual insulin store with L D M G L U T 4 content, and between p lasma insulin levels and P M G L U T 4 content 174 Append ix 1 A 1 . 1 . Effect of 25 and 50 ul of C E P on insulin standard curves 219 A 1 . 2 . Effect of addit ion of C E P to standard curves on p lasma insulin levels 222 A 1 . 3 . Effect of different amounts of charcoal in final separat ion step on standard curve and determination of p lasma insulin levels 224 A1 .4 . Effects of untreated rat p lasma on recovery of total insulin 226 A 1 . 5 . Reproducibi l i ty test of rad io immunoassay over 15 days 227 A1 .6 . Effect of source of p lasma sampl ing on determination of insulin 229 X V I I LIST OF ABBREVIATIONS A D P adenos ine d iphosphate A T P adenos ine tr iphosphate A U C g area under the curve of p lasma g lucose response during O G T T / I V G T T A U C j area under the curve of p lasma insulin response during O G T T / I V G T T B B B ioBreed ing B M O V bis(maltolato)oxovanadium IV Bo zero binding (of insulin-free buffer) Bp zero binding (of insulin-free or charcoal extracted p lasma) B S A bovine serum albumin B U N blood urea nitrogen C control c A M P 3',5'-cyclic adenos ine monophosphate C E P charcoal extracted p lasma c G M P cycl ic guanos ine monophosphate C H O Ch inese hamster ovary C M crude membrane C P M counts per minute C T control group treated with vanadyl sulfate C V control group treated with nagl ivan % C V coefficient of variation C y t P T K cytosol ic protein tyrosine k inase X V I I I D untreated diabetic group D-NR hyperglycemic untreated diabetic rats ("non-reverter") D-R normoglycemic untreated diabetic rats ("reverter") D A G diacylglycerol DI diabetic group treated with insulin D N A deoxyr ibonucleic acid D P diabetic pair-fed group D P - N R diabetic pair-fed group ("non-responder") D P - R diabetic pair-fed group ("responder") D T diabet ic group treated with vanad ium D T - N R diabetic group treated with naglivan ("non-responder") D T - R diabetic group treated with naglivan ("responder") D T 3 diabet ic group treated with vanadium from 3 days pos t -STZ injection DT10 diabetic group treated with vanad ium from 10 days pos t -STZ injection DT17 diabetic group treated with vanadium from 17 days pos t -STZ injection DVI diabet ic group treated with nagl ivan + insulin DVI -E diabetic group treated with nagl ivan + insulin ("euglycemic") DVI-H diabetic group treated with nagl ivan + insulin ("hyperglycemic") E euglycemic/near normal g lucose tolerance E D T A ethylenediaminetetraacet ic acid E G F epidermal growth factor E G T A ethyleneglycolbis(b-aminoethyl)ether-P-N,N,N'N'- tetraacet ic acid xix E L I S A enzyme- l inked immunosorbent assay E P R electron paramagnet ic resonance E R endop lasmic reticulum E S R electron spin resonance F F A free fatty ac ids % G H b glycosylated hemoglobin G I P gastr ic inhibitory polypeptide G L P - 1 glucagon-l ike peptide-1 G L U T 1 HepG2/erythrocyte type g lucose transporter G L U T 2 liver type g lucose transporter G L U T 4 muscle/fat insulin-regulatable g lucose transporter G O T glutamic oxaloacet ic t ransaminase G P T glutamic pyruvic t ransaminase G S H glutathione (reduced) G S S G glutathione (oxidized) H+GI hyperglycemic/glucose-intolerant H+GT hyperglycemic/near normal g lucose tolerance H 2 0 2 hydrogen peroxide H b A 1 c hemoglobin A 1c H C diabetic group treated with vanadyl ("high concentrat ion responder") H D M high density microsomal membrane i.p. intraperitoneal XX i.v. intravenous IDDM insul in-dependent d iabetes mellitus IGF-I insulin-like growth factor-1 IGF-II insulin-like growth factor-2 IR insulin receptor IRI immunoreact ive insulin IRS-1 insulin receptor substrate-1 IRTK insulin receptor tyrosine kinase IVGTT intravenous g lucose tolerance test Kg g lucose d isappearance rate K m affinity constant L C diabetic group treated with vanadyl ("low concentrat ion responder") L D M low density microsomal membrane M A P mitogen-activated protein m R N A messenger r ibonucleic acid N a 3 V 0 4 sodium orthovanadate N A D nicotinamide adenine dinucleotide (oxidized) N A D ( P ) H nicotinamide adenine dinucleot ide/(phosphate) (reduced) N a V 0 3 sodium metavanadate N I D D M Non- insul in-dependent d iabetes mellitus N O D nonobese diabetic O G T T oral g lucose tolerance test xxi P E P C K phosphoenolpyruvate carboxyk inase P F K - 2 6-phosphofructo-2-kinase P IC pancreat ic insulin content P K C protein k inase C P M p lasma membrane P P pancreat ic polypeptide pp15 15 kDa fatty acid binding protein 422(aP2) Ptd lns phosphatidyl inositol P T P a s e phosphotyrosine phosphatase pV peroxovanadium complexes RIA rad io immunoassay s.c. subcutaneous S E M standard error of mean S R sarcop lasmic reticulum S T Z streptozotocin t 1 / 2 half-life T 3 3,5,3' tri iodothyronine T 4 3,5,3',5' tetraiodothyronine, thyroxine v m a x maximum velocity V 0 2 + vanadyl ion V O 3 - metavanadate ion V O S O 4 vanadyl sulfate X X I I A C K N O W L E D G M E N T S I would like to sincerely thank my supervisor, Dr. John McNei l l , for his genuine interest, enthus iasm and cont inuous support without which this thesis would not have been possib le. I would a lso like to thank the members of my research committee, Drs. Roger Brownsey, A l ison Buchan , and R a y Pede rson , for their encouragement and adv ice throughout the duration of this work. Severa l people have contributed their t ime and energy to this work, and their efforts are greatly appreciated. They include: M s . Mary Battell, M s . Er ika V e r a , M s . S tephan ie L e e and M s . Violet Y u e n , whose technical ass is tance throughout the years were invaluable. The contributions of summer students M s . Jul ie Faun and M s . Paramjit Dhami are gratefully acknowledged. I would like to a lso thank Drs. Kathie Thompson and Gerard Cros for their ass is tance in measur ing vanad ium levels. Var ious technical aspects of this thesis would not have been possib le without the volunteered help of my co l leagues, especial ly Magg ie L i , S h u - C h a n H s u , Bob Winz , and S u e Curt is. I would a lso like to thank Drs. Stelvio Band iera , T o m C h a n g and R o b Th ies for their t ime and advice. Last but not least, I am deeply indebted to Dr. Br ian Rodr igues, whose constant encouragement , opt imism and generous support greatly ass is ted in the complet ion of this thesis. xxiii P R E F A C E The study presented in Chapter 2 has been publ ished as : C a m , M C , Pede rson R A , Brownsey R W , McNei l l J H : Long-term effect iveness of oral vanady l in streptozotocin- induced diabetes. Diabetologia 36 : 218-224, 1993. The pancreat ic perfusion and oral g lucose tolerance tests were performed in the laboratory of Dr. R a y Pede rson . The ad ipose t issue lipolysis studies were done under the supervis ion of Dr. Roger Brownsey. The study presented in Chapter 3 has been publ ished as : C a m M C , C r o s G H , Ser rano J J , Lazaro R, McNei l l J H : In vivo antidiabetic act ions of nagl ivan, an organic vanadyl compound in streptozotocin-induced diabetes. Diab Res Clin Prac 20 : 111-121, 1993. The nagl ivan compound was synthesized by Dr. R e n e Lazaro and initial testing performed by Drs. Cros and Serrano. The study presented in Chapter 4 has been publ ished as : C a m M C , Faun J , McNei l l J H : Concentrat ion-dependent glucose- lowering effects of oral vanady l are maintained following treatment withdrawal in streptozotocin-diabetic rats. Metabolism 44: 332-339, 1995. M s . Faun was responsible for routine blood ana lyses and treatment and care of the animals. The study presented in Append ix 1 has been publ ished as : C a m M C , McNei l l J H : A sensit ive rad io immunoassay optimized for reproducible measurement of rat p lasma insulin. J Pharm Toxicol Meth 35: 111-119, 1996. John H. McNei l l , P h . D . Superv isor 1 C H A P T E R 1 INTRODUCTION 1.1. DIABETES The pancreas is an organ composed of exocr ine (~ 98%) and endocr ine (~ 2%) cel ls. The islets of Langerhans are clusters of endocr ine t issue which are d ispersed throughout the exocr ine pancreas. There are four major cell types present within mammal ian islets. These are: ft-cells (insulin-producing), a-cel ls (g lucagon-producing), 8-cells (somatostat in-producing) and PP-ce l l s (pancreatic polypeptide). The critical role of the pancreas in d iabetes w a s not real ized until it w a s d iscovered that complete removal of this organ resulted in hyperglycemia in dogs (von Mer ing and Minkowsk i , 1889). Subsequent ly , the Nobel prize was awarded to researchers who d iscovered insulin from pancreat ic extracts which dramatical ly reduced hyperg lycemia in pancreatectomized dogs (Banting et a l , 1922). Gep ts (1965) later demonstrated speci f ic f i-cell abnormali t ies and inflammatory cel ls in the islets of Langerhans in recently d iagnosed insul in-dependent diabetic patients, and using quantitative morphometry, it was found that Type 1 diabetes was assoc ia ted with a speci f ic and complete loss of pancreat ic B-cells (Foul is and Stewart, 1984). G l u c o s e stimulates the ft-cells of the islets to re lease insulin, which then promotes g lucose uptake and storage in var ious t issues. Cons ider ing these effects, hyperglycemia was bel ieved to be due to insulin def ic iency and hypoglycemia due to insulin excess . However, with the advent of insulin rad io immunoassays , it b e c a m e apparent that the majority of patients with hyperglycemia were not completely insul in-dependent and in fact had normal or even elevated concentrat ions of circulating insulin. Thus , d iabetes mellitus is the name given to a multiple group of d isorders with different et iologies. It is character ized by derangements in carbohydrate, protein and fat metabol ism caused by the complete or relative insufficiency of insulin secret ion and/or 2 insulin act ion. T h e s e aberrat ions account for the acute (fatigue, polyuria, polydipsia, etc.) as well as chronic (retinopathy, neuropathy, nephropathy, peripheral vascu la r d i sease , heart failure, etc.) compl icat ions of the d i sease (Rubin et a l , 1994). 1.1.1. Classi f icat ion of diabetes The classif icat ion of d iabetes is based principally upon clinical symptoms and , when possib le, on more specif ic etiologic character izat ion. Tab le 1 summar izes the two major types of diabetes: a) d iabetes assoc ia ted with insul in-deficiency (Type 1, insul in-dependent IDDM; 5-10% of all cases ) and b) diabetes assoc ia ted with insulin res is tance (Type 2, non-insulin dependent N I D D M ; 90 -95% of all cases ) . Other types of d iabetes include gestat ional d iabetes, impaired g lucose tolerance and diabetes resulting from other condit ions or syndromes (National Diabetes Data Group , 1979). Type 1 Diabetes (IDDM). This d isease is assoc ia ted with a speci f ic and complete loss of pancreat ic f i-cel ls, leaving islets composed of an increased number of a , 5 , and P P cel ls. Thus , Type 1 diabetes can be thought of as a speci f ic fc-cytectomy, a phenomenon mimicked in animals with the use of chemica l agents like al loxan or streptozotocin. Auto immune destruction of pancreat ic f i-cel ls has been sugges ted to be the most c o m m o n cause of IDDM. Al though the factors that initiate this auto immune response are not completely understood, it is more frequent in patients with certain human leukocyte antigen t issue types. Other initiating factors include v i ruses (i.e., Coxsack i e B 4 ; Y o o n et a l , 1979) and chemical toxins. L e s s common c a u s e s of IDDM are condit ions that result in a reduction in the mass of islet cell t issue, such as may occur with severa l types of pancreatit is, pancreat ic carc inoma and pancreatectomy. Type 2 Diabetes (NIDDM). N I D D M patients represent 9 0 - 9 5 % of the diabet ic populat ion. Between 60 -90% of those having N I D D M are obese (Ekoe, 1988), often exhibiting hyperinsul inemia and assoc ia ted insulin resistance. Al though the primary c a u s e s of the d i sease have not been identified at the molecular level, current research 3 Table 1. Characteristic features of diabetes subtypes Characteristics TYPE I (IDDM) TYPE 2 (NIDDM) Symptoms Age Onset Nutritional Status Ketosis Insulin Diet fl-cells Islet Cell Antibodies Family History (Identical twins) Polyur ia, polydipsia, fatigue, weight loss < 35 (common in youth) Abrupt (days - weeks) Undernour ished Prone (unless diet, insulin coordinated) Mandatory Mandatory None (complete islet cell loss) Y e s + in 10% ~ 5 0 % concordance Often asymptomat ic in early years but may present with Type 1 symptoms especia l ly in advanced s tages >35 (frequent in adults) W e e k s - months - years Majority are overweight Resistant Requi red in <30% Controls 30 -50% c a s e s Var ies No + in 3 0 % ~ 100% concordance 4 strongly suggests that this d isease ar ises as a consequence of: a) failure of insulin action due to abnormali t ies at the cell surface (decreased affinity of the receptors for insulin) or within the cell (post-receptor defect) and b) def ic iency in insulin secret ion or a combinat ion of these p rocesses (Yki-Jarv inen, 1995). Al though the majority of patients with N I D D M are insulin-resistant, whether the primary molecular defect lies within the insulin s ignal transduction pathway or in fi-cell insulin secret ion remains undec ided (Taylor e t a l , 1994; Kahn , 1994). 1.1.2 Treatment of insulin-dependent diabetes mellitus (IDDM) Since the d iscovery of insulin in 1921, there has been little progress in the d iscovery of new and effective therapeutic agents for the treatment of d iabetes and the prevention of secondary compl icat ions. The apparent lack of growth in this a rea is of significant concern , consider ing the large number of diabetic patients (over 100-200 mill ion worldwide). Resul ts from the Diabetes Control and Compl icat ions Trial emphas ized that tight g lucose control with intensive insulin treatment improves the long-term outcome of Type 1 diabetic patients. B e c a u s e insulin is ineffective orally, an oral antidiabetic agent would be clearly advantageous with respect to patient compl iance and improved g lucose control in the diabetic populat ion. Oral ly effective insul in-sensit iz ing agents represent a new c lass of therapeutic agents which cou ld be useful in lowering insulin requirements and improving glucoregulat ion in diabetic patients. Included in this relatively new drug category are glucagon- l ike peptide-1 ( G L P - 1 ) and thiazol idinedione insulin-sensit izing agents: c igl i tazone, engl i tazone, piogl i tazone and troglitazone (Hofmann and C o l c a , 1992). Agen ts which contain chromium such as g lucose tolerance factor ( G T F ) and chromium (III) picol inate, which have been shown to lower g lycemia, are currently sold in health food stores. However , to date, no drug has yet been developed as an oral substitute for insul in, which remains the primary, if not the only, effective form of drug therapy in Type 1 d iabetes mellitus. 5 1.2. STREPTOZOTOCIN-DIABETES Insul in-dependent diabetes mellitus (IDDM) is caused by a marked reduction (> 90%) in the number of pancreat ic fi-cel ls (Gepts, 1965). Chemica l agents that produce d iabetes can be classif ied into three categor ies, and include agents that: a) specif ical ly d a m a g e fi -cel ls ; b) cause temporary inhibition of insulin production and/or secret ion and c) diminish the metabol ic eff icacy of insulin in target t issues. In genera l , chemica ls in the first category are of specif ic interest as they c losely reproduce les ions that occu r during fi-cel l destruction in IDDM. Moreover, these agents provide a relatively permanent d iabetes which is suitable for long term studies. A l loxan , a cycl ic urea ana log , w a s the first agent in this category that w a s reported to produce a permanent d iabetes in laboratory animals (Dunn et a l , 1943). Streptozotocin (STZ) (Rakieten et a l , 1963) has replaced al loxan as the principal agent used to produce exper imental d iabetes. Th is is due to: a) the greater selectivity of fi-cel ls for S T Z (Junod et a l , 1969); b) the lower mortality rate seen in STZ-d iabet ic animals (effective d iabetogenic dose of S T Z is 4-5 t imes less than its lethal dose ; Hoft iezer and Carpenter , 1973) and c) the longer half life of S T Z in the body (15 min; Agrawal , 1980). 1.2.1. Mechanism of the diabetogenic effect of STZ Streptozotocin (STZ) [2-deoxy-2-(3-methyl-3-nitrosourea) 1-D-glucopyranose] is a broad spectrum antibiotic which is produced from Streptomyces achromogenes. The diabetogenic response to S T Z was first detected by Upjohn Laborator ies during testing of potential antibiotics from this organism. However, Rakieten et al (1963) were the first to descr ibe that fi-cel l necrosis and the ensuing diabetic state could be produced after a single intravenous dose of S T Z in rats and dogs. The chemica l structure of S T Z compr ises a g lucose molecule with a highly reactive nitrosourea s ide chain which is thought to initiate its cytotoxic act ion. The g lucose moiety directs this agent to the pancreat ic fi-cel l (Johnson and Tjalve, 1978). 6 The deleter ious effect of S T Z results from the generat ion of highly reactive carbonium ions ( C H 3 + ) , formed from decomposi t ion of the nitroso moiety. The C H 3 + ions cause D N A breaks by alkylating D N A bases at var ious posit ions (Uchigata et a l , 1982), resulting in activation of the nuclear enzyme poly (ADP-r ibose) synthetase as part of the cell repair mechan ism. A s cellular pyridine nucleot ides, particularly N A D + are utilized as substrates for the nuclear enzyme, a profound decl ine in N A D + occurs within 20 minutes. In effect, a n abrupt and irreversible N A D + exhaust ion leads to cessat ion of N A D + - d e p e n d e n t energy and protein metabol ism, ultimately leading to cel l death. Inhibition of poly (ADP-r ibose) synthetase by agents like 3 -aminobenzamide and nicot inamide are known to protect f i-cells from N A D + deplet ion and cell death after S T Z exposure (Wilson et a l , 1984). Severa l mechan isms have been postulated to explain why these fatal events occur select ively in f i-cells: (i) a high affinity of S T Z for the ft-cell membrane, (ii) unique SH-groups which render the fi-cell membrane especia l ly sensi t ive to oxidative interactions, (iii) a low capaci ty of R-cel ls to s c a v e n g e free radicals and (iv) a low N A D + / D N A ratio in islets compared with other t issues. 1.2.2. Characterist ics of STZ-diabetes in rats Three methods of administration of S T Z to induce diabetes are commonly used . Single large dose. At a dose of - 1 0 0 mg/kg, S T Z c a u s e s an intense fi-cell necros is , absence of insulin in the Pi-cell and hyperglycemia within 24-72 hrs. Disintegration and phagocytos is of necrotic cel ls is rapid, with practical ly no ev idence of debris or inflammation visible after 3 days. In an identical rapid manner, blood g lucose va lues peak 1 to 2 days after S T Z administration and remain e levated. O n e compl icat ion with this dose of S T Z is the occurrence of non-speci f ic les ions which occur in cel ls which are in c lose contact to the necrotic fi cel ls. T h e s e diabet ic rats can survive for up to 4-7 days without exogenous insulin (Rerup, 1970) but eventual ly die due to the development of a severe ketotic state. 7 Neonatal diabetes. This mild and stable form of d iabetes, resembl ing Type II human d iabetes is produced by a single dose of S T Z (90 mg/kg i.v.) in 2-day-old neonatal rats (Bonner-Weir et a l , 1980). The induced fi-cel l injury is fol lowed by limited regenerat ion primarily as a result of ductal budding rather than mitosis of preexist ing f i -cel ls, creating a short-term normalization of g lycemia. At 6-15 weeks of age, the rats have a n impaired g lucose d isposa l rate and significant fi-cel l secretory dysfunct ion. Single moderate dose. A dose of about 45-75 mg/kg S T Z , either intravenously (i.v.) or intraperitoneally (i.p.) results in the most commonly used mode l of S T Z -diabetes. After a single injection, fi-cell necrosis can be detected within 2-4 hrs on ultrastructural examinat ion and within 24 hours by light microscopy (Junod et a l , 1967). Four days following administration of S T Z in adult rats, the remaining fi-cel ls appear degranulated, with ev idence of limited proliferation over severa l months, a probable consequence of pre-existing precursor fi-cel ls rather than a result of ductal or ac inar cel l transformation into fi -cel ls (Hamming and Reyno lds , 1977). A tr iphasic pattern of changes in blood g lucose begins within 45-60 min of injection with S T Z . A n initial hyperg lycemia which lasts for 1 hr is fol lowed by a critical period of marked hypoglycemia (lasting ~6 hrs) which is brought about by mass ive fi cell degranulat ion and an enormous re lease of pancreat ic insulin. Stab le hyperg lycemia deve lops within 24-48 hrs and remains 3-4 t imes higher than normal in concert with - 5 0 % reduction in p lasma insulin levels and a pancreat ic insulin content less than 5 % of control levels (Junod et a l , 1969). Al though these animals are insulin-deficient, they do not require insulin supplementat ion for survival and do not develop ketonuria. Seve ra l endocr ine abnormali t ies exist in chronic STZ-d iabe tes including: high circulating levels of g lucagon, somatostat in, vasopress in , cort icosterone, atrial natriuretic pept ide, and reduced levels of renin and angiotensin II, a ldosterone, and thyroid hormones T 4 and T3, wh ich in addit ion to hypoinsul inemia and hyperglycemia, contribute to the complex metabol ic and physiological features of STZ-d iabe tes (Toml inson et a l , 1992). 8 1.3. VANADIUM IN BIOLOGY 1.3.1. Introduction Vanad ium was first d iscovered in 1813 by the Span i sh mineralogist del R io , who gave it the name 'panchromium' because of its color changes when pass ing through var ious oxidation states. It was rediscovered in 1831 by a Swed ish chemist Nils Gabr ie l Sefs t rom, who appropriately named the compound after Vanad is , a n ickname of the German ic goddess of beauty (Freya or Frija). Vanad ium is a group V transitional element which is widely distributed in nature, in soi l , water, air, plants, and animals. It is the twenty-first most abundant e lement in the earth's crust; the average content (135 ppm) is 0 .014% and is c lose to that of z inc. In s e a water, it is less abundant (in the range of 2-30 ppb) but it is found in marine organisms, especia l ly in asc id ians (sea squirts) which concentrate vanad ium to levels > 1g/kg dry weight. Drinking water contains vanadium levels of 0-6 ppb, whereas plants and animals have levels of 0-5 ppm (Waters, 1977). Due to its wide use in industry as a catalyst, atmospher ic levels of vanad ium are increasing (Hudson, 1964). In humans, the total body pool of vanadium is est imated to be around 100-200 ug (Byrne and Kos ta , 1978). S ince it compr ises less than 0 . 0 1 % body weight, vanad ium is classif ied as a trace mineral. The approximate t issue vanad ium concentrat ions in humans are (ng/g): liver, 5-19; kidney, 3-7; bone and teeth, <1-8; sp leen and thyroid, 3-4; brain, fat, colostrum, bile and urine, <1; lungs and hair, 12-140 (Byrne and Kos ta , 1978). Physio logical concentrat ions of vanad ium in human serum measured by neutron activation analys is w a s est imated to range from 24 to 939 ng/l (Cornel is et a l , 1981) and from 260 to 1300 ng/l (Simonoff et a l , 1984) or in the range of ~ 1 0 _ 1 1 M. The daily intake of vanadium in humans is in the range of 10-60 ug (Byrne and Kos ta , 1979). There are several extensive review articles which d i scuss the role of vanad ium in biology (Macara , 1980; Nechay , 1984; Nechay et a l , 1986b; C h a s t e e n , 1990; Rehder , 1992). 9 1.3.2. Chemistry In common with most transitional metals, vanad ium can exist in severa l va lence states (-3, - 1 , 0, +1 to +5). Thus , when studying the effects of vanad ium in vitro, it is important to appreciate that the interconversion between the different spec ies in aqueous solution occurs with relative e a s e and is dependent on pH and concentrat ion. In biological sys tems, vanadium is found predominantly in vanadate (+5, V(V)) and vanady l (+4, V(IV)) forms. In aqueous solution at neutral p H , the predominant spec ies is the pentavalent vanadate (+5). At physiological concentrat ions of <10 u M , the monomer ic forms of tetravalently coordinated vanadate ( H 2 V 0 4 - and H V 0 4 2 - ) predominate at a pH of 6-8. Alternately, the form of vanadate which shares the c losest stereochemistry with phosphate, V 0 4 3 _ exists only at very bas ic pH (>12) where biological sys tems are inactive. In m M concentrat ions at pH 7, the cycl ic anion complex of four V 0 4 units ( H V 4 0 1 2 3 " ) can be found. Vanada te can be reduced to V 0 2 + by glutathione ( G S H ) , ascorbate and N A D H . Being a divalent cat ion, vanady l can compete for l igand binding sites in var ious proteins which are normally occup ied by other metals such as Z n 2 + , C a 2 + and M n 2 + (DeKoch et a l . , 1974). As ide from proteins, V 0 2 + binds readily to amino ac ids, nucleic ac ids, phosphates, phosphol ip ids, glutathione, citrate, oxalate, lactate, ascorbate and catechol (Nechay et a l , 1986). In chelated form, vanadyl is resistant to oxidation. A b o v e a pH of 2.3, hydrolyzed spec ies of V 0 2 + (VOOH+, ( V O O H ) 2 2 + ) can undergo air oxidation to form vanadates . At physiological pH , free vanadyl is probably nonexistent, because of its propensity to convert to vanadate, or to form stable complexes with proteins. The commerc ia l water soluble vanady l sulfate ( V O S 0 4 . - 2 H 2 0 ) gives a blue aqueous V O ( H 2 0 ) 5 2 + solut ion. 1.3.3. Absorpt ion, distribution and excretion It was est imated that only a smal l fraction of vanadium (>1%) ingested in the G l tract is actually absorbed (Byrne and Kos ta , 1978). However, studies in rats suggest 10 greater intestinal absorpt ion, in the range of 30 -40% when administered acutely or in the diet (Weigmann et a l , 1982; Bogden et a l , 1982). Mos t ingested vanad ium is transformed in the s tomach to V 0 2 + and remains in this form as it p a s s e s into the duodenum (Chas teen et al , 1986b). S ince vanadate is absorbed 3-5 t imes more effectively than V 0 2 + , the effect of dietary components (ascorbic ac id , E D T A , protein) which affect transition to vanadate limit its oral absorpt ion (Chas teen et a l , 1986b). Vanad ium is preferentially distributed in the bone, kidney and liver fol lowing an i.p. injection (Sharma et a l , 1980). Bone is the long-term storage depot for vanad ium, due to the element 's similarity to phosphate (Talvitie and Wagner , 1954). Consistent ly greater t issue vanad ium levels were detected in rats g iven N a 3 V 0 4 a s compared to V O S 0 4 in the drinking water (Parker and Sha rma , 1978). W h e r e a s significant t issue vanad ium w a s detected at 3 weeks after cessat ion of treatment, at 6 w e e k s of deplet ion, t issue vanadium levels of V O S 0 4 - t r e a t e d animals returned to control, except for kidney and bone, although it was still relatively high in N a 3 V 0 4 - t r e a t e d rats. The t 1 / 2 for el imination of vanad ium was determined to be 12 days after 3-week treatment with vanadyl sulfate (Ramanadham et al , 1991). Distribution patterns were identical following i.v. administration of various forms (+3 to +5) of vanad ium, suggest ing convers ion to a common spec ies in p lasma (Sabbioni et a l , 1978). T h e main route of excret ion of vanad ium is in the urine, where it is assoc ia ted mostly with low-molecular weight components (Talvitie and Wagner , 1954; Sabb ion i and Marafante, 1978). 1.3.4. Intracellular forms and levels In p lasma, vanadium probably exists in both oxidation states +5 ( V 0 3 * ) and +4 ( V 0 2 + ) as a result of oxygen tension and the presence of reducing agents (ascorbate and catecholamines) . Approximately 9 0 % of circulating vanad ium is bound to proteins (Nechay, 1984), preferentially with the iron transport protein transferrin (Harris et a l , 1984; Chas teen et a l , 1986a). At pharmacological levels of - 1 - 2 0 u M (after chronic 11 treatment), free p lasma vanadium is probably in the order of < 1Ch 6 M. Free vanadate enters into the cell via the anion transport system, similar to that used by phosphate (PO4-). Cel lu lar uptake of vanadate into red blood cel ls is rapid, and a constant cell-to-p lasma ratio is reached within 1 hour (Harris et a l , 1984). O n c e in the cytosol , vanadate is reduced nonenzymat ical ly by G S H to the vanadyl ( V 0 2 + ) ion with a t 1 / 2 of ~2 hours (Macara et a l , 1980). In red blood cel ls, intracellular V 0 2 + is bound to hemoglob in (Cant ley and A i s e n , 1979), whereas in rat adipocytes, it is complexed to G S H , mostly in a 1:1 V 0 2 + : G S H stoichiometry (Degani and Shechter , 1981). E S R spect roscopy of subcel lu lar liver fractions confirmed that vanadium exists in the +4 oxidation state following i.p. administration (Sakurai et a l , 1980). Complexat ion of V 0 2 + to l igands prevents its oxidation to vanadate, which would otherwise tend to occur in intracellular p H . Never the less, oxidation-reduction reactions can still be thought to result in formation of limited amounts of free vanadate. S ince most cel lular V 0 2 + is bound, an est imated <1% of intracellular V 0 2 + is free (Nechay et a l , 1986a). In musc le and liver following chronic treatment, vanadium levels are in the range of 1 0 - 6 to 1 0 - 5 M (Mongold et a l , 1990), in which case , free intracellular vanad ium is probably in the range of ~ 1 0 _ 8 - 1 0 - 7 M or 100 t imes the est imated physiological levels ( ~ 1 0 - 1 0 - 1 0 - 9 M) (Nechay et al , 1986b). Furthermore, most intracellular free vanad ium would be hydrolyzed V 0 2 + ( V O O H + and V O ( O H ) 2 2 + ) (Nechay et a l , 1986a). Thus , it is conce ivab le that the predominant intracellular spec ies of vanad ium, V 0 2 + - G S H may play an important role in the demonstrated insulin-like effects of vanad ium in vivo. 1.3.5. Nutritional role Although found in both plants and animals, the biological role of vanad ium is not completely understood. In rats, vanadium supplemented in the diet of subopt imal ly growing rats resulted in a positive growth response (Schwarz and Mi lne, 1971), suggest ing that vanad ium is essent ia l for normal growth. S igns of vanad ium def ic iency 12 were also descr ibed in chicks and rats maintained on diets of <30 or <100 ng/g vanad ium respectively (Mertz, 1974; Underwood, 1977). However , conflicting results were a lso reported. For instance, the sole obvious effects of vanad ium deprivat ion in rats w a s an impaired reproductive ability, a phenomenon which only occurred after the fourth generat ion (Hopkins and Mohr, 1974). However, more recent studies showed that goats fed a vanadium-def ic ient diet exhibited a higher abort ion rate, lower milk production, and a 3 0 % lower survival rate and skeletal deformities in newborn kids (Anke et a l , 1986). Similarly, rats fed low vanadium diet had an increased thyroid weight/body weight and lower growth (Uthus and Nie lsen, 1988). Hence , these def ic iency s igns suggest that vanadium is an essent ia l nutrient in higher an imals , and has a biological function either in thyroid hormone metabol ism or as a growth factor. A l though vanad ium def ic iency has not been descr ibed in humans, the dietary requirement of vanad ium was recommended to be smal ler than most essent ia l t race e lements, - 1 0 ug daily (Nielsen and Uthus, 1990), c lose to the est imated daily intake. 1.3.6. Effects on enzyme systems Vanada tes are known activators or inhibitors of numerous enzymes , including phosphoenzyme ion-transport A T P a s e s , r ibonuclease, acid and alkal ine phospha tases , adenylate cyc lase , and phosphotyrosyl phosphatases. It is important to appreciate the wide range of activities of vanad ium, documented in severa l review art icles (Macara , 1980; R a m a s a r m a and Crane , 1981; Jandhya la and Horn, 1983; Nechay , 1984). 1.3.6.1. ATPases Cant ley et al (1977) uncovered the potent N a + / K + - A T P a s e inhibitory activity of vanadate from a S i g m a grade A T P preparation. Inhibition by vanadate (Kj = 1 0 - 6 M) of purified N a + / K + - A T P a s e occurs from interaction with the cytoplasmic face of the enzyme (Cant ley et a l , 1978). Al though three t imes less potent, the inhibitory effects of 13 vanadate on C a 2 + / M g 2 + - A T P a s e have also been reported (Bond and Hudgins, 1980; Dupont and Bennett, 1982). B e c a u s e of structural similarity to P 0 4 - , vanadate is thought to act as an analog of inorganic phosphate. Thus , in intact red cel ls, an intracellular transformation to V 0 2 + greatly d iminishes its inhibitory activity (Cant ley and A i s e n , 1979). Indeed, vanadate up to a concentrat ion of 1 m M did not inhibit the N a + / K + - A T P a s e of intact adipocytes (Dubyak and Kleinzel ler, 1980; Shech te r and Kar l ish, 1980). Severa l other related A T P a s e s such as the H + / K + - A T P a s e (O 'Nea l et a l , 1979), dynein A T P a s e (Gibbons et al , 1978) and myosin-actomyosin A T P a s e s (Goodno , 1979) are also inhibited by vanadate, mostly at 1 0 - 3 M concentrat ions. 1.3.6.2. Adenylate cyclase and phosphodiesterase The dose-dependent activation of adenylate cyc lase by vanadate ( 1 0 - 5 - 1 0 - 3 M) was first descr ibed in rat ad ipose p lasma membranes (Schwabe et a l , 1979). Enhanced basa l and isoprenal ine-st imulated adenylate cyc lase activity was postulated to result from enzyme complex formation aided by the guanine nucleot ide regulatory protein (Krawietz et a l , 1982). Stimulation was not demonstrated with the V 0 2 + form (Schmitz et a l , 1982). In addit ion, vanadate and V 0 2 + - G S H at 1 0 " 6 M concentrat ions st imulated c A M P phosphodies terase activity in intact rat ad ipocytes ( S o u n e s s et a l , 1985). In card iac t issue, the net effect of vanadate was to increase intracellular c A M P levels (Grupp et al , 1979), but in rat hepatocytes, vanadate did not modify intracellular c A M P levels or cAMP-dependen t protein k inase activity (Vi l lar-Palasi et a l , 1989). 1.3.6.3. Miscellaneous Vanada te has been shown to be a potent inhibitor of severa l e n z y m e s (Kj ~ 1 0 - 6 M) whose mechan isms include a phosphorylated enzyme intermediate, including acid and alkal ine phosphatase (Van Etten et a l , 1974; Seargeant and S t inson , 1979). B e c a u s e the length of the V - 0 bond is not much greater than the P - 0 bond, 14 competit ion for the phosphate-binding site on the enzyme can result in formation of a stable transition state ana log (Gresser and Tracey, 1990). Vanada te ( 1 0 - 6 M) is a potent phosphotyrosine phosphatase ( P T P a s e ) inhibitor (Swarup et a l , 1982). S i nce inhibition of P T P a s e enhances the tyrosine k inase activity of the insulin receptor, this property has been proposed as a possible mechan ism of its insulin-like activity. 1.3.6.4. NAD(P)H Oxidation Vanada te has been shown to enhance the oxidation of reduced nicot inamide adenine nucleot ides, N A D ( P ) H , an effect not found to require the p resence of a speci f ic enzyme (Vyskoci l et a l , 1980). Instead, 0 2 " generated from N A D ( P ) H ox idase reacts with vanadate to yield a peroxovanadyl complex ( V 0 2 ) which ox id izes N A D ( P ) H v ia a free-radical chain reaction (Liochev and Fridovich, 1986). Interestingly, N A D P H is used in the reduction of ox id ized glutathione ( G S S G ) , whereas oxidation of reduced spec ies ( G S H ) occurs with the addit ion of H 2 0 2 . Thus , the following reactions have been demonstrated in erythrocytes (Wil l iams, 1980): glutathione reductase G S S G + N A D P H + H + > 2 G S H + NADP+ (1) glutathione peroxidase 2 G S H + H 2 0 2 > G S S G + 2 H 2 0 (2) Hence , addit ion of H 2 0 2 and the relative loss of N A D P H would lower cellular G S H , which is principally involved in reducing intracellular vanadate to V 0 2 + , and a major intracellular chelator of V 0 2 + , preventing its reoxidation to vanadate . S i nce most effects of vanad ium on enzymes have been attributed to vanadate, factors which tend to change the redox state of intracellular vanadium may also alter the effects of vanad ium in vitro, and perhaps in vivo. In support of this proposal , the addit ion of H 2 0 2 to vanadate w a s shown to potentiate its insulin-mimetic effects (Posner et a l , 1990). 15 1.3.7. Effects on physiological systems B e c a u s e of its wide-ranging effects on enzymes , vanad ium, perhaps not surprisingly, has also been shown to exert severa l effects in vitro and in vivo. Card iac responses to vanadium are dependent on spec ies and area of the heart (Akera et a l , 1983). Posit ive inotropic effects were observed in cat papil lary musc le , with maximal effects at 1 0 - 3 M vanadate (Hackbarth et a l , 1980). T h e s e inotropic effects were found to be unrelated to inhibitory effects on N a + / K + - A T P a s e , unlike with card iac g lycos ides. Other postulated mechan isms include the stimulation of adenylate cyc lase (Krawietz et a l , 1980), and an insulin-like effect in stimulating K + uptake in card iac myocytes (Werdan et a l , 1982). In contrast, negative inotropy w a s observed in gu inea pig and cat atria (Borchard et a l , 1979; Grupp et a l , 1979). The effects of vanadate on the kidney have been reviewed extensively (Phil l ips et a l , 1983). In rats, vanadate is a potent diuretic and natriuretic when administered i.v. in high doses , an effect attributed to the inhibition of N a + / K + - A T P a s e and tubular reabsorpt ion (Balfour et al , 1978). However, when chronical ly administered, the renal vanad ium concentrat ions ( 1 0 - 5 M) were not assoc ia ted with changes in N a + excret ion or N a + / K + - A T P a s e activity (Higashino et al , 1983). At high doses , vanadate induces vasoconstr ict ion (Hudgins and Bond , 1981), but sustained hypertension with chronic vanad ium treatment is demonstrated only in rats with diminished renal excretory function (Sus ie and Kentera , 1988). Sod ium orthovanadate ( V 0 4 - ) and to a lesser extent metavanadate ( V 0 3 - ) inhibited bone resorption induced by parathyroid hormone and prostaglandin E 2 in neonatal mouse calvarie (Krieger and Tashj ian, 1983). Vanada te at high concentrat ions ( 1 0 - 3 - 1 0 - 4 M) induced basal and glucose-st imulated insulin re lease from islets isolated from rats (Fagin et a l , 1987) and mice (Zhang et a l , 1991). Alternately, at lower concentrat ions (1-50 uM), vanadate inhibited both basa l and g lucose-st imulated insulin re lease in mouse islets (Voss et a l , 1992). 16 1.4. INSULIN-MIMETIC E F F E C T S OF VANADIUM IN VITRO The reports of various insulin-like act ions of vanad ium in vitro and subsequent ly in vivo has sparked interest in the possib le therapeutic value of vanad ium in d iabetes. There are severa l reviews summariz ing the insulin mimetic act ions of vanad ium in biological sys tems (Shechter et a l , 1990a,b; Orvig et a l , 1995) 1.4.1. Carbohydrate Metabolism To lman et al initially reported in 1979 that the addit ion of vanad ium in vitro to var ious t issues isolated from the rat resulted in a multitude of metabol ic effects on carbohydrate metabol ism similar to those exhibited with insulin. T h e s e effects were the activation of g lucose uptake and oxidation in adipocytes, stimulation of g lycogen synthesis in liver and d iaphragm, and inhibition of hepatic g luconeogenes is . Moreover , vanad ium enhanced the effects of submaximal concentrat ions of insulin. Subsequent ly , independent studies by Shech te r and Kar l ish (1980) and Dubyak and Kle inzel ler (1980) recognized that the insulin-mimetic effects of vanadium on g lucose transport and metabol ism in intact adipocytes were not linked to its wel l -documented inhibitory effects on N a + / K + - A T P a s e . The effects of vanadium on carbohydrate metabol ism in vitro are numerous (Table 2). A l though the large majority of effects are insulin-mimetic, they are mostly demonstrable at a high concentrat ion range ( 1 0 - 4 to 1 0 " 3 M). T h e s e effects include an increased g lucose transport, shown in rat adipocytes (Green , 1986; Pe rshads ingh , 1987), human and rat card iac myocytes (Werdan et a l , 1982), rat skeletal musc le strips (C lausen et a l , 1981; Clark et a l , 1985) and sarco lemmal ves ic les (Okumura and S h i m a z u , 1992). Vanada te a lso stimulated translocation of the insul in-regulatable g lucose transporter (GLUT4) in rat adipocytes (Paquet et a l , 1992), and increased g lucose transporter ( G L U T 1 ) express ion in vitro in NIH 3T3 mouse f ibroblasts, an effect attributed mostly to increased m R N A stabil ization (Mountjoy and Flier, 1990). 17 Table 2. In vitro effects of vanadium on carbohydrate metabolism Activity/ Effect/ Reference Concentration (M) Target Tissue G l u c o s e Transport Stimulated To lman e t a l , 1979 10-4 Rat ad ipocytes Dubyak and Kleinzel ler, 1980 1 0 - 4 - 1 0 - 2 G r e e n , 1986 10-4 Pershads ingh et al , 1987 10-3 C l a u s e n et a l , 1981 10 -4 -10 " 3 Rat skeletal musc le Clark e t a l , 1985 10-3 Okumura and S h i m a z u , 1992 10 -3 -10 -2 Rat sa rco lemma C a r e y e t a l , 1995 10-2 Human skeletal musc le G o m e z - F o i x et a l , 1988 10 -4 -10 -3 Rat hepatocytes Werdan et a l , 1982 10 - 5 -10 -3 Rat card iac myocytes Haj jaret a l , 1989 10 -4 -10 -3 Rat intestinal cel ls Unchanged Lonnroth e t a l , 1993 10-4 Human adipocytes Inhibited To lman et a l , 1979 10-4 Rat je junum Kellett and Barker, 1989 10-3 G l u c o s e Transporter Increased expression NIH3T3 mouse f ibroblasts Mountjoy and Flier, 1990 10-6 Increased translocation P a q u e t e t a l , 1992 10-3 Rat ad ipocytes Glyco lys is Stimulated G o m e z - F o i x e t a l , 1988 10 -4 -10 -3 Rat hepatocytes Duckworth et a l , 1988 10 -3 -10 -2 Rat adipocytes Clark e t a l , 1985 10-3 Rat skeletal musc le Inhibited B e n a b e et a l , 1987 10-6 Human erythrocytes G l u c o s e Oxidat ion Stimulated To lman e t a l , 1979 10 - 5 -10 -3 Rat ad ipocytes Dubyak and Kleinzel ler, 1980 10 -4 -10 -2 S h e c h t e r a n d Kar l ish, 1980 10-5 Degani et a l , 1981 10-4 Clark e t a l , 1985 10-3 Rat skeletal musc le G o m e z - F o i x et a l , 1988 10 -4 -10 -3 Rat hepatocytes G l u c o s e Output Inhibited Bruck e t a l , 1991 10-7 Rat liver Increased R o d e n e t a M 9 9 3 10 -5 -10 -3 Rat liver G lvcoqen Svnthes is Increased Rat hepatocytes To lman et a l , 1979 10-4 J a c k s o n et a l , 1988 10-5 Clark e t a l , 1985 10-3 Rat skeletal musc le Ueki e t a l , 1992 10 -4 -10 - 3 Rat ad ipose t issue 18 Vanada te was demonstrated to enhance g lucose oxidation in isolated rat ad ipocytes (Tolman et a l , 1979; Shechter and Kar l ish, 1980). The maximal effect of vanad ium on g lucose oxidation exceeded that of insulin, and was attributed to a select ive stimulation of the pentose phosphate shunt, while the max imal effect on glycolysis was equal to that of insulin (Duckworth et a l , 1988). Vanada te a lso stimulated glycolysis in hepatocytes isolated from control (Gomez -Fo i x et a l , 1988) and diabetic (Rodr iguez-Gi l et a l , 1991) rats, consistent with its effects on glycolytic enzymes (Table 3). T h e s e effects include the inhibition of g lucose 6-phosphatase and stimulation of 2,3-b isphosphoglycerate phosphatase, attributed to competi t ion for P 0 4 _ -binding sites (Singh et a l , 1981; Ninfali et a l , 1983). In rat hepatocytes, vanadate raised fructose 2,6-b isphosphate levels without concomitantly enhancing 6-phosphofructo-2-k inase (PFK-2 ) activity (Gomez-Fo ix et a l , 1988). This effect w a s subsequent ly proposed to ar ise from inhibition of f ructose-2,6-b isphosphatase activity, which w a s demonstrated in ch icken liver (Rider et a l , 1990). Vanada te inhibited the inactivation of P F K - 2 by g lucagon, by reducing intracellular c A M P levels (Miralpeix et a l , 1990; G o m e z - F o i x et a l , 1988). In addit ion, enzyme express ion is altered by vanad ium in an insulin-like manner. For instance, in rat hepatocytes, vanadate lowered basa l phosphoenolpyruvate carboxyk inase ( P E P C K ) m R N A levels (Bosch et a l , 1990) and increased the express ion of L-type pyruvate k inase gene (Miralpeix et a l , 1991). Vanada te was reported to stimulate g lycogen synthesis in isolated rat hepatocytes (Tolman et a l , 1979; J a c k s o n et a l , 1988) and in skeletal musc le (Clark et a l , 1985). In contrast, in rat d iaphragm, vanadate had no effect on g lycogen synthesis a lone or in the presence of insulin, but caused a several-fold increase in g lycogen synthes is in the p resence of IGF-I which, a lone, had no effect (Vandorpe et a l , 1992). Interestingly, vanadate at very low concentrat ions (0.5 - 1.0 uM) inhibited g lucose output by 50 -60% from the perfused rat liver, an effect which w a s not similarly demonstrated with insulin in the same preparation (Bruck et a l , 1991). 19 Table 3. In vitro vanadium effects on enzymes involved in g lucose metabol ism Activity/ Reference Effect/ Concentration (M) Target Tissue Glycogen Syn thase Tamura et a l , 1983 B o s c h e t a l , 1987 Rodr iguez-Gi l et a l , 1989 G l ycogen Phosphorv lase Rodr iguez-Gi l et a l , 1989 B o s c h e t a l , 1987 Glycera ldehyde 3-phosphate dehydrogenase B e n a b e e t a l , 1987 G lucose -6 -Phospha tase S ingh et a l , 1981 Yamaguch i et a l , 1989 G lucose-6 -phospha te -dehydrogenase Nour -E ldeen et a l , 1985 2,3-b isphosphoglycerate phosphatase (2 .3 -DPG) Car reras e t a l , 1982 Ninfali e t a l , 1983 M e n d z e t a l , 1990 6-Phosphofructo-2-k inase G o m e z - F o i x et a l , 1988 Rodr iguez-Gi l et a l , 1991 Miralpeix e t a l , 1989 Fructose-2,6-b isphosphatase R ider e t a l , 1990 Phosphog lucomutase Car reras et a l , 1988 Perc iva l e t a l , 1990 Pyruvate K inase Miralpeix et a l , 1991 P-enolpyruvate carboxyk inase B o s c h e t a l , 1990 Stimulated 10-4-10-3 Inactivated 10-4-10-3 10-3 Stimulated 10-3 10-4-10-3 Inhibited 10-6 Inhibited 10-6 1 0 - 5 - 1 0 - 4 Stimulated 10-4 Stimulated 10 -5 -10 -3 10 -6 -10 -5 10-4 Unchanged 10 -4 -10 -3 10 -4 -10 -3 10-3 Inhibited 10-5 Inhibited 10-6 10-9 Increased expression 10-5 Decreased expression 10 -4 -10 -3 Rat ad ipocytes Rat hepatocytes Rat hepatocytes Human erythrocytes Rat hepatic m ic rosomes Purif ied enzyme Purif ied enzyme Human erythrocytes Rat hepatocytes Diabet ic rat hepatocytes Ch icken liver Rabbi t musc le Purif ied enzyme Rat hepatocyte culture Rat hepatoma cel ls 20 1.4.2. Lipid metabolism The insulin-like effects of vanad ium a lso extend to the lipid metabol ic pathways (Table 4). Importantly, unlike its effects on carbohydrate metabol ism, these effects have been demonstrated at 1 0 - 5 M concentrat ions that can conceivably be ach ieved in vivo with chronic vanadium treatment. In rat adipocytes, vanadate w a s shown to inhibit epinephrine-st imulated lipolysis (Degani et a l , 1981). However, addit ion of G S H , which potentiated vanadate-st imulated g lucose oxidation, did not further enhance its antilipolytic effects. The degree of maximal inhibition of l ipolysis w a s found to be greater with vanadate relative to insulin (Duckworth et a l , 1988). Inhibition of the insul in-sensit ive cycl ic nucleotide phosphodiesterase (cyclic GMP- inh ib i tab le) did not affect anti l ipolysis induced by the V 0 2 + - G S H complex in rat ad ipose t issue (Brownsey and Dong, 1995). Interestingly, the antilipolytic effect of V 0 2 + w a s reversed by cytochalas in B, suggest ing that the effect of vanad ium in suppress ing F F A re lease could be secondary to the activation of g lucose uptake (Nakai et a l , 1995). Vanada te stimulated lipid synthesis to a level 2-3 t imes greater than insulin in isolated rat hepatocytes (Castro et a l , 1984; Agu is and Vaart jes, 1982). However , vanadate at a concentrat ion (10 uM) which inhibited apol ipoprotein B secret ion from rat hepatocytes, had no effect on l ipogenesis until higher concentrat ions (40-80 uM) were used , unlike insulin which affected both activities at similar concentrat ions (Jackson et a l , 1988). Low concentrat ions of vanadate (50 uM) potentiated insul in-st imulated l ipogenesis, which was preserved after removal of insulin (Fantus et a l , 1990). T h e s e effects were attributed to an enhanced affinity for the insulin receptor (IR) to insulin. Like insulin, vanadate stimulated the re lease of l ipoprotein l ipase activity from rat fat pads (Ueki et a l , 1989), an effect which was subsequent ly attributed to an increased phospho l ipase A 2 activity (Morita et a l , 1995). However , unlike insul in, vanadate a lso stimulated the re lease of rat hepatic l ipase activity from liver s l ices (Morita et a l , 1991). 21 Table 4. In vitro effects of vanadium on lipid metabolism Activity/ Reference Effect/ Concentration (M) Target Tissue Lipolvsis Degan i e t a l , 1981 Duckworth et a l , 1988 M o o n e y et a l , 1989 Sh i sheva and Shechter , 1993a Nakai e t a l , 1995 L ipoqenes is Shech te r and R o n , 1986 Duckworth e t a l , 1988 Sh i sheva and Shechter , 1992a Agu is and Vaart jes, 1982 Cast ro et a l , 1984 J a c k s o n et a l , 1988 Fatty Ac id Oxidat ion G u z m a n and Cast ro , 1990 Secret ion of Apol ipoprotein B J a c k s o n et a l , 1988 Lipoprotein L ipase Uek i e t a l , 1989 Morita et a l , 1993 Inhibited 10-5 10-4. 10-5 10-4 10-4 10-2 Stimulated 10 -5 -10 -3 10 -3 -10 -2 10-4 10 -4 -10 -3 10-3 10-5 Stimulated 10-3 Inhibited 10-5 Stimulated 10-4 10 -4 -10 -3 Rat adipocytes Rat adipocytes Rat hepatocytes Rat hepatocytes Rat hepatocytes Rat adipocytes Rat liver s l ices 22 1.4.3. Protein metabolism The effects of vanadium on protein metabol ism are listed on Tab le 5. In cultured calvar iae and chondrocytes, low concentrat ions of vanadate increased the synthesis of co l lagen and noncol lagen protein (Canal is , 1985). However, protein synthesis w a s shown to be unaffected in skeletal muscle (Clark et a l , 1985), despi te an enhanced amino acid transport in this t issue (Henr iksen, 1991). The effects of vanad ium on amino acid transport in rat jejunum are dose-dependent . A t low levels (10~ 4 M) , stimulation of transport occurs via the activation of adenylate cyc lase , while at higher concentrat ions, transport was reduced due to inhibition of N a + / K + - A T P a s e (Hajjar et a l , 1987). Vanada te inhibited protein degradat ion in liver homogenate only at high concentrat ions (10 mM) (Seglen and Gordon , 1981). 1.4.4. Mitogenic effects The mitogenic effects of vanadium, alone or in conjunction with other growth factors, have been reported at very low concentrat ions (~10- 6 - -10 - 5 M) (Table 5). For instance, vanadate (0.1-10 uM) increased D N A synthesis in cultured rat ca lvar iae, al though these effects were reversed at higher doses (100 uM) (Canal is , 1985). Either vanadyl or vanadate (5-50 uM) were mitogenic for Sw i ss mouse 3T3 and 3T6 cel ls , effects which were greatly potentiated by insulin (Smith, 1983). In cultured calvar iae, vanadate (5-15 uM) potentiated the mitogenic action of growth factors (insulin, E G F , IGF-I), increasing bone proliferation and differentiation (Lau et a l , 1988). Similar ly, vanadate enhanced insulin-stimulated D N A synthesis in cultured mouse mammary g land, al though it had no effects a lone (Hori and O k a , 1980), suggest ing s o m e difference. The mitogenic effects of vanadate and insulin a lso differed with respect to phosphorylat ion patterns in SV40- t ransformed 3T3 T cel ls (Wang et a l , 1994). A s most of the mitogenic effects of vanadium are demonstrated in cultured cell l ines, the re levance of these effects in vivo remains to be s e e n . 23 Table 5. In vitro effects of vanadium on protein metabolism and mitogenesis Activity/ Reference Effect/ Concentration (M) Target Tissue Protein Synthes is Cana l i s , 1985 Lau e t a l , 1988 Kato et a l , 1987 Marshal l and Monzon , 1987 Barnes et a l , 1995a Clark e t a l , 1985 Fantus et a l , 1989 Protein Degradat ion Seg len and Gordon , 1981 Clark e t a l , 1985 Amino Ac id Transport Henr iksen et a l , 1991 M u n o z e t a l , 1992 Ha j j a re ta l , 1987 Ha j j a re ta l , 1987 Stimulated -10-7-10-5 10 -6 -10" 5 10-6 10-6 10 -5 -10 -4 No Effect 10-3 10 -6 -10 -4 Inhibited 10-2 No Effect 10-3 Stimulated 10-4 10-3 10-4 Inhibited 10 -3 -10 -2 Cultured rat calvar ie Cultured calvarial cel ls Rabbi t chondrocyte culture Pr imary adipocyte culture X e n o p u s laevis oocytes Rat skeletal musc le Rat ad ipocytes Rat hepatocytes Rat skeletal musc le Rat skeletal musc le Rat jejunum Rat jejunum Mitogenic Activity Hori and O k a , 1980 Carpenter , 1981 Smi th , 1983 Cana l i s , 1985 Lau et a l , 1988 Montesano et a l , 1988 Maher , 1992 Increased 10-6 10 10 10 10 10 10 10-5 Cultured cel ls M o u s e mammary cel ls Human f ibroblasts S w i s s mouse 3T3/3T6 cell Rat calvarial cel ls Capi l lary endothel ium 24 1.4.5. NON-INSULIN MIMETIC E F F E C T S OF VANADIUM Not all reported effects of vanadium in vitro have been compat ib le with the metabol ic act ions of insulin. In some t issues, vanadium appears to be select ive for speci f ic act ions of insulin while failing to mimic others. For instance, in rat epitrochlearis musc le, vanadate increased g lycogen synthesis, and g lucose uptake and oxidat ion, but had no apparent effect on protein synthesis or degradat ion at the s a m e dose range (Clark et a l , 1985). In hepatocytes, opposing effects on carbohydrate metabol ism have been noted. Thus , despite the demonstrated insulin-like effects on glycolysis ( G o m e z -foix et a l , 1988) and g lycogen synthesis (Jackson et a l , 1988), non-insul in- l ike, glycogenolyt ic effects on phosphorylat ion and inactivation of g lycogen synthase were also found (Bosch et a l , 1987). This observat ion is in marked contrast to effects in rat adipocytes, wherein vanadate enhanced dephosphorylat ion and activation of g lycogen synthase (Tamura et a l , 1984), supporting the notion that the effects of vanad ium on s o m e e n z y m e s may be t issue-dependent. Unl ike its effects in rat hepatocytes, vanadate (10" 6 M) reduced the rate of glycolysis in erythrocytes by inhibiting g lycera ldehyde-3-P dehydrogenase, via the oxidation of cysteine groups on the enzyme (Benabe et a l , 1987). Vanada te was found to have no effect on pyruvate k inase in rat hepatocytes (Gomez-Fo ix et a l , 1988), despite competit ion for and binding to a divalent cation site occupied by M g 2 + (Lord and R e e d , 1990). There a lso appeared to be a lack of effect on g lucok inase or hexok inase activities (Singh et a l , 1981). S o m e effects of vanad ium are dose-dependent . Thus , infusion of vanadate (25 - 100 uM) in isolated perfused livers of rats increased hepatic g lucose output (Roden et a l , 1993), al though g lucose output was inhibited at lower concentrat ions (0.5 - 1.0 uM) (Bruck et a l , 1991). In rat smal l intestine, transmural transport of g lucose was diminished by vanadate , an effect l inked to the inhibition of N a + / K + - A T P a s e activity (Tolman et a l , 1979; Kellet and Barker, 1989) although at lower concentrat ions through chronic treatment, vanad ium enhanced g lucose transport (Hajjar et a l , 1989). 25 1.5. INSULIN-MIMETIC E F F E C T S OF VANADIUM IN VIVO 1.5.1. Control (nondiabetic) animals Chron ic treatment of control animals with vanad ium has been shown to lower p lasma insulin levels without affecting g lycemia (Heyl iger et a l , 1985; R a m a n a d h a m et al , 1989), suggest ing an improved insulin sensitivity and lowered insulin demand . In support, vanad ium treatment enhanced the hypoglycemic response to i.v. insulin (0.75 U/kg), an effect which was attributed to increased insulin sensitivity towards glycolysis and g lycogen synthesis observed in isolated so leus muscle from the vanadate- t reated rats (Chal iss et a l , 1987). Similarly, in vivo basa l hexose uptake into liver and musc le w a s a lso found to be enhanced in vanadium-treated control rats (Meyerovi tch et a l , 1987). A s wel l , vanadate administered i.p. into control rats was found to act ivate g lycogen synthesis by 2-fold in d iaphragm, and by 7-8 fold in heart and liver (Vandorpe et a l , 1992). However, using submaximal hyperinsul inemic c lamps, vanadium-treated control animals did not exhibit an enhanced insulin sensitivity in either peripheral g lucose utilization or hepatic g lucose production (Venkatesan et a l , 1991; B londel et a l , 1989). Similarly, in response to a high dose of i.v. insulin (10 U/kg), vanadate-t reated control rats did not demonstrate an enhanced hypoglycemic effect (Hei et a l , 1995). Centra l administration of vanad ium produced a transient sys temic hyperg lycemia in mice, an effect which was attributed to an increased g lucose uptake in neuronal cel ls , and enhanced sympathet ic outflow (Amir et al , 1987). The inhibition of food intake and body weight gain with 3-day oral vanadium treatment was similarly l inked to the stimulation of g lucose uptake in brain (Meyerovi tch et a l , 1989). W h e r e a s vanad ium treatment for 14 days reduced Na + - dependen t g lucose uptake in je junum and i leum (Madsen et a l , 1993), 30-day treatment enhanced the intestinal transport of g lucose , an effect attributed to increased g lucose metabol ism with prolonged exposure to vanad ium (Hajjar et a l , 1989a). P l a s m a fatty ac id , lipid and cholesterol synthesis have a lso been shown to be lowered in vanadium-fed chicks (Hafez and Kratzer, 1976). 26 1.5.2. Animal models of Type 1 diabetes Vanad ium treatment amel iorates symptoms of the diabetic state in animal models of Type 1 diabetes as summar ized below. The antidiabetic effects of vanad ium in vivo have been reviewed previously (Cros et a l , 1992; Brichard et a l , 1995). 1.5.2.1. Streptozotocin-diabetes The antidiabetic effects of vanad ium in vivo were first reported in 1985 by Heyl iger et a l . STZ-d iabet ic rats administered sodium orthovanadate ( N a 3 V 0 4 ) in the drinking water for 6 weeks exhibited normoglycemia and improved card iac function independent of any changes in p lasma insulin levels. E levated food and fluid intake in the diabetic animals were found to be reversed. Meyerovi tch et al (1987) subsequent ly reported that sod ium metavanadate ( N a V 0 3 ) lowered p lasma g lucose levels, and enhanced basa l hexose uptake by 2-fold in both liver and musc le . A s wel l , the reduced sensitivity of adipocytes to insulin-stimulated l ipogenesis was restored. S ince higher concentrat ions led to hypoglycemia and death in the animals, the authors reported optimal effects at circulating vanadium concentrat ions of 0.7-0.9 ug/ml (< 20 uM). Subsequent ly , Brichard et al (1988) demonstrated a concent ra t ion-dependence in the beneficial effects of vanadate, improving g lucose-to lerance and pancreat ic insulin content. G lycogen content was found to be increased in liver, but not in musc le . Vanad ium treatment has also been reported to alleviate the metabol ic defects assoc ia ted with insulin resistance in STZ-d iabe tes . A n enhanced insulin sensit ivity w a s reflected by the greater g lucose- lowering response of vanadium-treated animals to the acute administration of submaximal doses of insulin which had minimal effects in untreated diabetic rats (Ramanadham et al , 1990). In addit ion, hepat ic g lucose production, both in the basal state and in response to submaximal and maximal insulin was completely normal ized by vanadium treatment for 20 days (Blondel et a l , 1989). Per ipheral g lucose utilization, both basal ly and in response to submax imal insulin was 27 restored, al though maximal insulin response was still lower than control. In contrast, using a shorter treatment period (9-12 days) , vanadium was found to be ineffective in reversing insulin resistance at the level of hepatic g lucose production (Venkatesan et a l , 1990). Notably, steady-state insulin levels during the c lamp were 6 t imes higher in the earl ier study, which could have also contributed to dif ferences in detecting changes in insulin sensitivity. Per ipheral g lucose d isposal in response to submax ima l insulin w a s completely restored by vanadium treatment. The improved insulin sensitivity w a s dissoc iated from altered insulin receptor binding or tyrosine k inase ( IRTK) activity in musc le , suggest ing a post-receptor effect of vanad ium (Venkatesan et a l , 1990). Vanad ium treatment normal ized g lucose levels and reduced the signif icantly e levated hepatic P T P a s e activity in STZ-d iabet ic rats, suggest ing that inhibition of dephosphorylat ion of the IR, rather than direct activation of IRTK was the mechan i sm for the effects of vanadium (Meyerovitch et al , 1989). Enhanced insulin sensitivity in vanadium-treated rats was correlated with restoration of the diminished insul in-stimulated M A P and S 6 k inase activities in skeletal musc le (Hei et a l , 1994). Consis tent with the insulin-like effects on glycolytic enzymes in vitro, vanad ium treatment of STZ-d iabet ic rats reversed the reduced activities of P F K - 2 and g lucok inase in a t ime-dependent manner (Gi l et a l , 1988). However, unl ike the glycogenolyt ic anti-insulin-like act ions of vanadate in hepatocytes isolated from both control (Bosch et a l , 1987) and diabetic (Rodr iguez-Gi l et al , 1989) rats, the reduced g lycogen syn thase and g lycogen-synthase phosphatase activities in STZ-d iabet ic rats were restored by vanad ium treatment (Bol len et a l , 1990). The inconsistency between in vitro and in vivo effects likely results from the relatively high concentrat ions ( 1 0 _ 4 - 1 0 - 3 M) of vanadate employed in vitro. Reduced g lycogen synthase and phosphory lase activit ies in S T Z -diabetes were simultaneously corrected by vanadium treatment, suggest ing an improved g lycogen turnover rate, which was consistent with normal ized liver g lycogen content in the animals (Pugazhenth i and Khande lwa l , 1990). The restoration of normal 28 enzyme activities and m R N A levels supports a mechan ism for vanad ium in vivo at the pretranslational level. After 15 days of vanadium treatment, corrected L-pyruvate k inase and P F K - 2 activities were coupled with restored m R N A levels (Miralpeix et a l , 1992). However, complete restoration of enzyme express ion may be enzyme or t issue-specif ic. Hence , although hepatic m R N A levels and activities of L-pyruvate k inase and P E P C K were totally restored after 18 days of treatment, g lucok inase express ion and activity was only partially (40%) corrected at this time (Brichard et a l , 1993). Vanad ium treatment for 3 weeks a lso partially corrected (by > 50%) the d iminished express ion and activity of the l ipogenic enzymes ace ty l -CoA carboxy lase and fatty acid synthase in liver but not in ad ipose t issue (Brichard et a l , 1994). Aberrat ions in the t issue-speci f ic express ion of at least 2 isoforms of g lucose transporter in STZ-d iabe tes , which w a s enhanced in liver (GLUT2) and reduced in skeletal musc le ( G L U T 4 ) , were normal ized by 1-3 weeks of vanadium treatment (Brichard et a l , 1993; Strout et a l , 1990). A similar pattern in the express ion of P E P C K , G L U T 2 and 3-hydroxy-3-methylglutaryl-CoA ( H M G - C o A ) synthase gene in response to vanadium treatment suggests an effect of vanad ium in regulating a specif ic transcription factor in vivo (Valera et a l , 1993). Severa l t issue abnormali t ies have been demonstrated to be prevented by vanad ium treatment. T h e s e include card iac dysfunction as measured by the isolated working heart preparation in response to var ious left atrial filling pressures (Heyl iger et al , 1985), and in vivo in response to increased doses of norepinephrine (Pau lson et a l , 1978). In addit ion, the elevated ad ipose t issue lipolytic rates both in the basa l state and following inhibition by insulin were normal ized in vanadium-treated rats, effects a lso reflected in the corrected lipid profile in these animals ( R a m a n a d h a m et al , 1989). Moreover , vanad ium treatment preserved exocr ine pancreat ic function in STZ-d iabe t i c rats by restoring amylase content in pancreat ic acinar cel ls (Bendayan and Gr ingas , 1989), an effect attributed to increased transcription of the gene (Johnson et a l , 1990). 29 1.5.2.2. Spontaneously diabetic BioBreeding (BB) rats Chron ic vanad ium treatment reduced the amount of exogenous insulin required to prevent g lycosur ia in spontaneously diabetic (BB) rats ( R a m a n a d h a m et a l , 1990). The effect of vanad ium was dose-dependent and the required insulin d o s e to maintain normoglycemia was reduced up to 1/3, although total replacement of insulin w a s not ach ieved at vanad ium doses which restored normoglycemia in STZ-d iabe t i c rats (Battell et a l , 1992). Remova l of vanadium resulted in a gradual rise in insulin requirement. Vanad ium content in diabetic B B rat liver was lower than in nondiabet ic controls, suggest ing hormonal regulation of vanadium levels (Hamel et a l , 1993). 1.5.2.3. Partially pancreatectomized rats The beneficial effects of vanadium treatment has also been documented in 9 0 % pancreatectomized rats. Insulin res istance at the level of peripheral g lucose uptake was improved by 3-week vanadium treatment (Rossett i and Laughl in , 1989). Th is effect was mostly attributed to the correction of reduced g lycogen synthesis in skeletal musc le , which accompan ied supranormal g lycogen synthase activity. S i nce improvements in g lycogenes is were not observed with phlorizin treatment, the effects of vanad ium were d issociated from the elimination of hyperglycemia a lone. A l though no changes in hepatic IRTK activity were detected, consistent with the relative lack of insulin resistance at the level of hepatic g lucose production in this model , vanad ium (but not phlorizin) treatment was found to elevate basa l IRTK activity (Cordera et a l , 1990). Interestingly, addition of lithium to low dose vanad ium (0.05 mg/ml) in the drinking water restored insul in-mediated g lucose uptake by similarly correcting skeletal musc le g lycogenic rate. Moreover, the addit ion of lithium, z inc and magnes ium to vanad ium potentiated insulin-stimulated peripheral g lucose uptake to supranormal levels via activation of glycolytic flux which was not impaired in this mode l , support ing the speci f ic effects of z inc and magnes ium on this pathway (Rossett i et a l , 1990). 30 1.5.3. Animal models of Type 2 diabetes 1.5.3.1. Neonatal STZ-diabetic rats Neonata l rats administered S T Z on day 5 after birth exhibit severe insulin resistance and Pi-cell dysfunction as adults. In these rats, vanad ium treatment corrected hepatic g lucose production and peripheral g lucose utilization both basal ly and in response to submaximal and maximal insulin (Blondel et a l , 1990). Improved insulin sensitivity was independent of changes in hepatic IRTK activity, which w a s unaltered in the diabetic state, suggest ing the correction of a post-receptor defect. V a n a d i u m treatment did not improve Pi-cell insulin secretory function, either in the lack of response to g lucose or in hyperrespons iveness to arginine (Serradas et a l , 1991). 1.5.3.2. Genetically obese fa/fa rats and ob/ob mice Vanad ium treatment partially corrected the hyper insul inemia and impaired g lucose to lerance in genetical ly o b e s e fa/fa rats (Brichard et a l , 1989), improvements which were d issociated from decreased body weight or changes in counterregulatory hormones (Brichard et al , 1991). The effects of vanad ium in enhanc ing insulin sensitivity w a s attributed to an increased peripheral g lucose utilization in skeleta l musc le and heart, without changes in G L U T 4 m R N A or protein levels in these t issues (Brichard et a l , 1992a,b). Hence , the enhanced insulin sensitivity was postulated to be due to increased translocation or functional activity of p lasma membrane G L U T 4 . Treatment of obese , hyperglycemic, insulin resistant ob/ob mice with vanad ium lowered hyperglycemia, improved g lucose tolerance and hepatic g lycogen content, and prevented the exhaust ion of pancreat ic insulin stores (Brichard et a l , 1990). The reduction in g lucose levels induced by vanadium treatment w a s not assoc ia ted with correct ions in the reduced hepatic P T P a s e and IRTK activities (Meyerovi tch et a l , 1991), and occurred despite an increased P E P C K m R N A express ion and the a b s e n c e of effects in hepatic G L U T 2 express ion in these mice (Ferber et a l , 1994). 31 1.6. TOXICOLOGICAL E F F E C T S OF VANADIUM C O M P O U N D S IN DIABETES In control rats and mice, V O S 0 4 ( L D 5 0 > 1.5 mmol/kg) was found to be 2-3 t imes less toxic than N a V 0 3 when administered acutely by oral gavage (Llobet and Domingo, 1984). S ince the demonstrat ion of oral antidiabetic activity of vanad ium, there has been a renewed interest in the study of toxicity of vanad ium compounds following long-term treatment. Chron ic administration of vanadium in the drinking water to S T Z -diabetic rats was reported to induce several s igns of toxicity, including increased mortality, lower weight gain, and higher kidney:body weight ratio, independent of the salt administered (Domingo et al , 1991, 1994). Co-administrat ion of a chelator (tiron) with N a V 0 3 prevented the accumulat ion of vanadium in kidney and bone, which in that study, appeared to be the only sign of toxicity assoc ia ted with vanad ium treatment (Domingo et a l , 1992). Notably, vanadium treatment of diabetic rats in the previous studies was not effective in producing normoglycemia in the animals. W h e n administered to diabetic rats at d o s e s which correct the diabet ic state, a different picture can be observed. W h e n V O S 0 4 was administered for 39 days to rats, histopathological examinat ion revealed no morphological changes in liver, heart, s tomach and lungs (Mongold et a l , 1990). Epithelial cel lular swel l ing of distal tubules was demonstrated in all of the untreated diabetic rats, and w a s diminished with vanad ium treatment. Importantly, in control and diabetic rats, treatment with V O S 0 4 (0.16-0.71 mmol/kg/day) for one year did not induce any pathological changes in brain, thymus, heart, lung, liver, sp leen, intestine, kidney, or adrenal g land (Dai et a l , 1994). O n the other hand, diabetes-related pathologies in eye, heart, kidney and testis were prevented by treatment, which simultaneously normal ized g lycemic and lipid levels. Vanad ium treatment also significantly diminished the mortality rate of the diabet ic rats over this period (18.7% vs. 6 0 % for untreated rats). Thus , other than the occur rence of diarrhea and subsequent dehydrat ion observed with all of the vanad ium salts, there has been no consistent finding of pathology assoc ia ted with long-term treatment in rats. 32 1.7. MECHANISM OF INSULIN-MIMETIC E F F E C T OF VANADIUM 1.7.1. Insulin receptor A s severa l of vanadium's act ions appear to mimic insulin, it w a s hypothes ized that vanad ium exerts its effects via the insulin receptor. The IR is a t ransmembrane glycoprotein composed of two a and two ft subunits l inked by disulf ide bonds. The a subunits are extracellular and contain the insulin binding domain whereas the ft subunits span the membrane and possess an ATP-b ind ing site. Binding of insulin to its receptor results in autophosphorylat ion of the ft subunit and activation of its tyrosine k inase activity, effects which are essent ia l for insulin action (For review, see K a h n et a l , 1994). It w a s thus proposed that the insulin-mimetic effects of vanad ium may be l inked to its inhibitory effects on P T P a s e s (Swarup et a l , 1982). The reported effects of vanadium on the insulin signal ing pathway are listed on Tab le 6. Initially, vanadate was shown to activate the autophosphorylat ion of partially purified IR preparation that was free of P T P a s e activity, suggest ing a direct activation of the IR (Tamura et a l , 1984). However, although vanadate did not induce ser ine phosphorylat ion of partially purified IR, this was observed when a ser ine k inase known to phosphorylate the IR following IRTK activation was co-purif ied with the IR (Smith and S a l e , 1988). Vanada te a lso activated the turnover of pp15, a substrate of IRTK involved with stimulation of g lucose transport in 3T3-L1 adipocytes (Bernier et a l , 1988). It was also suggested that vanadate can enhance insulin receptor affinity and insulin sensitivity in rat adipocytes (Fantus et al , 1990). The observed 3-fold augmentat ion of insulin-stimulated IRTK activity by vanadate was assoc ia ted with enhanced IR autophosphorylat ion, and prolongation of the insulin effect on l ipogenesis (Fantus et a l , 1994). In addit ion, vanadate stimulated IR downregulat ion, and inhibited intracellular IR degradat ion in intact cel ls (Torossian et a l , 1988; Y u et a l , 1996). Interestingly, V 0 2 + , but not vanadate, maximal ly inhibited insulin-stimulated IR autophosphorylat ion and IRTK activity of partially purified IR (Elberg et a l , 1994). 33 Table 6. In vitro effects of vanadium on the insulin s ignal ing pathway Activity/ Reference Effect/ Concentration (M) Target Tissue Autophosphorvlat ion of IR Tamura et a l , 1984 Ueno e t a l , 1987 Gherz i e t a l , 1988 B e r n i e r e t a l , 1988 Smith and Sa le , 1988 Stimulated 10-3 10-4 10-5-10-3 10-3 10-5 Rat adipocytes/puri f ied IR Partial ly purified IR 3T3-L1 adipocytes Human placenta IR Tvros ine K inase Activitv Gherz i e t a l , 1988 Increased 10-5-10-3 Partially purified IR Down-requlat ion of IR Toross ian et a l , 1988 Marsal l and Monzon , 1987 Stimulated 10-4 10-6 Human lymphocytes Cultured rat adipocytes IR Bindinq Fantus e t a l , 1990 Er iksson et a l , 1992 Increased 10-5-10-4 10-4-10-3 Rat ad ipocytes Levin e t a l , 1988 Lonnroth e t a l , 1993 Inhibited 10-4 No effect 10-3 Human monocytes Human adipocytes IR Deqradat ion Toross ian et a l , 1988 Y u et a l , 1996 Inhibited 10-4 10-3 Human lymphocytes Rat ad ipocytes Marshal l e t a l , 1987 Increased 10-3 Rat ad ipocytes Protein Tvr K inase Activitv Klarlund e t a l , 1988 Brown and Gordon , 1984 Sh i sheva and Shechter , 1993a Elberg e t a l , 1994 Increased 10-5-10-4 10-6 10-4 10-6 NIH 3T3 f ibroblasts Ch icken embryo fibroblast Rat ad ipocytes Rat adipocytes, liver, brain Protein Ser /Thr K inase Activitv J e n o e t a l , 1990 D'Onofrio et a l , 1994 Increased 10-3 S 6 k inase 10" 5 M A P kinase Sw iss 3T3 cel ls C H O cel ls Phosohotvros ine Phospha tase S w a m p et a l , 1982 Klar lund, 1985 Liao and Lane , 1995 Inhibited 10-6 10-6-10-5 10-5 A-431 cel ls N R K - 1 cel ls 3T3-L1 preadipocytes Substrates Ryder and Gordon , 1987 Bern ie re t a l , 1988 Y a n g et a l , 1989 D'Onofrio et a l , 1994 Sch ieven et a l , 1995 Increased phosphorylation 10-6 pp60c-src Ch icken embryo fibroblast 10-4 p p 15 3T3-L1 adipocytes 10-6 p td lns-4-P Liver p lasma membranes 10-4 p42/44mapk C H O cel ls 1 0 - 5 P L C - y 2 B cel ls 34 1.7.2. Post-receptor mechanisms Whi le the former observat ions are consistent with the hypothesis of IRTK activation as a mechan ism of insulin-mimetic action of vanad ium, severa l studies have provided ev idence which do not support involvement of the IR. Stimulat ion of g lucose transport by vanadate in rat adipocytes was found to be unaffected by a loss of 6 0 % of IRs (Green , 1986). In addit ion, vanadate had profound insulin-like effects on g lycogen synthesis both in vitro and in vivo without activating IRTK (Jackson et a l , 1988; Strout et al , 1989). Similarly, in rat adipocytes, vanadate produced marked anti l ipolysis at a concentrat ion which had minor effects on tyrosine phosphorylat ion of the IR f i-subunit and intracellular proteins (Mooney et al , 1989). Furthermore, the stimulatory effects of vanadate on g lucose uptake and l ipogenesis in rat adipocytes were unaffected by IRTK inhibition, which fully blocked effects mediated by insulin (Sh isheva and Shechter , 1992a). Increased tyrosine kinase activity was assoc ia ted with a high level of phosphotyros ine in vanadate-treated cultured cel ls (Klarlund et a l , 1988), suggest ing that inhibition of P T P a s e s could enhance tyrosine phosphorylat ion and activation of severa l downst ream enzymes independent of any effects on the IR. In support, activation of M A P kinase by vanadate was not assoc ia ted with phosphorylat ion of the IR fi-subunit (D'Onofrio et a l , 1994). A cytosol ic protein tyrosine k inase (Cy tPTK) , distinct from the IRTK, was activated 3-5 fold in vanadate-treated rat ad ipocytes, but w a s unaffected by insulin (Sh isheva and Shechter , 1992b). Inhibition of C y t P T K but not of IRTK blocked the effects of vanadate on g lucose oxidation and lipid synthesis in intact rat adipocytes, but had no effects on g lucose uptake and inhibition of l ipolysis, suggest ing a select ive role for C y t P T K in some of the post-IR mechan isms of vanad ium (Sh isheva and Shechter , 1993a). Other P T P a s e inhibitors, tungstate and molybdate also s imul taneously activated C y t P T K and l ipogenesis in intact rat ad ipocytes (Elberg et al , 1994). Two other k inases, S 6 k inase (Jeno et a l , 1989), and case in k inase I (Villar-Pa las i et a l , 1989) were also found to be activated in vanadate-treated cel ls. 35 1.7.3. Other proposed second messengers Other second messengers have also been proposed as possib le mechan isms for the insulin-mimetic effects of vanad ium. For instance, the role of intracellular C a 2 + in the activation of g lucose transport has been proposed s ince a rise in cytoplasmic C a 2 + levels, perhaps due to inhibition of C a 2 + - A T P a s e by vanadate in S R or E R , w a s highly correlated with the increased g lucose transport into ad ipose t issue and skeletal musc le (C lausen et a l , 1981). Al though vanadate-st imulated g lucose uptake appeared to be dependent on extracellular C a 2 + (Yamanish i et a l , 1984; Pershads ingh et a l , 1987), it w a s found to be essent ia l for either insulin or vanadate only when cel ls were maintained in bicarbonate-free buffer (Shechter and R o n , 1986). Vanada te was a lso demonstrated to raise intracellular pH in human A431 cel ls (Casse l l et a l , 1984). The effect of vanadate in stimulating g lucose uptake was assoc ia ted with its insulin-l ike effect on stimulating K + uptake into rat heart cel ls (Werdan et a l , 1982). T h e stimulatory effects of vanadate or V 0 2 + - G S H on cycl ic A M P phosphodies terase activity, reported at concentrat ions a s low a s 5 u M (Souness et a l , 1985), could be relevant in the inhibition of g lucagon- induced increase in c A M P levels in rat hepatocytes (Villar-Pa las i et a l , 1989). In NIH 3T3 fibroblasts, vanadate w a s reported to increase phosphatidyl inositol and inositol phosphate content, secondary to the activation of phosphatidyl inositol-specif ic phosphol ipase C ( P t d l n s - P L C ) and of P td lns k inase ( R a n d a z z o et a l , 1992). Vanadate , like insulin, w a s also shown to activate the diacylglycerol (DAG)/protein k inase C ( P K C ) signal ing sys tem and the translocat ion of P K C - f i from the cytosol to the membrane fraction (Yamada et al , 1994). V 0 2 + has a lso been shown by E S R spect roscopy to bind to calmodul in in its C a 2 + binding si tes, with a stoichiometry of 4 mol V 0 2 + : m o l calmodul in (Nieves et a l , 1987). The effect of V 0 2 + binding on Ca 2 + / ca lmodu l i n -dependen t protein k inase (one of which is phosphory lase kinase) remains to be seen . It is a lso reasonable to a s s u m e that either V 0 2 + or V 0 2 + -G S H , which are the major intracellular spec ies , may be the more relevant forms in vivo. 36 1.8. PEROXOVANADIUM C O M P O U N D S The insulin-mimetic properties of H 2 0 2 have long been recognized ( C z e c h et a l , 1974). S ince insulin enhances intracellular H 2 0 2 production, it has been proposed as a second messenger in insulin signal ing (Kreiger-Brauer and Kather, 1992). Kado ta et al (1987a) initially d iscovered that the addit ion of H 2 0 2 to vanadate enhanced IGF-II receptor translocation and IRTK activity in a synergist ic manner in rat ad ipocytes. S ince these effects were abol ished by the addit ion of ca ta lase, the synerg ism w a s attributed to the formation of peroxide(s) of vanadate or pervanadate (pV) (Kadota et a l , 1987b). Furthermore, s ince pV was demonstrated to inhibit P T P a s e and activate IRTK in intact cel ls much more potently than vanadate, it exerts a fuller range of insul in-mimetic effects compared to vanadate at the same or at a lower concentrat ion range which is attainable in vivo ( 1 0 - 6 - 1 0 - 5 M, Table 7). Indeed, pV, but not vanadate , activated protein synthesis and IRTK activity in rat adipocytes (Fantus et a l , 1989). Moreover , pV(s) demonstrated insulin-like properties in isolated rat ad ipocytes with a potency of 1 0 2 (antilipolysis) to 1 0 3 ( l ipogenesis) fold greater than vanadate. In human adipocytes, pV, but not vanadate, exerted insulin-like effects on g lucose uptake and anti l ipolysis (Lonnroth et a l , 1993). Similarly, pV activated glycolysis, g lucose oxidat ion and g lycogen synthesis in so leus muscle preparations to the s a m e degree as insul in, whereas vanadate-st imulated g lycogenosis was only - 2 0 % of insulin-st imulated max imum (Leighton et a l , 1991). However, whereas insulin select ively st imulated phosphorylat ion of the IR B-subunit and IRS-1 , pV enhanced tyrosine phosphorylat ion of severa l addit ional proteins in C H O cel ls, demonstrat ing its potentially broad mechan ism of act ion. Never theless, the effect of p V on protein tyrosine phosphates was markedly diminished in mutant IR-transfected cel ls, arguing that the IR is a major target for P T P a s e and hence, of pV (Heffetz et al , 1992). Al though pV st imulated IR-tyrosine phosphorylat ion in intact C H O cel ls, increased IRS-1-assoc ia ted Ptd lns 3-k inase activity w a s only demonstrable in vitro (Wilden and Broadway, 1995). 37 Table 7. In vitro insulin-mimetic effects of peroxide(s) of vanadium Activity/ Reference Effect/ Concentration (M) Target Tissue G l u c o s e Transport S h i s h e v a and Shechter , 1993b Y u et a l , 1996 Lonnroth e t a l , 1993 Increased 10 - 6 - 10 - 5 1 0 - 4 - 1 0 - 3 10-4 Rat adipocytes Human adipocytes Glvco lvs is Foot e t a l , 1992 Increased 10-3 Rat skeletal musc le G lvcoqen Synthes is Tamura et a l , 1984 Leighton e t a l , 1991 Increased 10-3 10-3 Rat adipocytes Rat skeletal musc le L ipoqenes is Fantus et a l , 1989 P o s n e r e t a l , 1994 Stimulated 10-6-10-3 10-6-10-4 Rat adipocytes Lipolvsis Fantus e t a l , 1989 Er iksson et a l , 1996 Inhibited 10- 5 -10-3 10-5-10-3 Rat adipocytes Human adipocytes Protein Svnthes is Fantus et a l , 1989 Barnes et a l , 1995b Stimulated 1 0 - 8 - 1 0 - 5 10-5-10-4 Rat adipocytes X e n o p u s oocyte Mitoqenic Activitv Cort izo and Etcheverry, 1995 Stimulated 10-6-10-5 U M R 1 0 6 osteoblasts Phosphory lat ion of IR Fantus et a l , 1989 Sh i sheva and Shechter , 1993b Heffetz e t a l , 1992 Wi lden and Broadway, 1995 Stimulated 10-4 10-6-10-5 10-6 10-6 Rat adipocytes C H O cel ls Human platelets IR Tyros ine K inase Activitv Kadota e t a l , 1987 Fantus et a l , 1989 Er iksson et a l , 1996 P o s n e r e t a l , 1994 Increased 10-5-10-3 10-5-10-3 10-4 10-3 Rat adipocytes Human adipocytes Hepa toma cel ls IR Bindinq Y u e t a l , 1996 Increased 10-4-10-3 Rat ad ipocytes Phosohotvros ine Phospha tase Trudel e t a l , 1991 H e c h t a n d Zick, 1992 Sh i sheva and Shechter , 1993b P o s n e r e t a l , 1994 Inhibited 10-5-10-4 10-5-10-3 10-6-10-5 10-6 H L 6 0 cel ls Rat hepatoma (Fao) cel ls Rat ad ipocytes Hepa toma cel ls Subst ra tes Heffetz et a l , 1990 Wi lden and Broadway, 1995 Inazu e t a l , 1990 Increased phosphorylation 10-4 various Fao cel ls 10-6 IRS-1 C H O cel ls 10-5-10-4 var ious Human platelets 38 1.9. NEW ORGANIC VANADIUM C O M P O U N D S In an attempt to overcome the poor oral absorpt ion of vanad ium salts, and thus reduce the incidence of gastrointestinal s ide effects, more potent organic comp lexes of vanad ium have been formulated. A bis( l igand)oxovanadium(IV) complex, vanady l cyste ine methyl ester was effective in acutely lowering g lucose levels at a dose of 0.2 mmol/kg in STZ-d iabet ic rats (Sakurai et al , 1990). Bis(maltolato)oxovanadium(IV) ( B M O V ) , des igned to be a water-soluble complex of V 0 2 + , and readily absorbed through the gastrointestinal tract by pass ive diffusion, lowered g lucose and lipid levels in STZ-d iabet ic rats at a dose of 0.4 mmol/kg/day (McNei l l et a l , 1992). Treatment for 6 months with B M O V improved card iac function in STZ-d iabe t i c rats without any incidence of diarrhea, and other than bone, vanadium levels in all other t issues and p lasma were comparat ive to that obtained with V O S 0 4 or N a V 0 3 treatment (Yuen et a l , 1993). By oral gavage (0.55 mmol/kg) or i.p. injection (0.063 mmol/kg), B M O V w a s found to be 2-3 t imes more potent at g lucose- lowering than V O S 0 4 (Yuen et a l , 1994). Ineffective orally, pV, when administered i.p. (0.015 mmol/kg), lowered p lasma g lucose levels acutely in STZ-d iabet ic and control animals (Shish iva et a l , 1994). Severa l more stable b is-peroxovanadium complexes were synthes ized and tested for insulin-mimetic effects in vitro and in vivo (Posner et a l , 1994). Inhibition of IR dephosphorylat ion was strongly correlated with subsequent stimulation in vitro of IRTK activity by these compounds . W h e n administered i.v. (0.004-0.065 umol/kg) to control rats, pV compounds showed variable abilities to stimulate g lycogen synthes is in skeletal musc le , which reflected in their g lucose- lowering potency and were related to targeting by the ancil lary l igand (Bevan et a l , 1995). These compounds , administered s.c. were demonstrated to lower fasting p lasma g lucose and ketonuria in insul in-deprived diabetic B B rats for 3 days (Yale et a l , 1995). Synerg ism was reported between smal l d o s e s of pV compounds (0.2 umol/kg) and insulin at doses which a lone, had no hypoglycemic effects. The oral activity of pV compounds remains to be seen . 39 1.10. HUMAN TRIALS Recent ly , cl inical trials have been conducted to evaluate the ef fect iveness of vanad ium in Type I and Type II diabetic subjects. Treatment of six N I D D M subjects with oral V O S 0 4 H 2 0 (100 mg/day) for 3 weeks resulted in improved g lycemic levels, as reflected in reduced fasting p lasma g lucose and H b A 1 c va lues without concomitant changes in basa l p lasma insulin (Cohen et a l , 1995). However, est imates of oral g lucose tolerance and insulin secretory response such as p lasma insulin or C-pept ide concentrat ions after oral g lucose were not altered by vanad ium treatment. Improvements in insulin sensitivity were manifested in enhanced insul in-mediated g lucose uptake and inhibition of hepatic g lucose output following vanad ium treatment. It w a s determined that >80% of the increased g lucose utilization could be accounted for by an enhanced peripheral g lycogen synthesis, resulting from a 2 6 - 2 9 % reduction in K m in musc le g lycogen synthase both basal ly and during the post-c lamp period. Improvements in insulin sensitivity were found to persist for 2 w e e k s fol lowing cessa t ion of treatment. Mild gastrointestinal symptoms were reported during the first week of treatment, ranging from nausea to abdominal c ramps and mild diarrhea. In the second study, oral administration of N a V 0 3 (125 mg/day) for 2 weeks to 5 IDDM subjects lowered insulin requirements but had no effect on basa l g lucose or C -peptide levels (Goldf ine et a l , 1995). Al though 2 patients showed an improved g lucose utilization during the euglycemic c lamp following vanad ium treatment, on average, no significant improvement was detected. However, treatment of 5 N I D D M patients resulted in an improved insulin sensitivity which was accounted for by an enhanced nonoxidative g lucose d isposa l , with no changes in oxidative g lucose util ization. There were no significant effects of vanadium on hepatic g lucose production either in the basa l state or in response to insulin. Vanad ium treatment enhanced basa l S 6 and M A P kinase activity in monocytes but had no effect on insulin-stimulated activity of these enzymes . The major side effect was gastrointestinal intolerance and mild d iarrhea. 40 1.11. THESIS INVESTIGATION The aim of this thesis was to study the mechanism(s) of the antidiabetic act ion of oral vanad ium ( V O S 0 4 ) in STZ-d iabet ic animals. From the studies performed in vitro, it appears that vanad ium can have specif ic metabol ic effects which are similar to insulin. However, some studies conducted in vitro as well as in vivo suggest that the effects of vanad ium are different from, and may be limited in scope relative to that of insulin. Furthermore, the concentrat ions of vanadium in vitro which demonstrate insul in-mimetic effects appear to be far greater than that attainable in vivo in diabet ic rats chronical ly administered oral vanad ium. Hence , the mechanism(s) of vanad ium in ameliorat ing the diabetic state in vivo cannot be simply extrapolated from effects demonstrated in vitro. Moreover , the observed correction of metabol ic and physiologic derangements in S T Z -diabet ic rats treated with vanad ium becomes convoluted with the demonstrat ion that these rats remain normoglycemic for a prolonged period following removal of vanad ium therapy. It w a s thus hypothesized that a n important aspect of vanad ium therapy in diabetic animals could involve the protection of pancreat ic f i-cells from the cytotoxic effects of S T Z , and that the improvement of insul in-secretory function could subsequent ly lead to a partial recovery from the diabetic state. The overal l a im of the fol lowing ser ies of studies was to del ineate the multiple role(s) of vanad ium in ameliorating the diabetic state, by exerting insulin-like effects at the level of peripheral t issues, and by preserving fi-cell insulin secretory function and insulin stores. Initially, it was postulated that part of the effects of vanad ium could be attributed to the protection of pancreat ic R-cells shortly after the induction of the diabet ic state, s ince it w a s previously demonstrated that a significant number of ft-cells is still detectable at 3 days following S T Z (Pederson et a l , 1989). The aim of the first study was to compare the relative effect iveness of vanadium as a glucose- lower ing agent when treatment was initiated at de layed t imes (10 and 17 days) after the onset of d iabetes, when the cytotoxic effects of S T Z have been fully expressed (Chapter 2). 41 It appeared that administration of vanadyl sulfate in the drinking water to S T Z -diabetic rats somet imes resulted in severe gastrointestinal intolerance, d iarrhea and death due to dehydrat ion. S ince vanadium salts are ionic and are poorly absorbed through the gastrointestinal tract, it was hypothesized that a more lipid soluble complex of vanadyl could increase its bioavailability, and lower the effective dose of vanad ium, thus diminishing the incidence of diarrhea. The a im of the second study w a s to demonstrate the antidiabetic and cardioprotective effects of an organic vanady l compound , nagl ivan (50 mg/kg/day), administered by oral gavage (Chapter 3). B e c a u s e of the apparent inability in a subset of STZ-d iabet ic an imals to ach ieve normoglycemia to treatment with a single concentrat ion of V O S 0 4 (0.75 mg/ml) or nagl ivan (50 mg/kg/day), it w a s hypothesized that the lack of response w a s l inked to a more severe diabetic state induced by S T Z . The a im of the next study, presented in Chapter 4, w a s to determine whether an enhanced normoglycemic response could be induced by increasing the dose of either vanadyl sulfate or nagl ivan in diabetic an imals which did not initially respond to the doses used in the two previous studies. In addit ion, it was hypothesized that one mechan ism of maintenance of normoglycemia fol lowing withdrawal from long-term vanad ium treatment could be assoc ia ted with an improved insulin secretory function in some of the animals. Thus , vanadium-treated diabet ic an imals were monitored at var ious t imes up to 20-30 w e e k s fol lowing withdrawal from treatment to determine whether post-withdrawal normoglycemia w a s assoc ia ted with improvements in circulating insulin levels. To evaluate the insulin-like effects of vanadium per se both during treatment and after treatment withdrawal, it was essent ia l to determine if there were changes in circulating p lasma insulin. This was especial ly important s ince severa l commercia l ly avai lable a s s a y kits were not found to be reproducible enough for the number of samp les to be assayed over a long period of time. Hence , a sensit ive and reproducible insulin rad io immunoassay was establ ished, and is descr ibed in Append ix 1. 42 A lowering of insulin demand has been suggested to reduce the susceptibi l i ty of f i-cel ls to cytotoxic events (Spr ietsma and Schui tmakker , 1994). S ince vanad ium treatment lowers p lasma insulin levels in control animals, it was hypothesized that vanad ium might ameliorate the effects of S T Z by protecting f i-cel ls from the cytotoxic effects of the diabetogenic agent when given prior to the induction of d iabetes, and al low persistent normoglycemia even following withdrawal from treatment. The a im of the next study was to determine whether vanadium treatment prior to and for a short period following the induction of STZ-d iabe tes could result in the ameliorat ion of the diabetic state following withdrawal from treatment, and to observe if this phenomenon w a s linked to specif ic improvements in the pancreat ic insulin store (Chapter 5). Recent studies have proposed that the mechanism(s) of vanad ium in lowering g lycemia in STZ-d iabet ic rats could be secondary to its effects on reducing food intake (Malabu et a l , 1994, Domingo et al , 1994). S ince chronic food restriction has a lso been shown to enhance insul in-mediated g lucose uptake and reduce insulin secret ion, it w a s hypothesized that a reduction of food intake could contribute to the glucose- lower ing and fi-cell protective effects of vanadium treatment. Hence , the a im of the fol lowing study was to ascertain whether these effects of vanad ium could be attributed to the lowering of food intake per se by pair-feeding of diabetic rats (Chapter 6). Finally, in vitro studies have demonstrated that vanad ium alone, and in concert with insulin, can stimulate g lucose transport in a variety of t issues. A s improvements in g lucose to lerance were not assoc ia ted with increased insulin secret ion, it was postulated that the effects of vanadium on g lucose tolerance could be partly due to an increased sensitivity of peripheral t issues to insulin, by enhanc ing the translocat ion of G L U T 4 in vivo. Thus , the aim of the final study was to ascerta in whether vanad ium treatment could completely normal ize g lucose tolerance in STZ-d iabe t i c an imals , and whether this improvement was assoc ia ted with an enhanced presence of G L U T 4 at the ad ipose t issue cell surface in vivo in response to i.v. g lucose (Chapter 7). 43 Chapter 2 LONG-TERM EFFECTIVENESS OF O R A L V A N A D Y L S U L F A T E IN STREPTOZOTOCIN-DIABETIC RATS 2.1. INTRODUCTION A n interesting phenomenon is the maintenance of eug lycemia in STZ-d iabe t i c rats following withdrawal for 13 weeks after a period of vanad ium treatment ( R a m a n a d h a m et al , 1989). In these animals, morphological and immunohis tochemical analys is revealed that the number of insulin-staining beta cel ls was increased eight-fold compared to non-treated diabetic rats, though not completely restored to control levels (Pederson et a l , 1989). Further, the insulin-secretory response of the isolated perfused pancreas to g lucose, though suppressed compared to non-diabet ic control rats, w a s higher than in non-treated diabetic rats. S i n c e vanad ium treatment in these studies was initiated 3 days after the S T Z injection, a time when pancreat ic islet cell a rea and insulin content were not significantly d iminished, it is possib le that vanad ium treatment had al lowed some protection of the pancreat ic beta cel ls from STZ- i nduced cytotoxicity and in so doing, prevented a full express ion of the diabetic state. T h e s e findings suggest that the observed insulin-mimetic effects of vanad ium in our previous studies may in part be due to its act ions at the level of the pancreas . Thus , it was considered crucial to determine if the effect iveness of vanady l treatment depended on its p resence at a time when a critical number of remaining viable beta cel ls could be protected from further destruction by circulating S T Z and/or hyperglycemia. Therefore, the effects of vanadyl administration on both g lucose homeostas is and pancreat ic function when treatment w a s begun 3, 10 or 17 d a y s after the induction of d iabetes was investigated. Furthermore, vanad ium treatment w a s cont inued for 5 months to provide a further opportunity to observe poss ib le toxicological aspec ts of oral vanadyl treatment over an extended period. 44 2.2. MATERIALS AND METHODS 2.2.1. Treatment and maintenance of animals Male Wistar rats weighing between 200-250 g and fed ad libitum were used . D iabetes w a s induced by a n i.v. injection of streptozotocin (STZ , S i g m a C h e m i c a l C o . , St. Louis , Mo. , U S A ) at a dose of 55 mg/kg while control rats received only vehic le (NaC l 154 m M , pH 7.2). At 3 days following the S T Z injection, blood g lucose w a s checked by glucometer and animals exhibiting blood g lucose levels greater than 13.75 m M were cons idered diabetic. Control rats were divided into treated (CT, n=12) and non-treated (C , n=12) groups, while diabetic rats were divided into four groups wh ich were: non-treated (D, n=11), or treated with administration of vanadyl starting at 3 (DT3, n=12), 10 (DT10, n=11) and 17 (DT17, n=16) days following the S T Z injection. Al l treated animals were given vanadyl sulfate at a concentrat ion of 0.75 mg/ml in the drinking water ( V O S O 4 - 3 H 2 0 , F isher Scientif ic C o . , Fair Lawn, N e w Jersey , U S A ) . Vanady l solut ions were replaced twice weekly. The p H and color of the solut ion w a s checked and found to be unchanged over a two-week long period. The concentrat ion of vanadyl used in this study was based on our previous observat ions that a desi rable combinat ion of improved body weight and g lycemic control resulted when vanady l sulfate was provided at a concentrat ion of 0.75 mg/ml in the drinking water ( R a m a n a d h a m et a l , 1989a). Treatment for C T rats commenced at the s a m e time a s DT3 . For 15 weeks , p lasma g lucose, body weight, and food and fluid intake were c losely monitored. The daily vanadyl dose was calculated as the mean vo lume consumed per kg body weight multiplied by the concentrat ion of the solut ion. The treatment of animals was terminated at five months at which time p lasma samp les were obtained from nonfasted animals (in the afternoon) for the determinat ion of g lucose , triglyceride and cholesterol levels (Boehringer Mannhe im C a n a d a , Lava l , Quebec ) , insulin by RIA (Immunocorp, Montreal, Quebec) , g lutamic-oxaloacet ic t ransaminase ( G O T ) and blood urea nitrogen (BUN) by kits obtained from S i g m a . 45 2.2.2. Oral g lucose tolerance test (OGTT) At 5 months into the experiment, rats were fasted overnight and an oral g lucose dose (1 g/kg as a 4 0 % solution) was administered to the consc ious an imals by gavage . B lood samples were obtained before and at 10, 20, 30, and 60 min after g lucose administration and col lected into heparinized capil lary tubes from the tail ve in. Insulin was measured by RIA using purified rat insulin standard (Novo, C o p e n h a g e n , Denmark) and ant iserum raised in guinea pig. 2.2.3. Pancreatic perfusion Fol lowing an oral g lucose tolerance test, some rats were anaesthet ized with 60 mg/kg pentobarbital and the pancreas and assoc ia ted duodenum were isolated (n: C=5, CT=5, D=5, DT3=6, DT10=5, DT17=6). Insulin secret ion was measured in isolated pancreata perfused with 16.65 m M g lucose plus a gradient (0-1 ng/ml) of the insulinotropic hormone Gastr ic Inhibitory Polypept ide (GIP) a s descr ibed previously (Pederson et a l , 1982). Perfusate consisted of a modified Krebs-R inger b icarbonate buffer containing 3 % dextran and 0 .2% bovine serum albumin and g a s s e d with 9 5 % 02/5% CO2 to ach ieve a pH of 7.4. Portal outflow was col lected at 1-min intervals at a rate of 4 ml/min after a 10-min equil ibration. Perfusate samp les were subsequent ly ana lyzed for insulin content. 2.2.4. Glycerol output from isolated adipose t issue T h e s e exper iments were conducted on some of the animals (n: C=6, CT=6, D=5, DT3=6, DT10=6, DT17=5). At the time of killing, epididymal fat pads were removed and immediately incubated at 37°C in Krebs-Hensele i t b icarbonate buffer containing 2 mg/ml g lucose and defatted serum albumin (2% w/v). Fol lowing 30 min of incubat ion, fat pads were incubated in the p resence or a b s e n c e of insulin at a final concentrat ion of 1 mU/ml for 30 min, after which epinephrine w a s added to the 46 incubation medium for an addit ional 30 min at a concentrat ion of 1 ug/ml. At the end of the incubation period, pads were removed and we ighed. Incubation mixtures were heated at 95°C for 10 min and stored at -70°C for subsequent glycerol ana lys is , using commerc ia l kits (Boehringer). 2.2.5. T issue histopathology Liver and kidney s l ices were ana lyzed histologically for any apparent toxicological effects of the 5-month oral vanadyl treatment on the primary organs. T i ssues were fixed in 10% buffered formalin, dehydrated and embedded in paraffin. Sec t ions were stained in haematoxyl in and eos in . The histology s l ides were scored in a bl inded fashion, i.e. individual codes did not reflect the respect ive treatment groups. 2.2.6. Vanadium levels At termination, samples of t issue from kidney, liver, musc le and bone were ana lyzed for vanad ium levels using a Var ianAA-1275 spectrophotometer as descr ibed previously (Mongold et a l , 1990). 2.2.7. Statistical analysis All results are expressed as means + S E M . The data were ana lyzed using M A N O V A fol lowed by the Newman Keul 's or F isher test, p < 0.05 indicated statistical s igni f icance. 47 2.3. R E S U L T S 2.3.1. Effects of vanadyl treatment on body weight, food and fluid intake, and plasma g lucose Overal l increase in body weight over a 15-week period (Fig. 2.1 A ) w a s dep ressed in the STZ-d iabet ic animals and did not appear to be corrected by vanady l treatment. Vanady l intake in non-diabetic rats resulted in reduced weight gain compared with non-treated control rats. During the first week of vanady l treatment, mean body weight of the diabetic-treated (DT) groups dropped, al though recovery w a s evident by the following week. The induction of d iabetes produced the expected symptoms of hyperphagia as manifested by a two-fold increase in food intake (Fig. 2.1B). In all DT groups, a parallel lowering of food intake from diabetic to control levels was observed immediately following the onset of treatment. Thus , food intake (g/rat/day) among the D T groups at the end of the first week of treatment was not different (DT3= 19.2 ± 1 . 1 , DT10= 20.1 ± 1.4, DT17= 22.7 ± 1.2) and remained significantly lower than C (p < 0.05) until week 6, after which no difference was seen . Food intake was lowered significantly in C T animals throughout the duration of treatment (p < 0.05). Induction of the diabetic state produced an instantaneous two-fold higher fluid intake which gradual ly progressed to a four-fold rise in vo lume at 15 weeks (Fig. 2 .2C) . Administrat ion of vanadyl reduced fluid intake below control levels from the first day in both the DT and C T animals and the reduction was maintained throughout the treatment period. O n e rat in DT10 died of severe dehydrat ion apparently from refusal to drink the vanady l solut ion, while another in the D T 1 7 group deve loped severe diarrhea and weight loss and was subsequent ly removed from the experiment. O n e non-treated diabetic rat was similarly removed due to a severe catabol ic condit ion resulting from the S T Z injection. The daily vanadyl intake calculated over a 15-week period showed an overal l dec rease which was largely accounted for by an increase in 48 Weeks post-STZ Figure 2.1. Body weight and food intake over 15 weeks Body weight (A) and daily food intake (B) of control (C, O ) , control-treated (CT, • ) , diabetic (D, A), and diabetic rats treated with V O S 0 4 at 3 (DT3, A ) , 10 (DT10, • ) , and 17 (DT17, • ) days pos t -STZ injection. * p < 0.05 vs . all other groups. 4 9 0 2 4 6 8 10 12 14 16 Weeks post-STZ Figure 2.2. Fluid intake and vanadyl dose over 15 weeks Daily fluid intake (A) of control (C, O), control-treated (CT, • ) , diabetic (D, A), and diabetic rats treated with V O S 0 4 at 3 (DT3, A ) , 10 (DT10, • ) , and 17 (DT17, • ) days post-STZ injection. (B) Calculated V O S 0 4 dose for treated control and diabetic rats. * p < 0.05 vs. all other groups 50 body weight while total fluid intake remained quite constant (Fig. 2.2B). The d o s e s calculated at 15 weeks differed at most by 15% among the DT groups: in mg/kg/day [umol/kg/day] (DT3, 90.7 ± 2.3 [418 ± 11]; DT10, 75.4 ± 3.8 [348 ± 17]; DT17 , 87.2 ± 8.8 [402 ± 41]), and these va lues were all significantly higher (50-80%) than C T (50.6 ± 1.6 [233 ± 7]) (p < 0.05). Fol lowing treatment with S T Z , all diabetic an imals exhibited hyperg lycemia which ranged between 15-25 m M when measured at three days pos t -STZ injection. Administrat ion of vanadyl significantly and consistently reduced the average p lasma g lucose levels after one week of treatment in the DT groups (Fig. 2.3A). By the end of a 15-week maintenance period, the mean glycemic level of all DT groups was higher than that observed in non-diabetic control rats, but was significantly less than D and w a s not found to be different among treated groups. Further analys is revealed a strong negative correlation between body weight and g lycemia (r = -0.77) in the D T groups and in the D group but no apparent correlation was observed for C T rats (Fig. 2 .3B). C l o s e inspect ion of p lasma g lucose concentrat ions of the DT animals individually over the 15 weeks revealed a considerable range (normally distributed over 20 m M g lucose) of response to the glucose- lowering effects of vanadyl . Thus , while some DT animals had stable normoglycemia (exhibiting consistently normal p lasma g lucose levels (< 9.0 mM) as measured at 10.00 hours weekly over the duration of treatment), others had g lycemic levels which were lower than diabetic rats (16.9 ± 0.9 vs . 25.3 ± 1.2 mM) but which were still significantly higher than normal (Fig. 2 .4A-C) . Corresponding ly , treated an imals were subdiv ided into euglycemic (DT-E) and non-euglycemic ( D T - N E ) groups. The number of animals which were considered euglycemic (DT-E) were 4 of 12 (33%) in the DT3 group, 7 of 11 (64%) in the DT10 group, and 6 of 16 (38%) in the DT17 group. The mean body weight of D T - E rats did not differ significantly from C T at 15 weeks (429 ± 8 vs . 447 ± 8 g respectively) and was significantly higher than D T - N E or non-treated diabetic rats (353 ± 9 and 382 ± 10 g, respectively) (p < 0.05). 51 Body Weight (g) Figure 2.3. Effect of vanadium treatment on glycemia in diabetic rats (A) P l a s m a g lucose va lues of control (C , O ) , control-treated (CT , • ) , d iabet ic (D, A ) , and diabetic rats treated with V O S 0 4 at 3 (DT3, A ) , 10 (DT10, • ) , and 17 (DT17, • ) days pos t -STZ injection. * p < 0.05 vs . all other groups. (B) Correlat ion plot between p lasma g lucose and body weight at 5 months of untreated control, and diabet ic (treated and untreated) animals. Correlat ion coefficient for DT: r = -0.77. 52 5 </> o o 3 (5 £ (0 25 20 15 10 5 0 25 20 15 10 5 0 O E (n=4) • NE (n=8) DT3 l — i — i— r n— i — i — i — i — r — i — i — i — i — i — i 2 4 6 8 10 12 14 16 B O E (n=7) • NE (n=4) DT10 i—i—i—i—i—r 4 6 8 —l—l—l—r—I—l—l 10 12 14 16 O E (n=6) • NE (n=10) 7 l—i—i—i—i—i—i—i 8 10 12 14 16 Weeks post-STZ Figure 2.4. Diabetic subgroups according to hypoglycemic response to vanadyl (0.75 mg/ml) treatment over 15 weeks P l a s m a g lucose va lues of the subgroups of diabet ic an imals treated with V O S 0 4 at days 3 (A, DT3), 10 (B, DT10) and 17 (C, DT17) pos t -STZ injection. E a c h plot indicates the number of eug lycemic (E, 0) and non-eug lycemic ( N E , 4 ) an imals in a treatment group. The time at which treatment was initiated is indicated by an arrow (t) . 53 2.3.2. Effects of vanadyl on plasma parameters after 5 months of treatment The induction of STZ-d iabe tes resulted in reduced circulating insulin in the fed state, which was not altered by vanadyl treatment over 5 months (Table 8). P l a s m a insulin in C T was significantly lower than C (p < 0.05). Diabetic rats had e levated triglyceride and cholesterol levels which were corrected by vanad ium treatment, in agreement with previous studies (Ramanadham et a l , 1989; Mongo ld et a l , 1990). In addit ion, when the DT animals were pooled from DT3, DT10 and DT17 groups and ana lyzed according to level of g lycemia attained (DT-E vs. D T - N E ) , no signif icant di f ferences were s e e n . P l a s m a G O T was significantly increased in D and was lowered to control levels with treatment. There were no significant changes in B U N levels between the groups. S ince these and all subsequent parameters tested in this study were unaffected by delayed treatment with vanadium (i.e. not significantly different between the D T 3 , DT10 and D T 1 7 groups), the results on g lucose to lerance, pancreat ic and ad ipose t issue function in the DT3 , DT10 and DT17 groups were pooled and ana lyzed according to the glycemic levels ach ieved in response to vanad ium treatment (i.e. D T - E vs. DT -NE) . 2.3.3. Oral g lucose tolerance test ' Fast ing p lasma g lucose levels of both D T - E (n=17) and D T - N E (n=22) rats were not significantly different from control and were markedly lower than D (Fig. 2 .5A) . P l a s m a insulin concentrat ions did not differ significantly between the D and DT in fasted animals or fol lowing a g lucose load while the peak insulin of C T was signif icantly lowered to a level not different from the D and DT groups (Fig. 2 .5B, p < 0.05). Al though fasting p lasma g lucose levels in D T - E rats were similar to D T - N E , a greater improvement in g lucose tolerance was apparent in the D T - E group compared to D T - N E (t = 30 min, 16.8 ± 0.9 vs. 21.7 ± 0.7 mM). However, the in vivo insulin response to g lucose was not different between D T - E and D T - N E as judged by circulating insulin. 54 Table 8. P lasma parameters of various groups at 5 months Groups Insulin Cholestero l Triglyceride B U N G O T (n) (uU/ml) (mM) (mM) (mM) (U/l) c 55 2.0 1.6 7.0 44 (12) (4) (0.1) (0.1) (0.3) (3) C T 44+ 1.7 1.4 7.5 36 (12) (4) (0.1) (0.2) (0.2) (2) D 30+ 2.8* 3.3* 8.1 74* (11) (4) (0.2) (0.5) (0.2) (18) DT3 37+ 1.9 1.5 8.2 36 (12) (4) (0.1) (0.2) (0.5) (3) DT10 3 6 + 1.8 1.6 8.2 40 (11) (4) (0.1) (0.1) (0.4) (5) DT17 3 2 + 1.6 1.4 8.4 32 (16) (3) (0.1) (0.1) (0.4) (2) + significantly different from C (p < 0.05) Data is expressed as mean ± ( S E M ) . 55 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (min) Figure 2.5. Oral g lucose tolerance test at 5 months of study P l a s m a g lucose (A) and insulin (B) response to an oral g lucose cha l lenge (1 g/kg) in control (C, O ) , control-treated (CT, • ) , d iabet ic (D, A), and diabet ic rats treated with V O S 0 4 . Vanadyl - t reated diabet ic an imals (DT3, DT10 , DT17) were poo led and c lass i f ied accord ing to g lucose- lower ing response to treatment: eug lycemic (DT-E , 0) and non-eug lycemic ( D T - N E , • ) . * p < 0.05 vs. D and C. 56 2.3.4. Response of isolated pancreas to 16.65 mM glucose + 0-1 ng/ml GIP The effects of vanadium treatment of animals on the response of the perfused pancreas to g lucose plus a G I P gradient is shown in Figure 2.6. R e l e a s e of immunoreact ive insulin (IRI) from the diabetic perfused pancreas w a s so low as to be below the limit of detect ion, while the response from C T animals w a s not significantly different from non-treated control rats. Insulin re lease from the pancreas of treated diabetic rats pooled from 3 treatment groups (n=17) was markedly reduced compared to control rats (-10%) but was clearly detectable and significantly elevated compared to re lease from diabetic rats. S ince IRI re lease was not significantly different between the D T 3 (n=6), DT10 (n=5) and D T 1 7 (n=6) groups, the data were ana lyzed accord ing to g lycemic response to treatment. Thus , further expans ion of the sca le of the figure (inset, F ig . 2.6) clearly reveals a greater basa l insulin re lease by the D T - E an imals (n=6) as compared to D T - N E (n=11). Insulin re lease from the D T - N E group w a s consistent ly low throughout the period of perfusion. In contrast, re lease of IRI f rom the D T - E group showed an early peak which was absent from the D T - N E group and cont inued to rise slowly and progressively throughout the period of the perfusion. 2.3.5. Glycerol output in isolated adipose t issue Figure 2.7 shows the effects of insulin and/or epinephrine on glycerol output from epididymal fat pads isolated at the time of killing. Basa l and epinephrine-st imulated glycerol output was significantly higher in D and D T - N E (n=10) as compared to control rats. In contrast, glycerol re leased in the basal state was not different between C , C T and D T - E (n=7). Incubation of t issues in the presence of insulin had little effect on low basa l rates of glycerol output from C , C T , or D T - E groups whereas more pronounced inhibition of l ipolysis in D and D T - N E was apparent. In the p resence of epinephr ine (with or without insulin), glycerol output in the C T and D T - E groups were greater than C although significantly less than D and D T - N E . Fat pad weights were not 57 300 0 10 20 30 40 50 Time (min) Figure 2.6. In situ pancreatic perfusion at 5 months of study Immunoreactive insulin response to 16.65 m M g lucose + a gradient of 0-1 ng/ml G I P from isolated perfused pancreas of control (C, O ) , control-treated (CT, • ) , diabet ic (D, A ) , and diabetic rats treated with V O S 0 4 (DT, • ) . Inset shows enhanced Y -ax i s for diabetic-treated rats (DT3, DT10, DT17) pooled and classi f ied accord ing to g lucose-lowering response to treatment: euglycemic (DT-E , 0) and non-euglycemic ( D T - N E , • ) . 58 I I basal + insulin + epinephrine + epinephrine + insulin Figure 2.7. Effects of vanadium on adipose t issue lipolytic rates in diabetic rats Glycero l output from isolated ad ipose t issue of control (C), control-treated (CT) , diabet ic (D), and diabetic rats treated with V O S 0 4 (DT3, DT10, DT17), pooled and classi f ied according to hypoglycemic response to treatment: euglycemic (DT-E) and non-euglycemic (DT-NE) . Ad ipose t issue was incubated in the presence or a b s e n c e of insulin (1 mU/ml) fol lowed by epinephrine (1 ug/ml). * p < o.05 vs . C , # p < 0.05 vs . D. 59 altered by vanadium treatment in control animals. P a d weights in the D T - E rats were not significantly different from control and were significantly higher than in the D and D T - N E rats (p < 0.05). 2.3.6. Histopathology results Histological s l ides of the kidney showed moderately extensive vacuol izat ion and swel l ing of epithelial cel ls of the distal convoluted tubules in five out of six non-treated diabetic animals as compared to none in control (Fig. 2 .8A-C) . In contrast, analys is of 5-month vanadyl-treated diabetic rats revealed very little to no alterations in structure of kidney except for a smal l renal cell carc inoma in one DT3 animal . The inc idence of mild cellular infiltration seen in the medul la of diabetic rats was not altered by vanadyl treatment and was also observed to a similar extent in treated control an imals . There were no morphological changes in the liver of any animals . 2.3.7. Vanadium levels Vanad ium levels in t issues ana lyzed in the non-treated control and diabet ic groups were <0.1 ug/g wet weight. In the treated groups, vanad ium w a s found a s fol lows (ug/g): bone (4.95 ± 0.38 or 97.3 ± 7.5 nmol/g) > kidney (2.42 ± 0.24 or 47 .5 ± 4.7 nmol/g) > liver (0.95 ± 0.13 or 18.7 ± 2.6 nmol/g) > musc le (0.19 ± 0.03 or 3.7 ± 0.6 nmol/g). There was no correlation between vanadium levels in the t issues studied and the degree to which g lucose was lowered in response to treatment in the diabet ic animals. A s wel l , there were no significant dif ferences in t issue levels of vanad ium between D T - E and D T - N E rats. Vanad ium levels in musc le and liver agree with the va lues previously reported when VOSO4 was suppl ied in the drinking water at a concentrat ion of 0.75 mg/ml (Mongold et al , 1990), although the levels in bone and kidney are lower, possibly because rats were fasted overnight prior to killing and vanady l intake was consequent ly depressed . 6 0 B C Figure 2 . 8 . Prevention of morphological changes in kidney of diabetic rats with vanadium treatment Histological s l ides of kidney from control (A), diabetic untreated (B) and treated with vanadyl (C) at 5 months, demonstrat ing an overall dec rease in swell ing and vacuol izat ion of epithelial cel ls in the distal renal tubules of diabetic an imals with vanad ium treatment. Microbar length = 53 urn. 61 2.4. DISCUSSION The present study demonstrates that the eff icacy of oral vanad ium as a g lucose- lower ing agent is not attenuated by delaying the start of treatment from 3 to 10 or 17 days after the induction of d iabetes. Vanad ium treatment led to complete normal izat ion of p lasma triglyceride and cholesterol levels and markedly reduced g lucose levels to the s a m e extent in all the DT groups. In contrast to the previous study in which rats were administered similar concentrat ions (0.75 mg/ml) of V O S 0 4 in the drinking water (Ramanadham et a l , 1989b), not all animals became completely eug lycemic in the present study. Instead, one subgroup of treated animals cont inued to exhibit hyperglycemia (DT-NE) , while in another subgroup (DT-E) , p lasma g lucose w a s in the normal range (< 9.0 mM) throughout the entire study. The proportion of treated animals which deve loped normoglycemia was not lessened in the DT10 and DT17 groups relative to the DT3 group. This phenomenon of a partial response w a s previously reported in a study by Bendayan and Gr ingas (1989). In that study, pancreat ic amy lase activity was completely normal ized in a subset of diabetic an imals which became normoglycemic in response to treatment, it w a s only partially restored in treated animals which did not ach ieve normoglycemia. This apparent variability in response to vanad ium may have resulted from varying degrees of S T Z - i n d u c e d beta cell cytotoxicity and/or insulin resistance in the individual an imals s ince in subsequent studies, the number of completely euglycemic animals is progressively increased by increasing the concentrat ion of V O S 0 4 administered in the drinking water. Th is dose -dependent progression of D T - N E animals towards the point of true eug lycemia occurred to a similar extent independently of the time at which treatment with vanad ium was initiated. T h e s e studies demonstrate that the anti-diabetic eff icacy of vanady l is similarly effective in producing a euglycemic state despite delaying treatment after the induction of d iabetes. It is therefore most unlikely that the antidiabetic eff icacy of vanadyl depends on its ability to directly inhibit the cytotoxic effects of S T Z . 62 In v iew of the disparity in the glucose- lowering response to vanady l , it w a s important to determine if any correlation existed between pancreat ic function and the degree to which p lasma g lucose was reduced in the DT animals . Stud ies have indicated conflicting results in which treatment of STZ-d iabet ic rats with vanadate , which began between 7-10 days after the S T Z injection, led to either no changes in insulin-staining cel ls (Bendayan and Gr ingas, 1989), or modest increases in pancreat ic insulin content, which reached 6-7% (Brichard et a l , 1988) and 3 4 % (Blondel et a l , 1989) of control va lues. In the present study, the animals which exhibited eug lycemia under basa l condit ions (DT-E) had fasting and g lucose- induced circulating IRI which was markedly lower than C and indist inguishable from D or D T - N E . Despi te these observat ions, the D T - E group demonstrated markedly enhanced g lucose to lerance compared to both D T - N E and D (though still not equivalent to control rats). Furthermore, insulin secret ion in vitro from perfused pancreata of D T - E an imals w a s significantly higher than in D T - N E or D though, again , substantial ly lower than in non-diabet ic control rats. Of particular note w a s the gradual increase in the rate of insulin secret ion in D T - E throughout the period of perfusion. O n the bas is of IRI secret ion from the perfused pancreas, some improvement in pancreat ic function is seen in response to vanady l treatment which is not apparent from the determination of circulating IRI in vivo but which is nevertheless reflected in improved g lucose tolerance. S ince these responses to vanadium are achieved after the cytotoxic action of S T Z is complete (Junod et a l , 1967), it appears that a subsequent , albeit limited, recovery of pancreat ic function occurred in the D T - E group. Al though this effect might be attributed to the alleviation of chronic hyperglycemia (Rossett i and Laughl in, 1989; Leahy and Weir , 1991), chronic treatment of neonatal STZ- in jected rats with vanadate did not improve B-cel l response to g lucose, despite inducing normoglycemia (Serradas et a l , 1990). Thus , addit ional studies will be required to establ ish if the observed residual pancreat ic function is a cause or an effect of a full response to vanad ium treatment. 63 The observat ion that the D T - E group displays basa l eug lycemia despi te circulating insulin similar to va lues in D T - N E or D groups suggests enhanced sensitivity of the D T - E group (relative resistance of D and D T - N E ) to the residual endogenous insulin and/or the avai lable vanadium present throughout treatment. In agreement with previous reports (Ramanadham et al , 1989a,b), vanadyl treatment led to normal ized ad ipose t issue lipolytic function, as ev idenced by a correction in basa l glycerol re lease rate from ad ipose t issue isolated from D T - E . However, there was no improvement in basa l or catecholamine- induced lipolytic rates in the D T - N E group. T h e s e results are consistent with the hypothesis that the conservat ion of t issue function may be secondary to the amelioration of the hyperglycemic state. However , the responses of peripheral t issues to vanadium may be more complex. For instance, defects in musc le g lycogen synthesis which do not appear to be secondary to g lycemic status have been reported to be improved with vanadate treatment (Rosett i and Laughl in, 1989). Resu l ts from this study a lso confirm previous f indings with the treatment of control animals with vanad ium. For instance, chronic vanad ium treatment has previously been shown to lower the body weight, food and fluid intake of control rats ( R a m a n a d h a m et a l , 1989a; Blondel et a l , 1989). Circulat ing insulin levels in control an imals have also been reported to be reduced by vanad ium treatment (Heyl iger et la, 1985; R a m a n a d h a m et a l , 1989a). W e also observed that C T rats have lowered p lasma insulin levels and insulin secret ion in response to an oral g lucose cha l lenge without affecting either fasting g lycemia or g lucose to lerance. T a k e n together, these studies suggest that vanadium treatment may enhance insulin sensitivity in the control animals. O n the other hand, it is a lso possible that vanad ium treatment affects the intestinal re lease of incretins such as G I P and G L P - 1 which enhance the fi-cell insulin secretory response (Schauder et a l , 1975; Fridolf and Ahren , 1991). In addit ion, vanad ium treatment could have also reduced intestinal g lucose transport v ia downregulat ion of the N a + - d e p e n d e n t g lucose cotransporter (Madsen et a l , 1993). 64 This study also demonstrates the long-term toxicological profile of control and diabetic animals treated with vanad ium. Treatment did not appear to adverse ly affect the morphology of the liver and indeed prevented some morphological changes in the kidney which had appeared in the non-treated diabetic animals. Th is protective effect of vanad ium treatment on the kidney was not secondary to a correction in g lycemic levels s ince both D T - E and D T - N E animals exhibited a similar lack of pathological lesions. T h e s e findings support a previous study (Mongold et a l , 1989) in which the occur rence of cellular swell ing in the kidney was diminished markedly with administration of V O S 0 4 to STZ-d iabet ic rats for 39 days. However , discrete patches of cel lular infiltration in the medul la of treated control animals may suggest the p resence of mild inf lammation, and whether this phenomenon is a manifestat ion of a toxic effect of vanadyl and is reversible upon withdrawal from treatment requires further examinat ion. Notably, the high inc idence of cataracts observed in the untreated diabetic rats at 5 months was eliminated with vanadyl treatment in 38/39 rats. In summary, severa l important points were estab l ished in this study. Delaying vanadyl treatment for up to 2 weeks following the induction of STZ-d iabe tes did not impair the ability of vanadyl to exert its g lucose- lower ing effects. It s e e m s most likely that the effects of oral vanadyl in the STZ-d iabet ic rat involve direct effects on the peripheral target t issues at the level of insulin receptor or post-receptor events which are not dependent upon normal ized beta-cell function. In addit ion, chronic vanad ium treatment may also lead to some restoration of pancreat ic insulin secretory function which is not apparent in vivo but is more sensit ively detected v ia the pancreat ic perfusion method. The modest improvement of insulin availability may, in combinat ion with vanad ium, be responsible for the significant ameliorat ion of the diabet ic state for a prolonged period. The prevention of most abnormali t ies in the diabet ic kidney in 5 months treatment provides an addit ional impetus to explore the mechanism(s) of action of vanad ium in the STZ-d iabet ic rat and in other avai lable models of d iabetes. 65 Chapter 3 IN VIVO ANTIDIABETIC ACTIONS OF NAGLIVAN, A N ORGANIC V A N A D Y L COMPOUND IN STREPTOZOTOCIN- INDUCED DIABETES 3.1. INTRODUCTION Although it has been suggested that the degree of toxicity of vanad ium is independent of the oxide form (Domingo et a l , 1991), the inc idence of toxic s ide effects with vanad ium salts was reported to increase with va lency, vanadyl (+IV) being less toxic than vanadate (+V) (Rosch in et a l , 1980). Chron ic treatment with oral V O S 0 4 (0.1 mmol/day) in STZ-d iabet ic rats was not assoc ia ted with morphological changes in severa l organs (Mongold et a l , 1990). However , despi te these results and the lowered incidence of gastrointestinal toxicity as compared to vanadate, administration of oral V O S 0 4 has resulted in severe diarrhea at high d o s e s in s o m e an imals ( R a m a n a d h a m et a l , 1989). Thus , alternative formulat ions of vanady l which are better tolerated are currently being studied. The effective dose of inorganic vanadium made avai lable in the drinking water in STZ-d iabet ic rats ranges between 0.20-0.65 mmol/kg/day for N a 3 V 0 4 (Heyl iger et a l , 1985; Brichard et a l , 1988), 0.26-1.02 mmol/kg/day for N a V 0 3 (Meyerovi tch et a l , 1987), and 0.46-0.69 mmol/kg/day for VOSO4 ( R a m a n a d h a m et al , 1989). Al though the absorpt ion of vanadium was reported to be < 1 0 % (Byrne and Kos ta , 1978), some studies have indicated it to be as high as 4 0 % (Bogden et al , 1982). Never theless, because of the gastrointestinal s ide effects demonstrated with inorganic vanad ium, it was hypothesized that the gastrointestinal absorpt ion of vanad ium could be improved by increasing its lipophilicity. This could potentially lower the effective oral dose , and hence the incidence of diarrhea. Hence , the oral ef fect iveness of an organic vanadyl compound, nagl ivan (Fig. 1), on g lycemia and card iac dysfunct ion was studied in the STZ-d iabet ic rat. Figure 3.1. Bis(cysteine, amide N-octyl)oxovanadium IV (naglivan). 67 3.2. MATERIALS AND METHODS 3.2.1. Formula and characterization of the compound The complex Bis(cysteine, amide N-octyl)oxovanadium IV (naglivan) w a s prepared according to Lazaro et al (1988) and was generous ly suppl ied by P a n m e d i c a (Carros, France). Briefly, in a preliminary step, commercia l ly avai lable tert iobutyloxycarbonyl (Boc)-cysteine was l inked to octy lamide using overnight benzotriazol-1 -yl-oxy-tr is(dimethylamino)phosphonium hexaf luoro-phosphate ( B O P ) activation in dimethylacetamide. After evaporat ion of the solvent under vacuum, the residue was washed and recrystall ized by ethylacetate giving pure d iBoc-cys te ine di-N,N'-octy lamide ( M P = 125°C). This product was then deprotected by a solution of trif luoroacetic acid in dichloromethane. After wash ing, the trif luoroacetate salt of cyste ine di-N,N'octy lamide was obtained. This product was further neutral ized by diluted ammonium hydroxide and reduced by sodium borohydride in d ioxane, giving cyste ine N-octylamide which was reacted with vanadyl sulfate, leading to the oxovanad ium IV complex. The amount of vanad ium of this complex (9.6%) fitted well with a 2:1 l igand:metal stoichiometric ratio. E P R analys is showed a V 4 + ( V 0 2 + ) oxidation state for the complexed metal ion. 3.2.2. Treatment and maintenance of animals Male Wistar rats between 200-250 g were used . O n day 0, d iabetes w a s induced by a single i.v. injection of 55 mg/kg streptozotocin (S igma Chemica l C o . St. Lou is , Mo.) d isso lved in 0 .9% sal ine while control rats received the vehic le only. O n days 2 and 3, following the S T Z injection, blood g lucose was checked by a g lucometer from blood obtained from the tail vein and animals exhibit ing blood g lucose levels greater than 13.75 m M were considered diabetic. O n day 4, treatment was started in the var ious groups. The compound w a s made up as a suspens ion (10 mg/ml) with acac ia (3% w/v). Control rats were treated ( C V , n=8) or 68 not treated (C, n=8) with nagl ivan. Diabetic rats were divided into three groups: untreated (D, n=8), treated with insulin (DI, n=8), and treated with nagl ivan plus insulin (DVI, n=8). In the DVI and C V animals, 50 mg/kg nagl ivan (equivalent to 0.06 mmol vanadium/kg) was administered by oral gavage on day 4 . S i n c e no significant g lucose- lower ing effects were seen at this dose (as determined by the average blood g lucose levels 6 hours after administration), the dose was increased in DVI and C V groups to 100 and 150 mg/kg/day (0.12 and 0.18 mmol vanadium/kg) on days 5 and 6, respectively. The dose of nagl ivan w a s then restored to 50 mg/kg/day on day 7 in both groups. In addit ion, in the DVI group, nagl ivan treatment w a s supplemented with insulin (Protamine Z inc, a gift of Eli Lilly, C a n a d a ) . The dose of insulin was initiated at 4.5 U/kg/day, s.c. for 3 days and subsequent ly was adjusted daily to regulate p lasma g lucose at normal levels until day 21 . During the s a m e per iod, insulin treatment was initiated at 9.0 U/kg/day and subsequent ly titrated to maintain euglycemia in the DI group. O n day 21 , insulin treatment in the diabet ic groups was terminated. However, C V and DVI were maintained on nagl ivan 50 mg/kg/day for an addit ional 5 weeks . Food and water were provided ad libitum and p lasma g lucose, body weight, and food and fluid intake were monitored during the treatment period. At the time of termination, blood w a s col lected for determination of p lasma triglycerides, cholesterol , free fatty ac ids , insulin, %glycosylated hemoglobin (%GHb) , vanadium levels, g lutamate:oxalacetate t ransaminase (G O T) and urea. 3.2.3. Isolated working heart study At the time of termination, animals were killed by decapitat ion and hearts were immediately isolated. Card iac function was a s s e s s e d by the isolated working heart method as descr ibed previously (Vadlamudi et a l , 1982). Briefly, the heart was paced at 300 beats/minute and perfused with buffer at 37°C, and equil ibrated at 69 15 c m H 2 0 of left atrial filling pressure until a s teady state performance was ach ieved. Measurements of rate of contraction (+dP/dt), rate of relaxation (-dP/dt) and left ventricular developed pressure ( L V D P ) were obtained at a range of filling pressures (7.5 - 22.5 cm H 2 0 ) . After determining heart function, hearts were removed, blotted and weighed. 3.2.4. P lasma Ana lys is Kits for p lasma g lucose, triglycerides, cholesterol , and free fatty acid determinations were obtained from Boehr inger Mannhe im (Laval , Quebec ) . G lycosy la ted hemoglobin was ana lyzed using kits obtained from Isolab (Akron, Ohio). P l a s m a insulin was measured from a kit obtained from Immunocorp (Montreal, Quebec) . P l a s m a urea, creatinine and G O T were ana lyzed by kits obtained from S i g m a . P l a s m a vanad ium levels were detected by previously publ ished methods (Mongold et al , 1990). 3.2.5. Statistical Ana lys is All results are expressed as means + S E M . The data were ana lyzed using M A N O V A fol lowed by the Newman Keul 's test, p < 0.05 indicated statistical s igni f icance. 70 3.3. R E S U L T S 3.3.1. Effects of naglivan and/or insulin treatment on body weight, food and fluid intake. Overal l weight gain in the STZ-d iabet ic animals w a s significantly depressed (Fig. 3.2A). Short-term treatment with insulin or nagl ivan + insulin enhanced weight gain in the diabetic rats, al though after withdrawal from insulin treatment (w), the increase in body weight of the DVI and DI groups w a s simi lar to untreated diabet ics. Nagl ivan treatment did not affect weight gain in control rats. Symptoms of hyperphagia were initially observed in all the diabet ic an imals . In the untreated diabet ics, food intake gradual ly increased over 8 w e e k s to twice that of control animals (Fig. 3.2B). Administrat ion of insulin or nagl ivan + insulin in diabetic animals significantly reduced food intake to a level not different from control on weeks 2 and 3. After insulin treatment was withdrawn however, food consumpt ion in the DI group progressively increased over the duration of the study and by week 8 was similar to D. However, mean food intake in the DVI group remained at 6 7 % of D at the final week. Administrat ion of nagl ivan initially reduced food intake for 2 weeks in control animals, but this was normal ized for the most part of the treatment period until the final week. The induction of STZ-d iabe tes resulted in an elevated fluid intake which like food intake, gradual ly increased throughout the 8-week period in the D group (Fig. 3 .2C) . The polydipsia w a s initially corrected with the administrat ion of insulin or nagl ivan + insulin. Fol lowing withdrawal from insulin treatment, the DI group consumed progressively greater amounts of fluid which approached diabet ic levels, al though still significantly less than D by 8 weeks (DI = 250 ± 4 vs . D = 286 ± 6 ml/rat/day, p < 0.05). However, mean fluid intake of the DVI group remained at less than half the diabetic levels by week 8 (121 ± 2 ml/rat/day). Administrat ion of nagl ivan did not affect fluid intake in control animals. 71 72 3.3.2. Effects of naglivan and insulin treatment on plasma g lucose Diabet ic an imals exhibited hyperglycemia which averaged - 2 0 m M at four days pos t -STZ injection (Fig. 3.3). Oral administration of 50 mg/kg nagl ivan in the DVI group on day 4 did not lower mean g lycemia acutely as measured 6 hours after nagl ivan was administered. Similarly, increasing the nagl ivan d o s e to 100 and 150 mg/kg on subsequent days did not produce an acute g lucose- lower ing response in the diabetic animals over 3 days. Oral nagl ivan administration did not affect p lasma g lucose levels in control animals. Initially for 3 weeks , the administrat ion of long-act ing insulin in the morning to DVI and DI animals was found to maintain normoglycemia, when measured the following morning (24 hours after the last dose) , however p lasma g lucose was consistent ly lower than control levels when measured in the afternoon. A t 21 days pos t -STZ, when insulin treatment was withdrawn, mean g lycemia of the DI group progressively increased and was subsequent ly not different from the untreated diabet ic group by week 8. F rom the DVI group which cont inued to receive nagl ivan (50 mg/kg) daily, four animals were found to remain in a eug lycemic (DVI-E; p lasma g lucose < 9.0 mM) state after insulin treatment had c e a s e d , while the remaining four reverted to a hyperglycemic (DVI-H; p lasma g lucose > 9.0 mM) state in a manner similar to the DI group, thus resulting in a mean p lasma g lucose level which w a s intermediate between control and diabetic levels (Fig. 3.3, inset). 3.3.3. Effects of naglivan treatment on exogenous insul in requirement Initially, insulin was administered daily to maintain eug lycemia in the DVI and DI groups. The dai ly insulin dose w a s initiated at 9.0 U/kg/day for the DI group and either increased or reduced to ach ieve a stable normal A M g lucose reading prior to receiving the next insulin dose on the following day (Fig. 3 .4A,B) . The initial insulin dose used for the DVI group w a s substantial ly lower (4.5 U/kg/day) and w a s similarly adjusted thereafter to . maintain A M euglycemia. Throughout the insulin 73 Figure 3.3. Effect of naglivan and/or short-term insulin treatment on g lycemia P l a s m a g lucose levels of control (C , O ) , control treated with nagl ivan ( C V , • ) , d iabet ic untreated (D, A ) and treated with daily insulin (DI, • ) or nagl ivan + insulin (DVI, • ) from 4 days pos t -STZ injection fol lowed by withdrawal (W) from exogenous insulin at 3 weeks . Inset shows euglycemic (DVI-E, 0) and hyperglycemic (DVI-H, • ) nagl ivan-treated diabetic rats. 74 15 -, 10 -0 5 10 15 20 r 20 0 5 -10 15 20 0 5 10 15 20 0 5 10 15 20 Days post-STZ Figure 3.4. Reduced exogenous insulin requirement with naglivan treatment Insulin dose (A) and A . M . g lycemia (B) of diabetic rats treated with daily insulin (DI, O ) , or nagl ivan + insulin (DVI, • ) from 4 to 20 days pos t -STZ injection. Insets show va lues for eug lycemic (DVI-E, n=4, 0) and hyperglycemic (DVI-H, n=4, • ) nagl ivan-treated diabetic rats. 75 treatment period, the insulin dose was significantly lower in DVI than in diabet ic rats on insulin a lone (day 20, DVI = 5.31 ± 0.45 vs . DI = 9.69 ± 0.27 U/kg/day, p < 0.05), despi te A M blood g lucose levels not significantly different between both groups (day 20, DI = 9.36 ± 0.75 vs . DVI = 7.61 ± 2.03 m M , p > 0.05, F ig . 3.4B). Retrospect ive ana lys is indicated that at an insulin dose of 4.5 U/kg/day, the DVI-H group remained hyperglycemic, whereas this low dose of insulin was sufficient to maintain normoglycemia in the DVI-E group (Fig. 3.4, inset). Hence , when the dose of insulin was titrated to maintain normal p lasma g lucose levels, insulin requirement w a s significantly greater in the DVI-H animals than the DVI -E group. Never the less, the required insulin d o s e of the DVI -H group remained significantly less than that of the DI animals (day 20, DVI-H = 6.38 ± 0.27 vs . DI = 9.69 ± 0.27 U/kg/day, p < 0.05). 3.3.4. Effects of naglivan and insul in treatment on plasma l ipids, GOT, creatinine, BUN, insulin and % glycosylated hemoglobin at eight weeks At time of termination, various p lasma parameters were measured in all the groups (Table 9). The induction of d iabetes resulted in elevated lipids (triglycerides and free fatty acids), which were still significantly reduced after 5 weeks of withdrawal from insulin in both DVI and DI groups. However, p lasma cholesterol a m o n g the diabet ic groups w a s reduced only in the DVI-E group to levels not different from control at this t ime. P l a s m a G O T was not different in the D group from control but w a s lowered by treatment of control and diabetic animals with nagl ivan, a phenomenon which has been previously reported in vanadium-treated animals (Al-Bayat i et a l , 1990). There w a s no significant difference in urea and creatinine levels in all the groups. P l a s m a vanad ium levels in the C V and DVI rats were (ug/ml): 0.15 ± 0.03 (2.9 ± 0.5 uM) and 0.17 ± 0.06 (3.3 ± 1.2 uM), respectively, similar to those previously reported in rats treated with V O S 0 4 at varying concentrat ions in the drinking water (Mongold et a l , 1990), but considerably lower than other reported va lues using either N a V 0 3 76 Table 9. Effects of naglivan and insulin treatment on various plasma parameters at 8 weeks after the STZ injection Parameter C C V D DI DVI -E DVI-H (n=8) (n=8) (n=8) (n=8) (n=4) (n=4) Free Fatty A c i d s 0.66 0.53 1.12* 0.54 0.56 0.62 (mM) (.11) (.05) (.21) (.05) (.04) (.10) Cholestero l 1.32 1.24 2.12* 1.76* 1.45 1.85* (mM) (.09) (.06) (.13) (-11) (.08) (.05) Triglyceride 1.43 1.32 2.95* 1.08 1.12 1.04 (mM) (.10) (.19) (.47) (.16) (.20) (.15) G O T 57.84 33.97* 49.61 34.69* 22 .77* 28 .80* (IU/L) (3-41) (3.14) (6.23) (4.59) (2.65) (6.53) Urea Nitrogen 6.58 6.13 7.93 7.18 5.41 6.65 (mM) (.55) (.45) (.53) (.63) (.34) (.96) Creat in ine 0.48 0.47 0.42 0.35 0.39 0.39 (mM) (.03) (.02) (.04) (.03) (.06) (.06) * significantly different from C , p < 0.05 Data is expressed as mean (± S . E . M . ) 77 u/qoiBoLUdj-j pa}eiAsooAiQo/o t — CO CO Tt CM O I I I I I + * H i - 0 0 C O C M o I * ^ l l l l l t l t l + * IB Q UJ • Q o o co i — r o o o C M •« -—I 1 1 1 1 1 I o o o o o o o o S (C Ifl ^ M N r (liu/nri) uijnsui euiseid S Q O O CO o "O CD -*—» CO CD CD - Q CO > o c CO > co c § 5 <3 CD c co CO CD CO CO c > D o o c d •B v •g + N ^ I S E © cu -= *- 2 * J «J c _ o .= o ° O i i c o •o " ® o JH c >» 5 v> o |g> CD re i5 . _ CO — o 3 O CO >, .E TO re vp £ ^ CO ^ CO c CL d CO V % * CD * 0 0 & £ ? CO CD 0 3 J Q CD CO E =5 > £ a s c -V — c 3 CO CO > c = — cn c l 1 5 ra> co Q c ' >- 9 ° E ^ CD O & .E a5 in co CO = I O) jro i l o_ D CO c " ~ X 3 >, c = CO CO I 5 £ Q I I _>» •a C D c 3 co cu 78 (Meyerovi tch et a l , 1987) or V O S 0 4 (Ramanadham et a l , 1990). There was no difference in p lasma vanad ium levels between the DVI -E and DVI -H groups. At 8 weeks , diabetic rats had reduced circulating insulin in the fed state (Fig. 3.5). P l a s m a insulin levels were significantly higher in the DVI and DI groups compared to D, but were less than 5 0 % of control levels. M e a n p lasma insulin levels were not different between the insulin and vanadium-treated diabetic groups. M e a n % glycosylated hemoglobin (%GHb) levels were significantly e levated in the D and DI groups relative to control. However, in agreement with p lasma g lucose va lues , the % G H b of DVI -E was not different from control while that of DVI-H was not different from untreated diabet ics, despite similar p lasma insulin va lues between both the DVI -H and DVI -E groups (Fig. 3.5, inset). 3.3.5. Effects of naglivan and insul in treatment on heart.body weight and heart function at eight weeks At eight weeks after the S T Z injection, body weight in all the diabet ic groups was significantly lower than control (Table 10). A l though heart weight w a s not significantly different among the different groups, the heart:body weight ratios in those groups which were hyperglycemic at 8 weeks (D, DI and DVI-H) were signif icantly higher than control. However, the h e a i t b o d y weight ratio of DVI -E w a s not signif icantly different from control. Card iac function at 8 weeks w a s a s s e s s e d by measur ing the rate of contraction (+dP/dt), rate of relaxation (-dP/dt), and left ventricular deve loped pressure ( L V D P ) using the isolated working heart apparatus (Fig. 3 .6A-C) . Heart function was significantly depressed in D and DI groups, whereas this w a s intermediate between control and diabetic levels in the DVI group. W h e n ana lyzed accord ing to response to treatment, the normoglycemic DVI -E group had improved heart function which was not different from the control groups, whereas heart function in DVI -H w a s not different from D. 79 Table 10. Heart and body weight of the various groups at 8 weeks after the STZ injection C C V D DI DVI -E DVI -H (n=8) (n=8) (n=8) (n=8) (n=4) (n=4) Body weight 459 468 353* 385* 4 0 3 * 388* (BW)(g) (15) (17) (11) (7) (20) (16) Heart weight 1.66 1.67 1.52 1.56 1.52 1.49 (HW)(g) (0.09) (0.06) (0.05) (0.03) (0.06) (0.07) Ratio 3.45 3.49 * 4.10 3.91* 3.60 * 3.72 (H:BW) ( x10 3 ) (0.10) (0.11) (0.19) (0.22) (0.06) (0.04) Data is expressed as mean (± S .E .M . ) *significantly different from C , p < 0.05 80 o ^ g 00 m CM o CN m o m o o to o o CM o o o 00 o CO + S CO o o o © o © CO ~~r o o o CM m CM o CM m o o o in CM o CM m o r o o o 10 — r o o o —r o o o CO —r o o o CM o o o © CM a: 5 o 3 </> 9> ^ CL •2 CD " O CD o 00 Q > _ i CD i _ 00 CO CD " O * ; CD C Q . 0) o E CD "S > £ -o 1_ > o CD TO (0 = > CO ~ o -o o ro .a .55 ^ "O 5 C D_ — -a c ^ .2 c o -2 3 X CD 8 2 ra —* -£ ^ 0 c CO > co s s < q — 0 Q v CO CD T 3 CD T 3 ^ 0 0 0 S * "V co 3 . 0 - - Q CD II X I C CO -> o' •a g S &? 1 £ C T 3 O C O CO o 5 c Q . .2 y CO CD (0 c o 2 2 0- c o o . o <° cn CO CD CD "CO 3 * d IT 2 ul c ^ 0 < 0 Q 1 ^ 2 1 to CD > - CO 1 5 81 3.4. DISCUSSION This study demonstrates the chronic insulin-like activity of an organic vanady l complex in vivo in STZ-d iabe tes . Al though nagl ivan (50-150 mg/kg) a lone over three days was not effective in lowering hyperglycemia acutely as measured 6 hours after administrat ion, supplement ing suboptimal doses of insulin during the initial s tages of treatment was sufficient to normalize g lycemia in the naglivan-treated DVI group to levels which were not different from DI animals (treated with insulin a lone) which required much higher doses of insulin. In addit ion, the insulin requirement by poorly responding DVI-H (hyperglycemic, treated) animals, al though higher than DVI -E (euglycemic, treated), remained significantly lower than DI. This agrees with previous f indings in which oral V O S 0 4 enhanced the in vivo sensitivity to insulin by lowering the exogenous insulin requirement of STZ-d iabet ic and spontaneous ly diabetic BB-Wis ta r rats ( R a m a n a d h a m et al , 1990). In a subsequent experiment, when diabet ic rats were administered nagl ivan a lone without insulin at a starting d o s e of 50 mg/kg/day, the correction in p lasma g lucose was seen only after repeated doses over a period of one week. A s has been previously observed with oral V O S 0 4 in STZ-d iabe tes , a subpopulat ion of diabetic animals deve loped normoglycemia in response to the administered dose of nagl ivan. Thus , after withdrawal from insulin treatment, this group (DVI-E) w a s consistently euglycemic or had near-normal p lasma g lucose as verif ied by the % G H b levels at the time of sacri f ice, despi te the lack of a significant improvement in p lasma insulin levels. From retrospective analys is of the data, it w a s found that by the second week of treatment, the insulin requirement of the DVI -E group w a s distinctly lower than in DVI-H animals. Furthermore, in a separate experiment, when diabet ic an imals were chronical ly treated with nagl ivan alone at a dose of 50 mg/kg/day, those animals which had not deve loped euglycemia exhibited normal p lasma g lucose levels when the dose was subsequent ly increased to 100 and 150 mg/kg/day. Thus , var iances 82 in the glucose- lower ing response to a single dose of nagl ivan tested in this study may be due to insufficient dosing in the diabetic subgroup exhibiting a greater degree of insulin resistance. Hence vanadyl , in the form of nagl ivan, has a g lucose- lower ing effect in STZ-d iabe tes at a minimum oral dose of 0.06 mmol/kg/day, making it 7.6 t imes more potent than vanadyl sulfate, which has a minimum effective dose of ~0.46 mmol/kg/day (Ramanadham et a l , 1989a). In isolated adipocytes, nagl ivan w a s a lso found to be more potent than either V O S 0 4 or N a V 0 3 in inhibiting norepinephr ine-stimulated l ipolysis, indicating that nagl ivan could be more lipophilic and hence penetrate more easi ly into the cel l . A s with chronic V O S 0 4 (Mongold et a l , 1990), nagl ivan treatment led to no adverse effects on renal function as reflected in p lasma creatinine and B U N levels after 8 weeks of nagl ivan intake. However, the diarrhea seen with V O S 0 4 treatment w a s not observed with nagl ivan at the dose administered. Vanady l toxicity in rats has been documented in some studies, although there are inconsistencies. For instance, although V O S 0 4 in the drinking water of STZ-d iabet ic animals at a concentrat ion of 1.1 mg/ml for two weeks did not alter urea, creatinine, and haematocri t (Domingo et a l , 1991a), the very s a m e laboratory reported that administration of lower concentrat ions of vanad ium (0.31 mg/ml) for four weeks led to increased urea and creatinine (Domingo et a l , 1991b), which the authors suggested was ev idence for the unsuitability of vanad ium in the treatment of STZ-d iabe tes . Interestingly, p lasma g lucose was reduced to control levels by vanadyl treatment only in the former study whereas hyperg lycemia persisted in diabetic rats treated with vanadyl in the latter study, thus suggest ing that the diabet ic state itself might be a deciding factor. Moreover, there were no comparat ive data avai lable for vanadyl-treated nondiabetic controls. In the present study, no apparent s igns of vanad ium toxicity occurred in the naglivan-treated animals and there were no deaths in any naglivan-treated group. 83 Administrat ion of V O S 0 4 in the drinking water has been shown to result in dec reased weight gain in control animals (Ramanadham et a l , 1989, A l -Bayat i et a l , 1990). However, in the present study, mean body weight in control an imals w a s not affected by nagl ivan treatment over the 8-week period. In addit ion, unlike treatment with V O S 0 4 which results in dec reased food and fluid intake in controls, nagl ivan treatment for the most part did not affect these parameters in the control group. It is plausible that dec reased food intake leads to reduced body weight in vanadium-treated control rats and that the absence of reduction in both parameters may have resulted from the significantly lower dose of vanadium used in this study. Addit ional ly, whi le reduced circulating insulin levels have been reported for control an imals on oral VOSO4 (Heyl iger et a l , 1985; R a m a n a d h a m et a l , 1989a), the dose of vanad ium used in this study appeared to be insufficient to significantly lower p lasma insulin levels in control an imals . Interestingly, unlike previous studies which showed that vanad ium treatment did not improve p lasma insulin levels in diabetic rats, p lasma insulin levels in both the DI and DVI groups were significantly greater than D at 8 weeks , indicating that the removal of chronic hyperglycemia during the 3-week period of insulin treatment may have prevented the addit ional loss of beta cel l insulin store and funct ion. Insulin treatment of obese patients with N IDDM for one month w a s demonstrated to improve insulin secret ion, which persisted for at least 2 weeks after insulin w a s withdrawn (Andrew et a l , 1984). T h e s e results a lso further corroborate the f inding in which normalizat ion of p lasma g lucose levels in a subset of animals with chronic (5-month) VOSO4 treatment led to a limited protection of pancreat ic insulin secretory function in STZ-d iabe t i c animals. In this study, it appears that short-term treatment with insul in or vanad ium with low dose insulin to normal ize g lycemia at least during the early s tages of STZ-d iabe tes can result in significantly improved insulin levels, perhaps resulting from the preservat ion of insulin stores which is apparent even after a prolonged period (5 weeks) of severe hyperglycemia following cessat ion of insulin treatment. 84 Chron ic STZ-d iabe tes has been shown to result in a cardiomyopathy, which is evident by 6 weeks after the induction of d iabetes and reversed by chronic insulin treatment (Tahil iani et al , 1982). Improved heart function has been demonstrated in STZ-d iabet ic animals treated with N a 3 V 0 4 (Heyl iger et a l , 1985) or V O S 0 4 ( R a m a n a d h a m e t a l , 1989a), independent of changes in circulating insulin levels. In the present study, although p lasma insulin levels were significantly higher in the DVI group relative to D, cardiomyopathy was absent only in those animals which had maintained eug lycemia in response to chronic vanadium treatment. B e c a u s e other parameters, including circulating insulin levels, free fatty ac ids and triglyceride levels were similar in the DVI-H and DVI -E groups, it appears that the removal of metabol ic defects produced by hyperglycemia per se may have alleviated the d iabetes- induced card iac dysfunct ion. S i n c e the short-term removal of hyperglycemia a lone in DI and DVI -H appeared to be insufficient to prevent the onset of card iac dysfunction in the diabetic rats, it appears that the maintenance of normal cardiac function may be more critically dependent on the long-term control of normoglycemia attained with cont inued vanad ium treatment. In summary, the antidiabetic effect iveness of an organic derivative of vanady l has been establ ished. A s in the case of V O S 0 4 , the dose of insulin required for maintaining euglycemia was reduced, abnormal p lasma lipid levels were corrected, and card iac function was preserved in animals which had responded to treatment with nagl ivan. In addit ion, the positive effects of nagl ivan treatment were not l inked to improved p lasma insulin levels per se . This study also demonstrates the ef fect iveness of vanad ium using a once-dai ly dosing regimen, in compar ison to the sel f -administered route v ia the drinking water. Importantly, the reduced oral dose of vanad ium required for a g lucose- lower ing effect coupled with the absence of diarrhea or any s igns of liver or kidney toxicity in the naglivan-treated animals suggests that nagl ivan may be a more therapeutical ly desirable form of vanadyl . 85 Chapter 4 CONCENTRATION-DEPENDENT G L U C O S E - L O W E R I N G E F F E C T S OF O R A L V A N A D Y L A R E MAINTAINED FOLLOWING TREATMENT WITHDRAWAL IN STZ -DIABETIC RATS 4.1. INTRODUCTION Since treatment with 0.75 mg/ml V O S 0 4 in the drinking water does not a lways produce euglycemia in all of the treated animals, it w a s postulated that the relative eff icacy of vanadium treatment may depend in part on a critical level of circulating insulin and preservat ion of a limited number of functional f i ce l ls . Th is is because rats made diabetic with 55 mg/kg S T Z , although hypoinsul inemic, still have measurab le amounts of circulating insulin (Junod et a l , 1967). In addit ion, as the d iabetogenic action of S T Z is markedly inf luenced by severa l factors such as age , sex and metabol ic status, this could result in diabetic rats with var ious degrees of hypoinsul inemia and hyperglycemia (Dhal la et a l , 1985). Hence , the apparent lack of a g lucose- lower ing response to vanadium in some diabetic.rats may have been due to a more severe diabetic state and lower levels of circulating insulin, which could be enhanced or complemented by increasing vanadium intake. The importance of endogenous insulin is c lear from experiments with spontaneous ly diabetic B ioBreed ing (BB) Wistar rats which have no measurab le circulating insulin (Marl iss et a l , 1983). Treatment of B B rats with vanadyl diminishes the requirement for insulin, but d o e s not replace insulin therapy altogether (Battell et a l , 1992). O n e distinctive finding is that normal p lasma g lucose, and card iac and ad ipose t issue function are maintained in STZ-d iabet ic rats up to 13 w e e k s after vanad ium treatment is withdrawn (Ramanadham et a l , 1989). However , treatment w a s started 3 days after S T Z injection, a point at which it could be argued that its ft-cytotoxic effects may not have yet significantly reduced islet insulin content. Th is would al low vanad ium to exert early protective effects on the insul in-producing cel ls, which could 86 subsequent ly produce enough insulin to bring about a quasi -permanent eug lycemic state after treatment is s topped. Indeed, in an imals in which eug lycemia w a s maintained, the area occupied by insulin-staining f i-cells w a s increased 8-fold when compared to non-treated diabetic rats (Pederson et a l , 1989), which suggests that vanad ium treatment may have prevented the cont inued destruction of f i -cel ls by hyperglycemia. Hence , the purpose of the present study w a s threefold: 1) to examine the ex is tence of an in vivo dose-dependent effect of vanady l , 2) to examine if persistent eug lycemia after termination of vanadium treatment could be observed when treatment of diabetic rats was delayed up to 17 days after the S T Z injection, and 3) to extend the observat ion of post-withdrawal euglycemia to a longer period. 4.2. MATERIALS AND METHODS 4.2.1. Treatment and maintenance of animals. Male Wistar rats were injected with a s ingle dose of S T Z (55 mg/kg) in the tail ve in. Three days after the S T Z injection, diabetic rats (p lasma g lucose > 13 mM) were divided into 3 groups according to the initiation of vanad ium treatment: at 3 (DT3, n=8), 10 (DT10, n=6), and 17 (DT17, n=5) days after d iabetes was induced. The initial concentrat ion of vanad ium ( V O S 0 4 - 3 H 2 0 ) suppl ied in the drinking water w a s 0.75 mg/ml and was increased by 0.25 mg/ml increments on a weekly bas is in those an imals which did not ach ieve euglycemia (p lasma g lucose > 9.0 mM) until 1.50 mg/ml. A control group (CT, n=3) was administered 1.00 mg/ml V O S 0 4 . At 10 w e e k s of treatment, vanad ium was withdrawn and p lasma obtained for cholesterol , tr iglyceride and vanad ium analys is . After withdrawal, the animals were monitored at regular intervals for 5 months. P l a s m a samples were periodically col lected at 10.00 h. A separate group of animals were made diabetic with S T Z (55 mg/kg) and treated with nagl ivan from 4 days after S T Z . Nagl ivan (10 mg/ml in 3 % acac ia ) w a s administered at a dose of 50 mg/kg/day by oral gavage for 5 weeks at which time the 87 dose w a s increased to 100 mg/kg/day in those animals which remained hyperglycemic. After an addit ional 3 weeks , the dose was raised to 150 mg/kg/day in non-euglycemic animals. B e c a u s e of the limited supply of nagl ivan, the dose w a s not further increased in those rats which remained hyperglycemic. At 10 weeks , nagl ivan treatment w a s withdrawn and animals were monitored up to 30 weeks after the withdrawal date. 4.2.2. Analyt ical Methods O G T T s were performed prior to treatment withdrawal, and at 3 and 20 w e e k s after withdrawal from vanad ium treatment. Fo r nagl ivan-treated rats, the O G T T w a s done at 30 weeks after withdrawal. After an overnight fast, and prior to administer ing g lucose, rats were anaesthet ized with sodium pentobarbital (20 mg/kg, i.p.), which w a s found not to affect the kinetics of g lucose and insulin re lease in response to oral g lucose. A r e a s under the curve were calculated for g lucose ( A U C g ) and insulin (AUCj ) responses over 120 minutes via the trapezoidal method, using the Scient i f ic F ig . P rocesso r (Fig.P® Software Corporat ion, B I O S O F T , Durham, N C ) . Multiple regress ion analys is was performed using the least squares regression method via the Number Cruncher Statist ical Sys tem (NCSS®, Kaysvi l le, Utah). At var ious t imes, the following parameters were measured : p lasma vanad ium, g lucose, cholesterol and triglycerides, urea nitrogen and glutamic oxaloacet ic t ransaminase (GOT) . P l a s m a insulin was measured via RIA with rat insulin standards (Novo). The RIA method al lows for measurement of smal l vo lumes of 25 pi with an inter- and intra-assay C V of < 1 5 % and is sensit ive to 7 uU/ml (see Append ix 1). 4.2.3. Statist ical Ana lys is Data are expressed as mean ± S E M . Statist ical s igni f icance w a s evaluated by one-way analys is of var iance ( A N O V A ) fol lowed by Newman-Keu ls , or by Student 's paired t-test, where appropriate, p < 0.05 was considered significant. 88 4.3. R E S U L T S 4.3.1. Var ious parameters of diabetic animals treated with vanadyl sulfate. After a 10-week treatment per iod, vanadyl sulfate at concentrat ions of 0.75-1.50 mg/ml in the drinking water resulted in normoglycemia (nonfasted p lasma g lucose < 9.0 m M ; Tab le 11) in all diabetic animals. A s there w a s no signif icant di f ference in severa l parameters according to the time of initiation of treatment, the data were pooled and ana lyzed retrospectively according to the vanadyl concentrat ion at wh ich p l asma g lucose was normal ized. This resulted in four concentrat ion groups (in mg/ml): [0.75] (n=4), [1.00] (n=9), [1.25] (n=3), [1.50] (n=3). Delaying the start of treatment to 17 days after the S T Z injection did not significantly affect the dose of vanadyl required to ach ieve eug lycemia (Table 11). T h e initial vanadyl dose (loading dose during the first week of the highest concentrat ion administered) w a s equivalent among concentrat ion groups, amount ing to - 1 4 0 - 1 5 0 mg/kg/day or -0 .65 -0 .7 mmol/kg/day (Fig. 4.1). Over severa l weeks , there w a s a reduction in the calculated dose of vanadyl , mostly accounted for by a n increase in body weight. The dose curve for C T (administered 1.00 mg/ml) w a s similar to that for diabet ic rats treated at the s a m e concentrat ion, [1.00] (data not shown). A t week 10, there w a s a concentrat ion-dependent increase in the vanadyl dose : [0.75] < [1.00] < [1.25] » [1.50]. Food and fluid intake at 10 weeks w a s signif icantly lower only in the [1.50] group when compared to C T (Table 12). P l a s m a cholesterol and triglyceride levels were not different from control in most concentrat ion groups, with the except ion of [1.50], in which p lasma cholesterol levels remained e levated. No correlat ions were observed between the final V O S 0 4 concentrat ion administered and body weight ga in , or with p lasma vanad ium levels, which were in the range of 0.26-1.05 ug/ml (5.1 - 20.6 uM) (F ig. 4 .2A) . Al though not statistically significant, it appeared that lowered fed p lasma insulin levels were assoc ia ted with higher p lasma vanad ium levels fol lowing 10 weeks of treatment (r = -0.40, p = 0.06, F ig . 4 .2B) . 89 Table 11. Number of euglycemic animals at the end of 10-week treatment period according to vanadyl concentration in the drinking water C o n e (mg/ml): [0.75] [1.00] [1.25] , n-so] DT3 (n = 8) 2 5 1 D T 1 0 ( n = 6) 1 1 1 3 D T 1 7 ( n = 5) 1 3 1 Total (N = 19) 4 9 3 3 90 160 -i 0 2 4 6 8 10 Weeks Figure 4.1. Calculated vanadium dose in STZ-diabetic rats on var ious V O S 0 4 concentrations Calcu la ted daily dose of vanadyl (mg/kg/day and mmol/kg/day) in diabet ic an imals normal ized with var ious concentrat ions of V O S 0 4 in the drinking water for 10 w e e k s (mg/ml): • [0.75], • [1.00], O [1.25], • [1.50]. * p < 0.05 vs . [1.50]. 91 Table 12. Var ious characterist ics of animals at the end of a 10-week treatment period according to vanadyl concentration in the drinking water Control Diabetic V O S 0 4 [1.00] [ 0.75] [1.00] [1.25] [1.50] (mg/ml) (n=3) (n=4) (n=9) (n=3) (n=3) Body Weight 151 173 168 136 149 Ga in (g) (12) (22) (16) (18) (22) Food Intake 26 25 24 24 22* (g/day) (1) (1) (1) (1) (2) Fluid Intake 31 31 32 28 24* (ml/day) (2) (3) (2) (3) (2) G l u c o s e 5.7 7.4* 8.2* 8.6* 7.5* (mM) (0.1) (0.2) (0.3) (0.1) (0.4) Insulin 28 27 24 27 21 (ul l /ml) (10) (3) (2) (4) (4) Cholestero l 1.5 1.4 1.5 1.5 1.8* (mM) (0.1) (0.1) (0.1) (0.1) (0.1) Tr iglycer ides 1.3 1.4 1.3 1.4 1.1 (mM) (0.2) (0.4) (0.2) (0.1) (0.1) Data is mean (± S E M ) * p < 0.05 vs . Control 92 E co c e 20 A A 15 10 5 H • • 1 1 1 1 [0.75] [1.00] [1.25] [1.50] VOS04 Concentration (mg/ml) 40 B 3 (Q E to 30 H 20 H 10 0 r = -0.40 1 5 10 15 20 Plasma Vanadium 1/JM) Figure 4.2. P lasma vanadium levels of various diabetic groups at 10 weeks (A) P l a s m a vanad ium levels at 10 weeks of treatment in D T animals accord ing to the concentrat ion of V O S 0 4 in the drinking water. (B) Correlat ion plot between p lasma insulin and p lasma vanadium levels at 10 weeks of treatment (r = -0.40, p = 0.06), accord ing to V O S 0 4 concentrat ion at which eug lycemia was ach ieved (mg/ml): • [0.75], • [1.00], O [1.25], • [1.50]. 93 4.3.2. P lasma g lucose and insulin profile before and after 10 week treatment. A range in the severity of d iabetes induced by 55 mg/kg S T Z w a s reflected in a gradient of p lasma insulin and g lucose levels prior to vanad ium treatment, which w a s negatively correlated (r = -0.70, p < 0.001) (Fig. 4 .3A,B) . There appeared to be a normal distribution in the degree of d iabetes among the DT groups. However , when retrospectively examined, the more severely diabetic an imals (more pronounced hyperglycemia and hypoinsul inemia prior to treatment) appeared to require higher concentrat ions of vanadyl to ach ieve euglycemia (Fig. 4 .4A,B) . Thus , when diabet ic an imals were grouped accord ing to the vanady l concentrat ion at which g lucose levels were normal ized, i.e. low-concentrat ion (0.75-1.0 mg/ml, L C ) and high-concentrat ion (1.25-1.50 mg/ml, H C ) responders, it was found that H C had significantly higher p lasma g lucose (HC : 19.6 ± 0.8 vs . L C : 16.4 ± 0.5 m M , F ig. 4 .5A) and correspondingly, lower p lasma insulin levels (HC: 25.6 ± 3.3 vs . L C : 36.0 ± 2.2 uU/ml , F ig . 4 .5B) relative to L C prior to vanad ium treatment. However, after the 10-week treatment period, there were no significant di f ferences in either p lasma g lucose and insulin levels between L C and H C . W h e r e a s chronic vanadium treatment reduced p lasma insulin in L C , no further reduction was observed in the H C group. 4.3.3. P lasma glucose and insulin after withdrawal from vanadium treatment At 3 weeks after withdrawal from vanadyl , there were no di f ferences in either mean p lasma g lucose or insulin levels in either L C or H C groups compared to the t ime prior to withdrawal. M e a n p lasma g lucose levels in [0.75] and [1.00] were not different from C T up to 5 months after treatment (Fig. 4 .5A, 4 .6A) . However , a return to hyperglycemia was observed in the H C groups by the first ([1.50]) and fifth ([1.25]) months. Thus , at 20 weeks after withdrawal from treatment, mean g lycemia in the H C group had returned to pre-treatment levels. After treatment withdrawal, there w a s a gradual rise in p lasma insulin in C T , which reached - 1 0 0 uU/ml by 20 w e e k s 94 Plasma Insulin (fjU/ml) Plasma Insulin (fjU/ml) Figure 4.3. Variance in severity of diabetic state induced by 55 mg/kg STZ Correlat ion between p lasma g lucose and insulin va lues before treatment with V O S 0 4 (r = -0.70, p = 0.01). (A) Accord ing to when treatment was started: at 3 (DT3, O ) , 10 (DT10, • ) , and 17 (DT17, • ) . days after the S T Z injection. (B) Accord ing to the concentrat ions of V O S 0 4 in the drinking water at which eug lycemia w a s ach ieved (mg/ml): • [0.75], • [1.00], O [1.25], • [1.50]. 95 2 5 ~i I I Before Treatment After Treatment 5 E o o s E <o 2 0 1 5 1 0 0.75 1.00 1.25 1.50 E s: 0 . 7 5 1.00 1.25 1.50 (n=4) ln=11) (n=3) (n=4) V0S04 Concentration (mg/ml) Figure 4.4. Effect of severity of the diabetic state, a s s e s s e d by pretreatment p lasma g lucose and insul in levels, on subsequent vanadyl requirement P l a s m a g lucose (A) and insulin (B) va lues before and after vanad ium treatment accord ing to the concentrat ions of V O S 0 4 (mg/ml) in the drinking water at which eug lycemia w a s ach ieved. 96 5 CD vt O O 3 E 5: • LC (n=13) MM HC (n=6) Before After 3 wks 20 wks Vanadyl Treatment After Withdrawal 60 ^ 50 H =3. 3 . C 6 vt TO 4 0 H 30 20 H 10 0 Before After 3 wks 20 wks Vanadyl Treatment After Withdrawal Figure 4.5. P lasma glucose and insulin profile before and after vanadyl treatment P l a s m a g lucose (A) and insulin (B) levels at various t imes: before and after a 10-week period of treatment with vanadyl and at 3 and 20 weeks after withdrawal from treatment in diabetic rats normal ized with low (LC, [0.75] to [1.00]) and high (HC , [1.25] to [1.50]) concentrat ions (mg/ml) of V O S 0 4 in the drinking water. A p < 0 .05 vs . before treatment, * p < 0.05 vs . L C . 97 CD «o O O 5 e s; 25 20 H 15 10 -5 -- f — i 1 1 1 r WD 1 2 3 4 5 =3. 3 E 5: 120 100 80 60 40 20 0 B 1 1 1 1 r-WD ^ 2 3 4 5 Months Figure 4.6. P lasma glucose and insulin levels in control and diabetic rats over 5 months fol lowing withdrawal from vanadyl treatment P l a s m a g lucose (A) and insulin (B) levels in control (CT, A ) and diabet ic rats for 5 months after withdrawal (WD) from treatment according to the concentrat ion of V O S 0 4 (mg/ml): ( • [0.75], • [1.00], O [1.25], • [1.50]) in the drinking water at which eug lycemia w a s ach ieved. * p < 0.05 vs . all. 98 (Fig. 4 .5B , 4 .6B) . Alternately in L C , there was a more moderate rise in p lasma insulin levels, reaching twice that of initial pre-withdrawal levels (54.1 ± 4.9 vs . 24.3 ± 1.4 uU/ml) by 20 weeks while in H C , this was approximately half the level in L C and w a s not significantly different from pre-withdrawal levels (27.5 ± 5.6 vs . 25.6 ± 3.4 uU/ml). At 5 months after withdrawal from treatment, severa l parameters were e levated in the [1.50] group relative to C T , assoc ia ted with severe hyperg lycemia in this group relative to all other groups at this time. Thus , p lasma cholesterol (3.8 ± 0.5 vs . C T : 2.3 ± 0.2 mM) and triglycerides (3.1 ± 0.1 vs . C T : 1.4 ± 0.3 mM) were signif icantly higher only in [1.50] (p < 0.05). Similarly, p lasma urea nitrogen w a s higher in [1.50] relative to C T (11.5 ± 1.6 vs. C T : 6.7 ± 0.1 m M , p < 0.05). However , p lasma G O T w a s not different among the various groups at 20 weeks after withdrawal from vanady l . 4.3.4. P lasma g lucose and insul in after withdrawal from treatment in [1.50]. After withdrawal from vanadium treatment, six of 19 diabetic an imals reverted to the hyperglycemic state. Notably, this group included all of the [1.50] group and two of three from the [1.25] group. The individual results after withdrawal from treatment in the [1.50] group are shown in Figure 4.7. In these animals, p lasma insulin levels appeared to influence coexist ing g lucose levels. For instance, in Figure 4 .7A, where in hyperglycemia recurred within the first week, there w a s no apprec iab le change in p lasma insulin va lues from pre-withdrawal levels. In Figure 4 .7B , an 8-week period of normoglycemia was observed, during which time circulating insulin w a s stable for the first 5 w e e k s and subsequent ly increased two-fold over 3 weeks . After 8 w e e k s , a reduction in insulin accompan ied a rapid increase in g lucose, whereas a further decl ine in insulin levels over 10 weeks reflected steady development of hyperg lycemia. In Figure 4 . 7 C , normal g lucose was maintained for 8 weeks , accompan ied by a marked three-fold increase in insulin. However, concurrent with the return to hyperg lycemia, p lasma insulin dec reased gradually over 10 weeks to prewithdrawal levels. 99 30 - i 25 -E: » 20 -Vi O 5 15 -e 10 -Q_ O 5 -0 -9-Q. a • o-e ./ 0 t WD 5 10 15 Weeks Post-Withdrawal r 80 60 E 3i h 40 20 m S a. Figure 4.7. Assoc iat ion between insul in and maintenance of normoglycemia in rats reverting to hyperglycemia after withdrawal from vanadium treatment P l a s m a g lucose and insulin levels over 5 months following withdrawal (WD) from vanadyl treatment in 3 diabetic rats (A-C) normal ized on [ 1.50] mg/ml V O S 0 4 . 100 4.3.5. OGTTs at 10 weeks of treatment and after treatment withdrawal. To investigate the sustained effects of vanadium on g lucose to lerance after withdrawal from treatment, O G T T s were conducted at 10 weeks of treatment, prior to withdrawal ( O G T T 0), and at 3 weeks (short-term, O G T T 1) and 20 weeks (long-term, O G T T 2) after vanadium treatment was terminated. For these exper iments, untreated age- and weight-matched control rats (C, n=4) were added for compar ison . After 10-week treatment (Fig. 4 .8A,D) , C T rats had lower insulin response than C without al tered g lucose to lerance, as demonstrated previously. G l u c o s e response in the L C and H C groups were abnormal but only significantly different from control at 60 min (p < 0.05). Fast ing and peak insulin levels were significantly lower in L C and H C than in C T . At the 3 week-withdrawal period ( O G T T 1), fasting g lucose and insulin in L C and H C did not differ significantly from C T (Fig. 4 .8B ,E) . G l u c o s e response in L C and H C w a s still abnormal but not significantly higher than C T until 60-120 minutes. P l a s m a insulin peaks of the diabetic groups were approximately 5 0 % (LC) and 2 5 % (HC) of C T , which itself was - 6 0 % of C . P e a k insulin was significantly higher in L C at O G T T 1 relative to O G T T 0 ( 1 : 40.4 ± 2.3 vs . 0: 30.8 ± 3.7 uU/ml), but was not different in H C (1: 19.7 ± 2.3 vs . 0: 22.0 ± 1.8 uU/ml). Al though g lucose to lerance between L C and H C was similar in O G T T 1, peak insulin in H C was significantly lower than L C . At 20 weeks after withdrawal ( O G T T 2), g lucose to lerance of L C w a s not significantly different from C T at any time point, although insulin response w a s still very much subdued relative to C (Fig. 4 .8C ,F ) . Alternately, H C appeared to be markedly glucose-intolerant, with no significant insulin response. Both basa l and g lucose-stimulated insulin response in C T was greater than O G T T 1. Similarly, peak insulin in L C was significantly higher at O G T T 2 than during O G T T 1, al though there w a s no difference in peak insulin in H C . W h e n the H C group was examined more c losely, g lucose to lerance was more impaired in the [1.50] than the [1.25] group, a l though the insulin responses were not significantly different between the two groups. 101 Plasma Glucose (mM) 20 15 OGTT 0 10 OGTT 1 OGTT 2 ~ 1 — l — i — i — i — i — l — 0 10 20 30 40 50 60 120 20 15 H 10 5 H B - i — i — i — i — i — i — i — 0 10 20 30 40 50 60 120 20 15 10 5 H "1—I—I—i—I—I—I— 0 10 20 30 40 50 60 Time (min) 120 Plasma Insulin (fjU/ml) - i — i — i — i — i — i — r 0 10 20 30 40 50 60 120 T — i — I — I — r 0 10 20 30 40 50 60 140 120 100 80 H 60 40 20 H 0 1—i—i—I—I—I—I ll 1 0 10 20 30 40 50 60 120 Time (min) Figure 4.8. OGTTs prior to and at 3 and 20 weeks after treatment withdrawal P l a s m a g lucose (A-C) and insulin (D-F) response to a 1 g/kg oral g lucose d o s e prior to ( O G T T 0, A,D), and at 3 ( O G T T 1, B ,E) and 20 weeks ( O G T T 2, C,F) after withdrawal from treatment in control (C, O ) , control-treated (CT, • ) , and diabet ic rats normal ized on low (LC , • ) and high (HC, • ) V O S 0 4 concentrat ions (* p < 0.05 vs . CT ) . 102 4.3.6. Integrated g lucose and insul in responses during OGTT 0-2. The a reas under the curve for g lucose ( A U C g ) and insulin (AUCj ) levels over 120 minutes during the ser ies of oral g lucose tolerance tests were calculated as an index of g lucose tolerance, and insulin secretory response. A t 10 w e e k s of treatment and prior to withdrawal from vanadyl ( O G T T 0), the A U C g levels in the L C and H C groups were significantly greater than in C T , but not different from one another, a pattern which w a s maintained at 3 weeks post-withdrawal ( O G T T 1) (Fig. 4 .9A) . After long-term (20 weeks) withdrawal from vanady l ( O G T T 2), integrated g lucose response in H C w a s significantly greater than C T and L C . At this t ime, A U C g of the L C group, albeit significantly different from C T , was not different from O G T T 0. Total insulin response (AUCj) was significantly lower in H C relative to L C at all t ime points (Fig. 4 .9B) . A U C j was not different between O G T T 0 to O G T T 1 in all the groups. However, A U C j in both L C and C T were significantly greater in O G T T 2 relative to O G T T 1, al though A U C j of L C remained significantly lower than C T at all t ime points. A U C j in H C did not differ at all t ime points and remained lower than C T and L C , which at 20 w e e k s w a s coincident with a marked loss of g lucose to lerance. The degree of g lucose tolerance after long-term withdrawal from vanad ium as measured by the A U C g w a s significantly correlated to fed g lucose levels at 20 w e e k s (r = 0.83, p < 0.0001) (Fig. 4.1 OA). A s wel l , insulin response to an oral g lucose load at O G T T 2 correlated significantly with fed insulin levels at 20 w e e k s (r = 0.69, p < 0.05) (Fig. 4 .10B) . To see whether g lucose tolerance was dependent on insulin response after a short and long-term withdrawal, the A U C g va lues were plotted against the A U C j responses during the O G T T s . Whe reas there was no significant correlation between the g lucose and insulin response in all the animals at the 3 week-wi thdrawal period ( O G T T 1, r = -0.17, p = 0.448) (Fig. 4.11 A) , at 20 weeks after withdrawal from treatment, there was a significant negative correlation (r = -0.74, p < 0.0001) between integrated g lucose and insulin response ( O G T T 2, F ig . 4 .11B) . 103 c £ o CM 5 E o "5 2500 H 2000 1500 1000 H 500 H WM OGTT 0 VA OGTT 1 HJ OGTT2 Figure 4 .9 . Integrated g lucose and insul in responses during OGTTs Integrated g lucose ( A U C g ) (A) and insulin (AUCj) (B) responses at 10 w e e k s of vanad ium treatment, prior to withdrawal ( O G T T 0), and at 3 ( O G T T 1) and 20 weeks ( O G T T 2) after withdrawal from vanadium treatment in control (CT) and diabet ic rats normal ized on low (LC , [0.75] to [1.00]) and high (HC , [1.25] to [1.50]) concentrat ions (mg/ml) of V O S 0 4 . + p< 0.05 vs . O G T T 1, * p < 0.05 vs . C T . 104 Plasma Insulin (fjU/ml) Figure 4.10. Relationship between glucose-tolerance and fed p lasma g lucose , and between insul in secretory function and fed plasma insul in levels at 20 weeks Correlat ion plots of integrated g lucose response ( A U C g ) in O G T T 2 vs . fed p lasma g lucose va lues (A, r = 0.83, p < 0.0001) and integrated insulin response (AUCj ) in O G T T 2 vs . fed p lasma insulin va lues (B, r = 0.69, p < 0.05) in control (CT , A ) and diabet ic rats according to V O S 0 4 concentrat ion ( • [0.75], O [1.00], • [1.25], • [1.50]). 105 c E o CM 5 E O 3000 -i 2500 H 2000 1500 H 1000 H 500 H r=-0.17 1 1 7500 9000 —I 1 1500 3000 4500 6000 AUC, (fjU/ml • 120 min) c E o 5 E O 3000 2500 2000 1500 H 1000 H 500 H B r=-0.74 —i 1 1 1 1 1 1500 3000 4500 6000 7500 9000 AUC, (fjU/ml • 120 min) Figure 4.11. Relationship between glucose-tolerance level and insul in secretory response during OGTT at 3 and 20 weeks after withdrawal from treatment Correlat ion plots of integrated g lucose ( A U C g ) vs . insulin (AUCj) response during the O G T T s at 3 (A, O G T T 1, r = -0.17, p = 0.448) and 20 (B, O G T T 2, r = -0.74, p < 0.0001) weeks after withdrawal from vanadium treatment in control (CT, A) and diabet ic rats accord ing to V O S 0 4 concentrat ion ( • [0.75], O [1.00], • [1.25], • [1.50]). 106 4.3.7. Glucose- lower ing effects of an organic vanadyl compound, naglivan. The administration of 50 mg/kg/day naglivan (0.06 mmol vanadium/kg/day) resulted in normal g lucose in 3 diabetic animals (Fig. 4 .12A) . After increasing the d o s e to 100 mg/kg/day in the 4 remaining non-euglycemic rats, only 1 deve loped eug lycemia . W h e n the nagl ivan dose was further increased to 150 mg/kg/day in the 3 which remained non-euglycemic, there was a slight lowering of p lasma g lucose, but these rats remained hyperglycemic. The animals were ana lyzed accord ing to response to treatment: euglycemic (responders, DT-R , n=4) and hyperglycemic (non-responders, D T - N R , n=3). After naglivan (50 mg/kg/day) had been administered for 5 days , p lasma insulin va lues were found to be significantly higher in D T - R (Fig. 4 .12B , DT -R : 35.5 ± 0 •1.9 vs . D T - N R : 24.2^1 3.6 uU/ml) at a time point where p lasma g lucose levels were reduced but had not yet reached control levels (DT-R: 16.7 ± 2.2 vs . D T - N R : 25.1 ± 1.7 mM). P l a s m a insulin in D T - R was significantly reduced by nagl ivan to levels which were not different from D T - N R at 10 weeks (25.0 ± 3.6 uuVml), whereas no further reduction was seen in D T - N R which had received the highest dose of 150 mg/kg/day. After treatment was withdrawn, p lasma insulin levels in D T - R rebounded and remained significantly higher than D T - N R , which showed no change in insulin levels. The an imals which responded to a dose of 50 mg/kg/day maintained stable eug lycemia , whi le one rat which had required 100 mg/kg/day showed a temporary reversion to mild hyperglycemia, which returned to normal at 30 weeks . The moderate reduction in g lucose in D T - N R was reversed to pre-treatment levels after treatment w a s withdrawn. At 30 weeks after withdrawal from nagl ivan treatment, D T - R had normal g lucose tolerance, as ev idenced by the integrated g lucose response which was not signif icantly higher than C T at O G T T 2 (Fig. 4 .13A). Paral lel to what w a s previously s e e n with the L C and H C groups at O G T T 2, peak and total p lasma insulin re lease in D T - R w a s significantly higher than D T - N R (Fig. 4 .13B) . The insulin responses of D T - R and DT-N R were similar to L C and H C at O G T T 2, respectively. 107 Figure 4.12. Effects of chronic naglivan treatment in STZ-diabetic rats P l a s m a g lucose (A) and insulin (B) levels in diabetic rats administered increasing d o s e s of nagl ivan: 50 ( • ) , 100 (A), and 150 (• ) mg/kg/day by gavage from 4 days after the S T Z injection. Treatment was withdrawn after 10 weeks and animals were monitored for addit ional 30 weeks . 108 «3 E M a . 60 40 20 H B 0 i — i — i — i — i — i — i — 0 10 20 30 40 50 60 120 DT-R DT-NR ln=3) Time (min) Figure 4.13. OGTT in naglivan-treated rats at 30 weeks after treatment withdrawal P l a s m a g lucose (A) and insulin (B) levels in fasted rats in response to a 1 g/kg oral g lucose dose at 30 weeks after withdrawal from nagl ivan treatment. Diabet ic rats were pooled and classi f ied as responders (DT-R, • ) and non-responders ( D T - N R , • ) . Insets show corresponding integrated g lucose ( A U C g ) and insulin (AUCj ) response . Dotted line represents C T va lues from O G T T 2. * p < 0.05 vs . DT -R . 109 4.4. DISCUSSION From the correlative levels of p lasma g lucose and insulin in this study, it appears that 55 mg/kg S T Z produced animals with a range of severity in the diabet ic state. The significantly lower insulin and higher g lucose levels in H C relative to L C suggest that the residual circulating insulin determined the relative requirement for vanad ium. B e c a u s e the comparat ively lower insulin levels in the H C animals was apparent ly offset by addit ional vanadium intake, it appears that vanad ium could act in a complementary or synergist ic manner to the attenuated levels of endogenous insulin in the diabetic animals. Similarly, in naglivan-treated animals, p lasma insulin was found to be significantly higher in D T - R (responders) relative to D T - N R . Interestingly, initial insulin levels in the L C and D T - R groups were not significantly different ( LC : 36.0 ± 2.2 vs . DT-R : 35.5 ± 1.9 uU/ml) whereas H C and D T - N R also showed similar va lues : ( H C : 25.6 ± 3.3 vs . D T - N R : 24.2 ± 3.6 uU/ml). Hence it can be postulated that even higher d o s e s of nagl ivan could have resulted in euglycemia in the D T - N R animals . Seve ra l s tudies have reported the vanadium- induced reduction in p lasma insul in in control an imals , support ing the notion of an enhanced t issue sensitivity to endogenous insulin (Heyl iger et a l , 1985; R a m a n a d h a m et a l , 1989; Pugazhenth i and Khande lwa l , 1990). In this study, treatment of L C animals resulted in a drop in p lasma insulin to levels which were not different from H C at 10 weeks , suggest ing a similar reduct ion in insulin demand in the long-term. Success fu l lowering of p lasma g lucose in the nagl ivan-treated D T - R rats was also accompan ied by a downward trend in p lasma insulin wh ich was not different from D T - N R by 10 weeks . There appeared to be a min imum requirement for a basa l level of p lasma insulin (~ 20-25 uU/ml) in the fed state, as there w a s no further lowering in insulin in rats treated with higher doses , and vanad ium treatment rarely reduced p lasma insulin beyond this level. The trend towards higher p lasma vanad ium with lower p lasma insulin at 10 weeks of treatment sugges ts that vanad ium may have supplemented the remaining avai lable circulating insul in. 110 Al though the phenomenon of post-withdrawal eug lycemia w a s reported in animals which had begun treatment at 3 days after the S T Z injection and for 13 w e e k s after withdrawal from vanadium treatment (Ramanadham et a l , 1989), the occur rence of prolonged eug lycemia was presently observed in diabetic an imals which had started treatment as late as 17 days after the S T Z injection, and has been extended to 20-30 weeks after withdrawal from treatment. The state of maintained eug lycemia in diabet ic an imals after withdrawal from vanadium treatment was accompan ied by a cont inuous increase in p lasma insulin levels over 20 weeks . This phenomenon occurred primarily in the L C group which had significantly more insulin prior to treatment, and sugges ted that the remaining functional fi cel ls were sufficient to respond to a higher insulin demand , when vanad ium was no longer being consumed . In C T , there w a s a dramat ic rise in circulating insulin, reaching 100 uU/ml at 20 weeks . It could be argued that the higher insulin level in C T is attributed in part to increased insulin res is tance, which has been reported in rats with increasing a g e and weight (Reaven and R e a v e n , 1981). However, the age and weight of C T were not different from L C at this t ime. Similar ly, p lasma insulin levels in naglivan-treated animals were higher by the first week after withdrawal from treatment. Overal l , circulating insulin appeared to inf luence g lucose levels after vanad ium treatment was withdrawn. The pers is tence of hyperg lycemia in the naglivan-treated D T - N R animals was assoc ia ted with hypoinsul inemia, which w a s unimproved after discontinuation of treatment. In addit ion, the observed transitory period of normoglycemia after treatment withdrawal was accompan ied by a gradual increase in p lasma insulin to 50-80 |aU/ml, which consistently decl ined to pre-withdrawal levels concurrent with the return to hyperglycemia. This reversal occurred primarily in the H C group, which had been notably more insul inopenic than L C prior to treatment. Thus , the apparent inability of the pancreas to re lease greater amounts of insulin in spite of a larger insulin demand is consistent with the notion of a gradual exhaust ion of the limited insulin stores in H C (Welsh and Hellerstrom, 1990). 111 The degree of g lucose tolerance and insulin response during an O G T T reflected the prevail ing status of the animals during the fed state. Thus at 3 weeks , when fed g lucose levels were normal in virtually all the diabetic groups, only a slight impairment in g lucose tolerance was observed. However, in accordance with g lycemic status at 20 weeks , a greatly impaired g lucose tolerance was observed in rats which had reverted to hyperglycemia, and was most distinct in the [1.50] group. Similar ly, insulin responses in the various groups correlated with fed insulin va lues. Hence , coincident with the elevated levels of circulating insulin in C T and L C at 20 weeks , insulin response in C T and L C was increased to 3- and 2-fold H C , respect ively. The insulin response was not significantly altered in H C at any t ime point from O G T T 0. The degree of g lucose tolerance was observed to be independent of insulin response at 3 weeks after withdrawal from vanadium (i.e. there was no correlat ion between A U C g and A U C j ) . Notably, g lucose tolerance was similar in H C and L C at 3 weeks despi te the comparat ively d iminished insulin response in H C relative to L C , which sugges ts pers is tence of an even more enhanced insulin sensitivity in H C at 3 w e e k s after treatment was c e a s e d . It is possib le that vanad ium is still avai lable from severa l storage sites at a relatively short (3 weeks) period of withdrawal (Parker and S h a r m a , 1978), al though it is undetectable in p lasma (Dai et a l , 1994). At 20 w e e k s after withdrawal, the degree of g lucose intolerance was proportionate to the concurrent insulin response to oral g lucose, suggest ing that the insulin re leased determined to a large extent the degree of g lucose tolerance at this time. However, eug lycemia w a s still maintained in the L C group despite insulin levels which remained at 5 0 % of C T in the fed state and at peak response to g lucose. Thus , al though it appears that the prolonged state of increased insulin sensitivity as a result of vanad ium treatment which was evident at 3 weeks after withdrawal from treatment was still present to s o m e extent at 20 weeks , at this prolonged time period, an improved insulin secretory capaci ty may have played a more important role in the maintenance of eug lycemia . 112 In conc lus ion, it appeared that in a hypoinsul inemic state, effects Of residual insulin can be augmented by increasing doses of vanad ium, which may act in a complementary or synergist ic manner to endogenous circulating insulin. Prev ious studies have presented ev idence that supports the notion of a mechan ism of vanad ium in increasing t issue sensitivity to exogenous ly administered insulin. For instance, using hyper insul inemic euglycemic c lamps, it was demonstrated that vanad ium enhanced hepatic and musc le insulin sensitivity in STZ-d iabet ic rats (Blondel et a l , 1989). In addit ion, vanad ium administered to control (Chal iss et a l , 1987) and STZ-d iabe t i c ( R a m a n a d h a m et al , 1990) animals increased the hypoglycemic response to exogenous insulin. Moreover , nagl ivan (50 mg/kg/day) reduced the insulin requirement by 5 0 % in untreated STZ-d iabet ic rats. Furthermore, in spontaneous ly diabet ic (BB) rats, vanad ium reduced the exogenous insulin required to maintain a n ag lycosur ic state (Battell et a l , 1992). From the current study, it appears that the relative eff icacy of vanad ium in the whole animal may in fact depend on the p resence of insulin and that dif ferences in the degree of severity of the diabetic state and the relative def ic iency in residual circulating insulin can ultimately determine the respons iveness to vanad ium treatment. With regard to the post-withdrawal effects of vanady l , it appeared that the susta ined normal g lucose homeostas is in the short-term may have resulted from a cont inued enhancement in insulin sensitivity, whereas in the long term, this effect w a s more c losely assoc ia ted with an improved pancreat ic function. It is poss ib le that the long-term elimination of hyperglycemia by vanadium may have resulted in preservat ion of s o m e fi cell function, s ince elevated g lucose levels have been reported to d a m a g e beta cell integrity further (Leahy et a l , 1986; Ziegler et a l , 1989). Indeed, it has been suggested that vanad ium, through a reduction in insulin demand , prevents the exhaust ion of insulin stores (Brichard et a l , 1990). Altogether, the results in this study confirm that improvements in the diabetic state may be extended over a prolonged period after treatment is ended with either vanadyl sulfate or nagl ivan. 113 Chapter 5 PARTIAL PRESERVATION OF PANCREATIC f i -CELLS B Y VANADIUM: EVIDENCE FOR A MECHANISM OF CHRONIC AMELIORATION OF DIABETES 5.1. INTRODUCTION There are two mechan isms proposed for long-term normoglycemia in S T Z -diabetic rats following the cessat ion of vanadium treatment. It is poss ib le that vanad ium can accumulate in various storage sites following chronic treatment and continue to exert its effects at least for a short period after vanad ium treatment is withdrawn. Another mechan ism involves preservation of fi-cell insulin stores by chronic vanad ium treatment, a notion supported by the near-normalizat ion of islet insulin a rea in the diabetic animals after a 13-week withdrawal from vanad ium treatment (Pederson et a l , 1989). A l though a role for fi-cell preservat ion by vanad ium treatment has been sugges ted , there is no ev idence so far to demonstrate a direct link between the degree of f i-cell protection induced by short-term vanad ium treatment and an ability to maintain normal g lucose homeostas is following chronic withdrawal from treatment. Recen t studies have suggested that a reduction in insulin b iosynthesis and secret ion can render f i-cells less suscept ib le to cytotoxic events (Spr ie tsma and Schui tmaker , 1994). Thus , insulin treatment prevented the onset of d iabetes in genetical ly suscept ib le B B rats (Gotf redsen et al , 1985) and N O D mice (Atk inson et a l , 1990). Converse ly , high concentrat ions of g lucose potentiated the d iabetogenic effects of S T Z in vitro (Eizirik et a l , 1988) and in vivo (Sandler and A n d e r s s o n , 1982). It w a s hypothesized that vanadium treatment, by lowering insulin secret ion, could exert a f i-cell protective effect when administered prior to and for a short period fol lowing the induction of STZ-d iabe tes . Thus , the a im of this study was to establ ish whether short-term vanad ium treatment might lead to some degree of ft-cell preservat ion which in turn, could result in the long-term amelioration of the diabetic state. 114 5.2. MATERIALS AND METHODS 5.2.1. Treatment and maintenance of animals Male Wistar rats (160-190 g) were obtained from Char les River (St. Constan te , Q u e b e c , Canada) . Rats were divided into 3 non-diabet ic groups [untreated (C, n=7), and 2 treated (CT3 , n=7 and C T 1 4 , n=7)] and 3 diabetic groups [untreated (D, n=10), and 2 treated (DT3, n=10 and DT14, n=10)]. Vanady l sulfate ( V O S 0 4 - 3 H 2 0 , F isher Scienti f ic C o . , Fair Lawn, New Jersey , U S A ) was administered at a concentrat ion of 0.75 mg/ml in the drinking water to the control and diabetic-treated groups for 3 days , fol lowed by 1.00 mg/ml for 4 days . A t 7 days , S T Z (55 mg/kg i.v., S igma) w a s administered to the diabetic groups, while control groups received vehic le ( N a C l , 154 m M , pH 7.2). Vanad ium treatment was continued at the s a m e concentrat ion for 3 days in C T 3 and DT3 , and for 14 days in C T 1 4 and DT14. The concentrat ion (1.0 mg/ml) of vanady l sulfate w a s chosen on the basis of previous work which showed that administrat ion of this dose for 10 weeks to rats made diabet ic with 55 mg/kg S T Z resulted in long-term amelioration of diabetic symptoms for up to 20 weeks following withdrawal of treatment (Chapter 4). P l a s m a g lucose, insulin, body weight, and food and fluid intake were monitored frequently during and after treatment. 5.2.2. P lasma and Vanadium Ana lyses The experiment was terminated at 5 weeks following the S T Z injection, and p lasma w a s obtained for the determination of g lucose, B U N , creatinine and S G O T using kits. P l a s m a insulin was measured via rad io immunoassay, employing rat insulin s tandards. The RIA al lows for measurement of p lasma or pancreat ic extracts using vo lumes of 25 pi with an inter- and intra-assay coefficient of variation of < 1 0 % and is sensit ive to 7 i^U/ml (See Append ix 1). At termination, samp les of kidney, liver, musc le and bone were ana lyzed for vanadium levels via atomic absorpt ion as previously descr ibed (Thompson and McNei l l , 1993). 115 5.2.3. Oral g lucose tolerance test (OGTT) At 4 weeks after S T Z , an oral g lucose tolerance test was conducted in overnight fasted animals which were lightly anesthet ized with sod ium pentobarbital (20 mg/kg, i.p.) as previously descr ibed. This treatment does not affect the kinetics of g lucose and insulin during an O G T T . The areas under the curve calculated over 60 minutes for g lucose ( A U C g ) and insulin (AUCj) levels include basa l re lease. 5.2.4. Pancreatic insulin extraction At termination, pancreata were d issec ted, c leared of lymph nodes and fat, blotted and we ighed. The pancreas was immediately homogen ized using a polytron homogen izer (position 5) in 5 ml of cold 2 N acet ic acid for 5 seconds , and boi led at 100 °C for 10 minutes. The extract was centrifuged at 15,000 rpm for 10 minutes at 4°C. The supernatant was frozen in liquid nitrogen and stored at -70°C for insulin ana lys is . 5.2.5. Histological analysis A portion of the pancreas was fixed in 2 % formalin for 1-2 days , dried and embedded in paraffin. Sect ions were stained for granulated f i-cel ls by the modif ied a ldehyde fuschin method (Mowry et a l , 1980) and examined by light microscopy. 5.2.6. Statistical analysis. O n e - or two-way analys is of var iance ( A N O V A ) w a s used , as appropriate, fol lowed by the Fisher 's L S D test. The Fisher 's Exact test was used to determine significant di f ferences in the proportions of diabetic rats between g lycemic subgroups. L inear and nonl inear regression analys is was performed using the F ig .P Scient i f ic Processor . The following nonl inear equation for asymmetr ic s igmoid w a s used : Min + (Max-Min)/1 + ( (X/X 5 0 ) _ P) . p < 0.05 was considered significant. Data are exp ressed as means ± S . E . M . unless otherwise speci f ied. 116 5.3. R E S U L T S 5.3.1. General characterist ics of diabetic rats treated with vanadium. Vanad ium treatment reduced daily food intake by 1 3 % (22.6 ± 0.4 vs . 25.9 ± 0.4 g/rat) and fluid intake by 3 6 % (29.8 ± 0.2 vs . 46.7 ± 1.7 ml/rat) prior to S T Z . Whi le food intake was not significantly elevated in the diabetic rats at 3 days after S T Z , at 2 weeks , it w a s 3 3 % greater than C . The self-administered dose of vanad ium prior to withdrawal was similar between the groups ( C T 3 : 0.55 ± 0.05; C T 1 4 : 0.54 ± 0.06; D T 3 : 0.56 ± 0.03; D T M : 0.57 ± 0.04 mmol/kg/day). O n withdrawal of vanad ium, food and fluid intake were reversed in DT3 to levels similar to D, al though these parameters in DT^4 were not significantly different from control (Table 13). Body weight w a s reduced by treatment with vanadium for one week prior to S T Z (247 ± 4 vs . 268 ± 7 g, p < 0.05). Vanad ium intake for two weeks pos t -STZ also reduced weight gain significantly in DT14 (by 10%) relative to D. At 5 weeks after S T Z , and after chronic withdrawal from vanad ium treatment, weight gain in the diabetic rats w a s similar and lower than control. Prior to the induction of STZ-d iabe tes , vanad ium treatment for 1 week reduced p lasma insulin levels by at least 4 0 % (DT3: 16.9 ± 2.6, DT14 : 18.5 ± 2.5 vs . D: 30.6 ± 3.8 pU/ml , p < 0.05), but did not significantly alter mean g lycemia. M e a n p lasma g lucose in D after S T Z was 15.5 ± 1.9 m M (day 1) and 20.2 ± 0.9 m M (day 2), and w a s reduced significantly by vanad ium treatment on both days (DT3: 11.4 ± 1.3 and 14.4 ± 1.2; DT14: 9.7 ± 1.2 and 16.1 ± 0.8 m M , p < 0.05). After chronic withdrawal of vanad ium treatment at 5 weeks after S T Z , mean p lasma g lucose levels of D and D T 3 groups were high (> 18 mM) whereas that of DT14 (12.8 ± 2.0 mM) w a s signif icantly lower (Table 13, p < 0.03). M e a n p lasma insulin levels were not significantly different between the diabetic groups, and were significantly lower than control. At 4 weeks after S T Z , vanad ium treatment had been withdrawn from the C T 3 and D T 3 groups for 3.5 weeks , and from C T 1 4 and DT14 for 2 weeks . T h e integrated area under the g lucose curve over 60 minutes ( A U C g ) of control w a s 445.2 ± 14.2 117 Table 13. Parameters of various groups at weeks 4-5. C CT3 CT14 D DT3 DT14 (n=7) (n=7) (n=7) (n=10) (n=10) (n=10) Body Weight G a i n (g) Food Intake (g/day) Fluid Intake (ml/day) P l a s m a G l u c o s e (mM) P l a s m a Insulin (uU7ml) (mM-60 min) A U C j (uUTml-60 min) 156.6 170.9 (6.2) (2.5) 28.4 29.3 (1.0) (1.4) 56.4 59.0 (3.8) (3.8) 6.2 6.5 (0.2) (0.1) 49.9 52.5 (3.8) (4.6) 445 .2 438 .9 (14.2) (16.1) 2611.7 2447.8 (425.6) (326.9) 162.3 115.4* (4.7) (7.5) 30.7 40 .3 * (0.4) (1.2) 64.2 125.7* (4.9) (12.4) 6.3 18.2* (0.1) (1.7) 42.2 32 .5* (5.7) (4.4) 443.6 766.6* (17.2) (88.6) 1726.6* 1131.5* (155.2) (114.2) 103.6* 126.0* (7.6) (7.5) 45 .1 * 34.7+ (3.7) (1.3) 168.2* 88.7+ (17.1) (13.6) 20.0* 12.8+ (1.6) (2.0) 23.0* 34.7* (4.2) (5.5) 804 .9* 619.6 (92.7) (50.8) 1088.3* 1161.4* (114.5) (116.1) Data is mean (± S E M ) * p < 0.05 vs . C , + p < 0.05 vs. D 118 (range: 363.5 - 519.6) mM-60 min (Table 13). M e a n A U C g was significantly greater in the D and DT3 groups relative to C (p < 0.05), whereas mean A U C g of DT14 w a s not significantly different from either C or D. The integrated insulin response (AUCj) , was significantly lower in C T 1 4 relative to C (p < 0.05). Al l the diabet ic groups had average A U C j va lues which were - 4 0 % of C and not different from one another. At termination, vanadium was not detectable in the p lasma of all untreated or treated rats. Vanad ium levels in bone and kidney of untreated rats were undetectable, and higher in DT14 (bone: 90.3 ± 5.9, kidney: 5.9 ± 0.6 nmol/g) than in DT3 (bone: 70.7 ± 7.9, kidney: 4.7 ± 0.6 nmol/g). T h e s e levels are lower than those reported in diabet ic rats after chronic (5-week) treatment (Mongold et a l , 1990), or at 16-week withdrawal period after 1-year treatment (Dai et a l , 1994). Vanad ium levels in the pancreas and liver were detectable but low (< 2.0 nmol/g). There were no di f ferences in p lasma B U N , creatinine or S G O T between control and diabetic groups. At a f ixed concentrat ion of V O S 0 4 (1.0 mg/ml) in the drinking water, no incidence of diarrhea w a s observed . 5.3.2. Chronic normoglycemia in diabetic rats after withdrawal from vanadium. W h e n individual rats were examined, chronic hyperglycemia w a s found in 9/10 an imals in D with 1 rat remaining eug lycemic (p lasma g lucose < 9.0 mM) by the end of the study (Fig. 5.1A). A similar proportion (1/10) of animals w a s found to be normoglycemic in DT3 (Fig. 5.1B). However, extending vanad ium treatment for 2 weeks after S T Z corrected p lasma g lucose in 5 0 % (5/10) of the rats, which further maintained normoglycemia for 3 weeks after treatment was withdrawn (Fig. 5 .1C) . In the remaining 5 rats in DT14, there was an intermediate reduction in g lucose levels during vanad ium treatment, and severe hyperglycemia recurred after withdrawal from treatment. The 5/10 normoglycemic rats in DT14 had significantly lower g lucose levels by day 2 after S T Z relative to rats in this group which had recurrent hyperg lycemia after treatment w a s withdrawn (14.1 ± 0.9 vs. 18.2 ± 0.3 m M , respectively; p < 0.05). 119 25 -i 20 -15 -10 -5 -0 -(n=9) (n=1) C/D (n=7) CD </> O O <5 6 CO s: 25 -, 20 15 10 5 -0 -25 20 15 -10 -5 0 (n=9) CT3/DT3 (n=5) (n=5) (n=7) CT14/DT14 1 -7 — i — 0 t STZ —I H 1 1 14 21 28 35 Days Figure 5.1. Effect of vanadium treatment fol lowed by withdrawal period on fed plasma g lucose levels in control and diabetic rats P l a s m a g lucose levels in STZ-d iabet ic rats ( O , * ) and respect ive controls (A) which were untreated: (A) C / D , or treated with V O S 0 4 for one week prior to S T Z injection and for 3 (B) C T 3 / D T 3 and 14 days (C) C T 1 4 / D T 1 4 after S T Z , showing eug lycemic ( O ) or hyperglycemic (•) animals in each diabetic group. Sol id bar = V O S 0 4 treatment. 120 5.3.3. Pancreatic insul in content. There was no difference in pancreat ic weight in the var ious groups. Vanad ium treatment did not alter pancreat ic insulin content in control an imals (Fig. 5.2). A t 5 w e e k s after S T Z , there w a s a marked reduction by >90% in the m e a n insul in content in the diabetic groups. Pancreat ic insulin content in the D T M group w a s significantly greater than either the D or DT3 groups (p < 0.05). 5.3.4. Classi f icat ion of diabetic animals. A s previously reported using a dose of 55 mg/kg S T Z , we observed a highly heterogeneous population of diabetic animals. In order to determine the response to vanad ium treatment and to elucidate the mechanism(s) underlying the progress ive s tages of severity in the diabetic state and apparent reversion to normoglycemia, all d iabet ic (untreated and treated, n=30) rats were pooled and c lassi f ied accord ing to their g lycemic status. This was done using p lasma g lucose levels, both in the fed state and in acute response to an oral g lucose chal lenge. There w a s a 3-fold range in nonfasted p lasma g lucose levels among the diabetic rats (7.0 - 24.7 mM). Hence , rats were cons idered eug lycemic (E) when nonfasted p lasma g lucose was < 9 m M ; mean g lycemia in this group (n = 7) was 7.5 ± 0.1 m M . In addit ion, results from the O G T T a lso indicated that there was a considerable range (4-fold) in g lucose to lerance ( A U C g : 451.1 - 1741.7 mM-60 min) among the diabetic rats. The E subgroup w a s found to have a near-normal g lucose tolerance, as only mean g lycemia at the 60 minute time point w a s significantly greater than control (Fig. 5.3A). Accord ingly , the mean A U C g in E was not significantly different from C (inset) and individual A U C g levels in this group did not e x c e e d 750 mM-60 min. Thus , the remaining hyperg lycemic rats (p lasma g lucose > 9.0, n = 23) with an A U C g > 750 mM-60 min were classi f ied as having severe g lucose intolerance (H+GI, n=13), whereas other hyperg lycemic rats with a n A U C g < 750 mM-60 min were considered to have near-normal g lucose to lerance (H+GT, n=10). 121 Figure 5.2. Effect of vanadium treatment on pancreatic insul in content Pancreat ic insulin content in control (C) and STZ-d iabet ic rats which were untreated (D) or treated with V O S 0 4 for one-week prior to S T Z injection and for 3 days ( C T 3 , DT3) and 14 days (CT14, DT14) after the S T Z injection. Pancreat ic insulin extracts were col lected at the end of the study (5 weeks post -STZ) , fol lowing chronic withdrawal f rom vanad ium treatment (* p < 0.05 vs . C , + p < 0.05 vs . D). 122 2 a> o> o u 3 O <o E 20 H 15 H 10 H 5 H C E H + G T H + G / n I I I I I I 0 10 20 30 40 50 60 3 ta E 5; 80 60 H 40 20 C E H+GT H+GI -1 1 1 1 1 1 1 0 10 20 30 40 50 60 Time (min) Figure 5.3. Oral g lucose tolerance test demonstrating the var iances in g lucose -tolerance and insulin response in the pooled diabetic animals P l a s m a g lucose (A) and insulin (B) response to an O G T T in diabetic rats: • , E; • , H+GT; A , H+GI; and O , untreated control. Bar graphs represent integrated g lucose (AUCg) (C) and insulin (AUCj) (D) response of control and diabet ic rats accord ing to g lycemic status. (* p < 0.05 vs . C , + p < 0.05 vs. E, A p < 0 . 0 5 v s . H+GT). 123 Thus , H+GT had a mean A U C g not different from C or E, whereas mean A U C g w a s significantly greater in H+GI than in C or E (Fig. 5.3A, inset). The g lucose-st imulated insulin response in diabetic rats was significantly less than control, and there w a s a downward trend according to severity of d iabetes (E > H+GT > H+GI; F ig . 5 .3B, inset). The proportion of rats in each category is shown in Tab le 14. The number of E rats w a s significant in DT14 (5/10) as compared to D (1/10) and D T 3 (1/10) (p = 0.01). Moreover , the total number of animals (E , H+GT) which had near-normal g lucose- to lerance ( A U C g < 750 mM-60 min) was higher in DT14 (9/10) relative to D (5/10) (p = 0.01) or DT3 (6/10) (p = 0.03). Despite marked hyperg lycemia, the mean nonfasted p lasma g lucose in H+GT (18.1 ± 1.1 mM) was lower than H+GI (22.3 ± 0.5 mM) (p < 0.05). Nonfasted p lasma insulin levels (uU/ml) in E (37.9 ± 2.9) and H+GT (37.2 ± 3.6) were significantly lower than C , and further reduced in H+GI (17.2 ± 2.9) (p < 0.05). Weight gain was significantly lower than control (156 ± 6 g) but highest in E (138 ± 6 g) fol lowed by H+GT (119 ± 5 g), and least in H+GI (93 ± 6 g). 5.3.5. Correlations between residual insulin content and g lycemic status. Figure 5.4A depicts pancreat ic insulin content of the diabet ic an imals when ana lyzed according to g lycemic status. The E group had an average insulin content of - 1 2 . 0 % (0.204 ± 0.021 U/g) of control (1.69 ± 0.48 U/g), which w a s 2 and 4 t imes higher than H+GT and H+GI, which had mean insulin contents of - 6 . 0 % (0.103 ± 0.013 U/g) and - 3 . 0 % (0.050 ± 0.008 U/g), respectively. S i n c e the severi ty of the diabet ic state appeared to be assoc ia ted with alterations in the insulin content, we quest ioned the extent to which the var iance in g lycemic status among the diabet ic an imals could be l inked to changes in residual insulin content. Interestingly, it w a s found that residual insulin content in the pooled diabetic animals (n=30) correlated very strongly with the 3-fold range in p lasma g lucose levels in the fed state (Fig. 5 .4B, r = -0 .91 , p < 0.0001). Thus , the H+GI rats which had the highest g lucose levels (range: 19.5-24.7 mM) had 124 Table 14. Classi f icat ion of STZ-diabetic animals according to g lycemic status. Group (n) E H+GT H+GI Fed P l a s m a G l u c o s e (mM): < 9.0 > 9.0 > 9.0 G l u c o s e To lerance ( A U C g ) : < 750 < 750 > 750 (mM-60 min) D ( 1 0 ) 1 4 5 D T 3 ( 1 0 ) 1 5 4 D T 1 4 ( 1 0 ) 5* 4 1 Total (30) 7 13 10 significantly different from D (p = 0.01). Legend : E = euglycemic/near-normal g lucose-to lerance H+GT = hyperglycemic and near-normal g lucose-to lerance H+GI = hyperglycemic and glucose-intolerant A U C g = A r e a under curve calculated for p lasma g lucose from 0-60 min in response to an oral g lucose load (1 g/kg) in overnight-fasted animals. 125 5 « o o 3 E 5: 25 n 20 15 10 5 -\ B r = -0.91 - i 1 1 1—II—i 1 0 100 200 300 17001800 25 20 15 10 5 H r = -0.95 - i 1 1 1 1 0 100 200 300 400 Pancreatic Insulin Content (mil/g) Figure 5.4. Relationship between glycemic status and residual insul in content in pooled diabetic animals at 5 weeks post-STZ. (A) Pancreat ic insulin content in diabetic rats, according to g lycemic status (p < 0.05 vs . E (+) or H+GT (A)) . Correlat ion between fed p lasma g lucose and pancreat ic insulin content in pooled diabetic (r = -0 .91, p < 0.0001) (B) and untreated diabet ic rats a lone (r = -0.95, p < 0.0001) (C) showing subgroups: » :E; D :H+GT; A :H+GI and O : C (n=7). 126 the lowest residual insulin stores (95%tile range: 2 .0-3.9% of control) whereas the eug lycemic subgroup which had the highest residual insulin content (8.6-15.5%) had correspondingly lower g lycemic levels (7.0-8.1 mM). Intermediate between these groups, the H+GT animals had g lucose levels which varied from 10.2 - 23.3 m M and a range in insulin content of 5.4-9.5%. Notably, this strong correlation persisted with the analys is of untreated diabetic rats a lone (Fig. 5 .4C, r = -0.95, p < 0.0001). Res idua l insulin content in the pooled diabet ic rats (n=30) w a s highly correlated with the 4-fold range in g lucose tolerance, in a negative, hyperbol ic manner ( A U C g , r = -0.84, p < 0.0001, F ig . 5.5A) and with a 4-fold range in insulin response in a posit ive manner (AUCj , r = 0.70, p < 0.0001, F ig . 5.5B). Both correlat ions persisted with the analys is of untreated diabetic rats a lone (insets). Seve re g lucose intolerance ( A U C g > 750 mM-60 min) was observed only below an insulin content of - 1 0 0 mU/g or - 6 % of control. Res idua l insulin store w a s also correlated with fed p lasma insulin levels (r = 0.54, p = 0.006), and body weight (r = 0.53, p = 0.004) (data not shown). 5.3.6. Histological examination of f i -cells. To ascerta in if changes in pancreat ic insulin content could be assoc ia ted with an altered number of histologically detectable f i-cel ls, pancreat ic sect ions were sta ined and the numbers of granulated f i-cells counted in five similarly s ized islets per sect ion. In control rats, severa l islets were found to contain a substantial number of wel l -granulated f i-cel ls, indicated by the darkly stained areas within each islet (Fig. 5.6A). In contrast, the number of granulated fi-cells per islet was markedly depleted in H+GT (4.7 ± 0.8, not shown), and almost undetectable in H+GI (1.1 ± 0.4, F ig . 5.6B). However , in sect ions taken from E rats, each islet contained a significant number of darkly stained f i-cel ls (16.8 ± 0.6) (Fig. 5.6C). However, the number of islets found in e a c h sect ion w a s d imin ished in E relative to C . The number of histologically detectable f i -cel ls per islet w a s found to be consistent in at least 25 islets per group. 127 3000 2500 H E 2 2000 ^ . 1500 O ^ 1000 B 500 r = 0.71 3 0 0 0 -2 5 0 0 -2 0 0 0 -1 5 0 0 1 0 0 0 5 0 0 H 0 r = 0.89 —\ 1 1 1 0 1 0 0 2 0 0 3 0 0 -1 1 1 1 II 1 1 0 100 200 300 1700 1800 Pancreatic Insulin Content (mU/g) Figure 5.5. Relationships between glucose-tolerance and insul in secretory function at 4 weeks post-STZ with residual insulin content in pooled diabetic rats. Correlat ion between (A) g lucose response ( A U C g , r = -0.84, p < 0.0001) and (B) insulin response (AUCj r = 0.71, p < 0.0001) during an OGTT at 4 weeks post -STZ for pooled diabetic rats and residual insulin content. Insets show similar relationship for untreated diabetic rats a lone. (Legend: • : E; • : H+GT; A : H+GI, O: control rats (n=7)). F igu re 5.6. Photomicrographs of islets, stained for fi-cells in untreated control (A), and in diabetic animals: H+GI (B) and E (C). (x 500) 129 5.4. DISCUSSION A previous study has shown that following chronic withdrawal from 3-week vanad ium treatment, STZ-d iabet ic rats maintained normoglycemia despi te only minor improvements in pancreat ic secretory function, amount ing to - 1 2 % of control (Pederson et a l , 1989). Susta ined normoglycemia was thought to depend upon increased sensitivity to circulating insulin in the vanadium-withdrawn an imals . In this study, al though vanad ium pretreatment for 1 week reduced p lasma insulin levels by 4 0 % , it did not prevent the STZ- induced deplet ion of insulin stores in vivo, thus ruling out any protective effects of vanad ium against STZ- i nduced fi-cytotoxicity. H e n c e , it was quest ioned whether vanadium treatment after S T Z could improve pancreat ic insulin content and function, and therefore induce a chronic ameliorat ion of the diabet ic state. A 2-week treatment period was chosen to eliminate the possibil ity that t issue vanad ium stores might contribute to persistent normoglycemia. Indeed at 5 weeks , when p lasma vanad ium levels were undetectable, DT14 rats showed an overal l improvement in the diabetic state. Food/f luid intake and g lycemia were significantly lower in this group, and the pancreat ic insulin store was greater than either D or DT3 . W e have reported that S T Z (55 mg/kg) produces a var iable severity of d iabetes in rats, judging by a wide range in p lasma g lucose and insulin levels. The subclassi f icat ion of diabetic rats in the present study w a s not accord ing to human diagnost ic criteria (National Diabetes Data Group, 1979), but it does serve to illustrate and dist inguish between progressive s tages of d iabetes. A similar heterogeneity in the diabetic state has been descr ibed in spontaneously diabetic B B rats (Lucke et a l , 1988), C h i n e s e hamsters (Nakaj ima et a l , 1994) and in early Type 1 d iabetes in humans (Tuomilehto and Wolf, 1987). Approximately 1 0 % of STZ-d iabe t i c rats (55 mg/kg S T Z ) fail to deve lop d iabetes, and of the diabetic animals, 100% become normoglycemic when treated with vanadyl concentrat ions of 0.75-1.5 mg/ml. In this study, at 1 mg/ml vanady l , a significant number of animals in the D T M group deve loped and maintained 130 normoglycemia following withdrawal from treatment. The 3-fold range in both g lycemia and g lucose to lerance in the pooled diabetic rats w a s strongly correlated to residual insulin content. A l though C T 1 4 had a lower A U C j , suggest ing cont inued enhancement of insulin act ion by vanad ium at week 4, the significant relat ionships persisted on analys is of untreated diabet ic rats a lone, support ing the idea that near-normal g lucose homeostas is in the diabetic rats w a s more likely dependent on residual insulin stores. Thus , the finding that diabet ic rats with a markedly depleted insulin store demonstrate normoglycemia and near-normal g lucose to lerance indicates that a modest (<5%) improvement in the pancreat ic insulin reserve is physiological ly relevant and can induce a chronic ameliorat ion of the diabet ic state. Two studies have reported total insulin content of pancreata from S T Z -diabetic rats treated with vanadium (Brichard et a l , 1988; B londel et a l , 1989). In these studies, residual insulin reserves increased from 2.5-fold to 8-fold in response to vanad ium treatment compared to untreated rats. Despi te these improvements, the authors noted that the overall changes in insulin store were relatively insubstantial when v iewed as a percentage of control levels and hence, downplayed the role for an improved insulin content in the regulation of normal g lucose homeostas is in the diabet ic rats. Interestingly, the pancreat ic insulin content found in vanadium-treated rats (~185 mU/g , Br ichard et a l , 1988) is within the range (150-250 mU/g) present ly found to support normoglycemia per se . A n even greater improvement in insulin store w a s found in diabetic rats in which insulin resistance w a s reversed by vanad ium treatment, amount ing to - 3 3 % of control (Blondel et a l , 1989). Thus , it is plausible that relatively smal l improvements in insulin stores in vanadium-treated rats could have contributed to the amel iorat ion of d iabetes in the long term and may have adequate ly sus ta ined chronic normoglycemia had treatment been withdrawn. Indeed, the current levels of pancreat ic insulin store (12%) in the E rats were found to match the residual secretory function in vanadium-withdrawn rats (Pederson et a l , 1989). 131 It has long been recognized that both humans (Child et a l , 1969) and rats (Kauffmann and Rodr iguez, 1984) develop frank d iabetes when >90% of the pancreas is removed, and that nonfasting hyperglycemia occurs in STZ- t reated mice only when the reduction in islet function is >90% (Bonnev ie-Nie lsen, 1981). In this study, within a narrow range of residual pancreat ic insulin, (1.3-16.5% of control), the range in g lycemic levels is considerable, from normal to severe hyperg lycemia. In contrast, Junod et al (1969) reported a correlation between g lycemia (from high to normal) and residual insulin store, but over a much wider range in insulin content (1-50% of control) at 24 hours after i.v. administration of S T Z . However , insulin stores were depleted over 28 days and although no correlation was reported at that time, normoglycemia w a s evident with an insulin content of - 2 0 % (Junod et a l , 1969). Present ly , despi te the notion that severa l regulatory factors could s imultaneously affect g lucose homeostas is , the significant l inear correlation between g lycemia and residual insulin store at 5 w e e k s pos t -STZ suggests that nonfasted p lasma g lucose can be an accurate index of the residual insulin content within the low range (below 10% of control) during a stable, chronic diabetic state. Furthermore, it suggests that smal l changes in insulin content which may be easi ly over looked could nevertheless be functionally significant. For instance, Le et al (1985) reported significantly lower g lycemia assoc ia ted with a - 2 . 3 % higher level of pancreat ic insulin store in cholesterol-pretreated STZ-d iabe t i c mice. Similarly, Ser radas et al (1989) found an improved fi-cell function assoc ia ted with - 1 0 % greater levels of insulin store in gl iclazide-treated STZ-d iabet ic rats. S i nce the changes in insulin store in both studies were statistically insignificant, their contribution to overal l improvements in the diabetic state was not cons idered. Thus , it may have been poss ib le to link these events to changes in insulin store, and detai led ana lys is of the diabetic groups may have revealed this associat ion. O n e plausible mechan ism of fi-cell preservat ion is that removal of hyperg lycemia by a peripheral action of vanadium reduced glucotoxicity and exhaust ion 132 of the f i-cel l . Indeed, early treatment of d iabetes with insulin has long-term beneficial effects on the insul in-secret ing cel ls (Shah et a l , 1989). Insulin treatment for 1 week following the S T Z injection, reversed the diabetic state for 3 months, correcting islet cell morphology and insulin secret ion, effects attributed to a reduction in insulin demand (Ar 'Rajab and Ah ren , 1993). Similarly, short-term insulin treatment fol lowing S T Z in the neonatal rat increased the spontaneous remission of d iabetes (Portha and P i c o n , 1982). D iazox ide, an inhibitor of insulin secret ion, lowered the inc idence of d iabetes in B B rats (V lahos et a l , 1991) and improved fi-cell g lucose- respons iveness and insul in content in partially pancreatectomized rats (Leahy et a l , 1994), support ing the notion that excess ive insulin secret ion by a compromised fi-cell mass leads to exhaust ion (Sako and Gri l l , 1990). Hence , an insul in- l ike/enhancing agent cou ld prevent res idual f i-cel ls from secret ing insulin in excess of its synthetic rate, thus al lowing the cel ls to refill or regenerate. Importantly, fi-cell protection may depend on a critical number of cel ls which initially survive S T Z toxicity. Thus , fol lowing a high d o s e of S T Z (75 mg/kg), hyperglycemia recurred in all the animals after vanad ium treatment w a s withdrawn (Bendayan and Gr ingas, 1989). Similarly, insulin treatment could not reverse the diabet ic state following high d o s e s of S T Z (>60 mg/kg) (Ar 'Rajab and A h r e n , 1993). In conc lus ion, while vanadium pretreatment did not prevent the onset of S T Z -diabetes, subsequent 2-week treatment after S T Z el iminated hyperg lycemia in a signif icant proportion of diabetic rats even after treatment w a s c e a s e d . Th is phenomenon w a s linked to a smal l but significant improvement in pancreat ic insulin content and an increased number of granulated f i-cells per islet. Thus , short-term vanad ium may induce a chronic ameliorat ion of the diabet ic state by the preservat ion of a critical f i-cell insulin store. In addit ion, these results support the content ion that nonfasted p lasma g lucose can be an effective index of residual insulin content (<10%) in chronic d iabetes and that modest changes in pancreat ic insul in store c a n have profound consequences on g lucose homeostas is in the long-term. 133 Chapter 6 THE ANTIDIABETIC E F F E C T S OF VANADIUM TREATMENT IN STZ-DIABETIC RATS A R E INDEPENDENT OF ITS E F F E C T S ON REDUCING FOOD INTAKE 6.1. INTRODUCTION Susta ined normoglycemia after vanadium treatment in STZ-d iabe t i c rats may be partly attributed to an improved pancreat ic secretory function. Intensive insulin treatment for 2 weeks following recent onset d iabetes w a s shown to preserve fi-cell function and insulin content, effects which were attributed to lowered insulin secretory activity (Shah et a l , 1989). A s well , inhibition of insulin secret ion by d iazox ide lowered the inc idence of d iabetes in B B rats (Vlahos et a l , 1991) and increased islet insulin content in partially pancreatectomized rats (Leahy et a l ; 1994). Thus , vanad ium may preserve insulin stores via a similar reduction of insulin secret ion in STZ-d iabe t i c rats. A n establ ished secondary outcome of vanad ium treatment is a reduct ion in food intake, an effect attributed to a specif ic anorexigenic st imulus in the central nervous sys tem (Meyerovi tch et a l , 1989). It was suggested that the ant ihyperglycemic effects of vanad ium treatment could be attributed entirely to its suppress ion of food intake (Malabu et a l , 1994; Domingo et a l , 1994), s ince a similar reduction in g lycemia was observed in vanadium-treated and pair-fed diabet ic groups. In support, the suppress ion of food intake by 4 0 % in STZ-d iabet ic rats reduced hyperg lycemia and increase insulin sensitivity during a hyperinsul inemic c lamp (Cameron-Smi th et a l , 1994). Furthermore, chronic food restriction in control rats reduced insulin secret ion (Chu et a l , 1991) and enhanced insul in-mediated g lucose uptake (Escr iva et a l , 1992). H e n c e , a possibil i ty exists that at least some of the antidiabetic act ions of vanad ium treatment may result from its auxil iary effects on food intake. In the present study, we examined the relative contribution of a reduction in food intake per se to the effects of vanad ium in inducing an overall amelioration of the diabetic state. 134 6.2. MATERIALS AND METHODS 6.2.1. Treatment and maintenance of animals Male Wistar rats (170-200 g) were obtained from Char les River (St. Constan te , Q u e b e c , Canada) . The animals were maintained on s tandard laboratory chow and housed in individual wire-bottom cages . The rats were divided into control (C, n=4) and 3 diabetic groups: untreated (D, n=10), vanadyl- treated (DT, n=10), and pair-fed (DP, n=10) groups. O n e week prior to the administrat ion of S T Z , vanady l sulfate w a s administered at a concentrat ion of 0.75 mg/ml in the drinking water to the D T group for 3 days , fol lowed by 1.00 mg/ml for 4 days . Th is treatment protocol w a s chosen s ince pretreatment with vanadium prior to S T Z fol lowed by 2-week treatment resulted in chronic amelioration of the diabetic state after treatment w a s c e a s e d , a phenomenon which was subsequent ly linked to a significant improvement in pancreat ic insulin stores. At 7 days , streptozotocin (STZ , 55 mg/kg i.v., S i g m a Chemica l C o . , St . Lou is , M O , U S A ) w a s administered to the diabetic groups, while control rece ived vehic le (NaC l , 154 mmol/ l , pH 7.2). Vanad ium treatment was cont inued at 1.0 mg/ml for 1 week. B e c a u s e of the low response rate (1/10) at this concentrat ion and s ince food intake was to be averaged over the entire group, the concentrat ion of vanad ium w a s increased by 0.25 mg/ml per week in all the DT animals, regardless of response , up to a max imum of 1.75 mg/ml which was maintained for the final 2 weeks . F lu ids were administered ad libitum. Fed p lasma g lucose, insulin, body weight, and dai ly food and fluid intake were monitored. Throughout the study, food intake in DT w a s measured twice daily: at 0900 h and 1800 h to account for nocturnal and dayt ime food consumpt ion, respectively. To mimic the feeding pattern of DT, an identical amount of food which had been consumed by the D T group was administered to the D P an imals during the respect ive time periods (at 0900 h and 1800 h) the following day. We igh ing trays were p laced underneath the feed troughs of the DT and D P groups to measure food which w a s administered but not consumed by either group. 135 The study was terminated at 5 weeks pos t -STZ, and p lasma obtained for measur ing g lucose, tr iglycerides, cholesterol (Boehr inger Mannhe im C a n a d a , Lava l , Q u e b e c , Canada ) , S G O T , S G P T , B U N , creatinine and albumin (Sigma). Insulin w a s measured via RIA, using rat insulin standards (Novo R e s e a r c h Institute, C o p e n h a g e n , Denmark) as descr ibed in Append ix 1. 6.2.2. Oral g lucose tolerance test (OGTT) At 5 weeks after S T Z , an O G T T was performed in overnight-fasted rats as previously descr ibed. The areas under the curve determined over 60 minutes for g lucose ( A U C g ) and insulin (AUCj) levels include basa l re lease. 6.2.3. Pancreatic insul in extraction At termination, pancreata were d issected, c leared of lymph nodes and fat, blotted and we ighed. The pancreas w a s immediately homogen ized in 5 ml cold (2N) acet ic ac id for 5 seconds , and boi led for 10 minutes. The extract w a s centr i fuged at 15,000 rpm for 10 minutes, and resulting supernatant f rozen at -70°C until further analys is of insulin. 6.2.4. Vanadium levels At termination, kidney, liver, musc le , bone, pancreas and p lasma samp les were ana lyzed for vanad ium levels via atomic absorpt ion using a method previously descr ibed (Mongold et a l , 1990). 6.2.5. Statistical analysis. One-way analys is of var iance ( A N O V A ) w a s used fol lowed by the N e w m a n -Keu ls test. Data are expressed as mean ± S . E . M . p < 0.05 was cons idered signif icant. 136 6.3. R E S U L T S 6.3.1. General characterist ics of animals Vanad ium treatment and pair-feeding for one week resulted in a similar reduction in body weight gain relative to untreated rats prior to the S T Z injection (Fig. 6.1 A) . No weight loss was observed in any of the diabetic groups throughout the study. However , over 5 weeks , weight gain was consistently lower in D T and D P relative to D. Moreover , despi te a similar daily food intake, there was a more pronounced reduction in weight gain in D P relative to DT. Throughout the study, an imals in the D P group appeared l ist less, with limited fat reserves and low musc le m a s s . In compar ison , it w a s observed that in DT, body weight, appearance and level of activity improved in e a c h animal once normoglycemia was ach ieved. Vanad ium treatment reduced fluid intake by 5 8 % (Fig. 6.1 B) and food intake by 2 0 % (Fig. 6.2A) during the week prior to S T Z . Diabetes caused an elevat ion of food and fluid intake by 2 and 4.3 t imes, respectively by 5 weeks . Food consumpt ion w a s significantly reduced by vanadium treatment to less than control throughout the study. Gradual ly increasing concentrat ions of vanadium to 1.75 mg/ml in all the DT an imals did not result in a further reduction in food intake. Fluid intake in DT w a s significantly lower than control va lues throughout the study. Fluid intake in D P appeared to rise fol lowing S T Z , but returned to control levels by the final week. Both measurement of food intake in DT and subsequent pair-feeding of D P were performed twice daily for the entire duration of the study. It w a s observed that approximately 1/3 of the daily food intake in DT would be consumed between 0900 h and 1800 h and - 2 / 3 between 1800 h and 0900 h. Al though the food a l lowance given to D P was intended to last for entire day/night-t ime periods, pair-fed an imals would consume their entire food rations within the first hour, and were hence without food for extended periods of time. Thus , the daily fasting periods for D P occurred between 1100-1800 h (7 hours) and from 1900-0900 h (14 hours). 137 7.00 1.25 1.50 1.75 mg/ml V (•) i 1 1 1 1 1 1 - 1 0 1 2 3 4 5 Weeks t STZ Figure 6.1. Effects of vanadium treatment and pair-feeding on body weight and fluid intake in STZ-diabetic rats over 6 weeks Body weight (A) and fluid intake (B) of control (C, O ) , diabet ic (D, • ) , d iabet ic vanadium-treated (DT, • ) and diabetic pair-fed (DP, • ) groups over 6 w e e k s . (* p < 0.05 vs . C , + p < 0.05 vs . D, A p < 0.05 vs . DT). V O S 0 4 concentrat ions administered are as indicated above the x-axis. 138 6.3.2. Effect of vanadium treatment and pair-feeding on glycemia S ince routine blood col lection occurred at 0900-1000 h, g lucose levels for D P are 14-hour fasted levels. Never theless, mean g lycemia in the vanadium-treated diabetic group was significantly reduced by the first day following S T Z relative to both untreated and pair-fed diabetic groups (Fig. 6.2B). Gradual ly raising the vanady l concentrat ions to 1.75 mg/ml in all the animals resulted in a progressively greater number of rats which deve loped normoglycemia (p lasma g lucose < 9.0 mM), such that at termination, 10/10 DT rats were normoglycemic. Importantly, food intake w a s not further reduced, and remained at - 7 0 % of control with higher concentrat ions of vanady l (F ig. 6.2A). Alternately, pair-feeding did not result in normoglycemia in any of the rats, even at the final week, when a 2 5 % reduction in mean g lycemia in D P w a s observed . A s observed previously, there were two subgroups of DT an imals which responded either to low (DT -LC , 1.00-1.25 mg/ml, n=4) or high concentrat ions ( D T - H C : 1.50-1.75 mg/ml, n=6) of vanadyl to ach ieve eug lycemia (Fig. 6.3B). A l though all vanadium-treated diabetic rats received higher concentrat ions of vanad ium regard less of response, vanad ium intake was consistently higher in the D T - H C group until week 4 (Fig. 6.3A). The greater self-administered dose in D T - H C resulted from the greater fluid intake in these animals prior to achieving stable normoglycemia. Interestingly, two subpopulat ions appeared in similar proportions in the pair-fed group, one having lower and fluctuating g lycemia, ("responder" or D P - R ) (Final p lasma g lucose: 13.7 ± 1 . 4 m M , n=4), and the other having severe hyperglycemia ("non-responder" or D P - N R ) (Final p lasma g lucose: 23.9 ± 1.3 m M , n=6) (Fig. 6 .3C). At termination, DT animals which had responded early to lower concentrat ions of vanad ium (DT-LC) had a signif icantly greater weight gain relative to D T - H C (DT-LC : 90 ± 8.3 g vs . D T - H C : 56 ± 12.1 g, p < 0.05). Similarly, pair-fed animals which had lower g lucose levels ( D P - R ) demonstrated more weight gain (60.5 ± 8.4 g), relative to those which were consistent ly more hyperglycemic ( D P - N R ) (20.3 ± 4.6 g) by the end of the study. 139 i 1 1 1 1 T 1 - 1 0 1 2 3 4 5 Weeks t STZ Figure 6.2. Effects of vanadium treatment and pair-feeding on food intake and plasma g lucose levels in STZ-diabetic rats over 6 weeks Food intake (A) and p lasma g lucose levels (B) of control (C, O ) , diabet ic (D, • ) , diabetic vanadium-treated (DT, • ) and diabetic pair-fed (DP, • ) groups over 6 weeks . (* p < 0.05 vs . C , + p < 0.05 vs . D, A p < 0.05 vs . DT). V O S 0 4 concentrat ions administered are as indicated above the x-axis. 140 mg/ml V 5 H - 1 0 0 10 20 30 Days f STZ Figure 6.3. P lasma glucose in vanadium-treated and pair-fed diabetic rats Vanad ium dose (A) and fed g lycemia (B) in diabetic-treated animals normal ized to low ( D T - L C : 1.0 - 1.25 mg/ml, • ) and high ( D T - H C : 1.50 - 1.75 mg/ml , O ) concentrat ions of V O S 0 4 . (C) P l a s m a g lucose levels in pair-fed diabetic animals, depict ing rats which had lower, fluctuating ("responder", D P - R , • ) and higher ("non-responder", D P - N R , O ) p lasma g lucose levels throughout the study. Untreated diabet ic ( • ) and control ( • ) groups are added for compar ison. VOSO4 concentrat ions are shown in panel A . 141 6.3.3. Effects of vanadium treatment and pair-feeding on plasma insul in Vanad ium treatment for 1 week significantly reduced p lasma insulin by 5 0 % prior to the induction of d iabetes, whereas in the D P group, this w a s not as marked (Fig. 6.4A). A l though p lasma insulin in vanadium-treated and pair-fed groups did not change at 3 days after S T Z , p lasma insulin in D was increased from day 3 and w a s higher than control at 1-2 weeks , but dec reased to the s a m e level as D P and DT ( - 5 0 % of control) by 5 weeks . At 1-2 weeks , p lasma insulin a lso rose in DT, but w a s - 5 0 % the levels of D and similar to the pair-fed group until week 5. In control rats, p lasma insulin levels rose steadily, paralleling weight gain ( -200 g) over the 5-week time per iod. Figure 6 .4B depicts a correlation between p lasma insulin and g lucose levels in the fed state, averaged over 4 readings in the final 2 weeks . A strong correlat ion between insulin and g lucose levels was shared by D and D P rats (r = -0.74, p = 0.0002). This relationship was distinct from the significant correlation obtained in DT (r = -0.68, p = 0.03) (inset). No significant correlation was observed in control group. 6.3.4. Oral G lucose Tolerance Test At 5 weeks , fasting g lycemia in DT was not significantly different from C (Fig. 6.5A). A l though C , D and DT rats were fasted for 16 hrs, D P rats consumed their dayt ime food ration within 1 hour, and were hence fasted for 22 hrs. Never the less , fasting g lycemia in D P was not different from D. Marked g lucose intolerance w a s observed in D P , similar to D, while mean peak g lucose in D T at 20-30 minutes w a s intermediate ( -15 mM) and returned to levels not significantly different from control by 60 minutes. Simi lar to fed g lycemic levels, A U C g in the DT groups w a s not signif icantly different from control, while it was higher in D P - N R and D P - R (Fig. 6 .5C) . A l though insulin response was not statistically different between the diabetic groups, the more severe ly diabetic subgroups ( D T - H C and D P - N R ) had A U C j levels which were 2 5 % lower than the more glucose-tolerant D T - L C and D P - R groups, respect ively (Fig 6.5D). 142 - r -GO o CO o I m o 0 0 m CM o t\l m ~ l o I in o m o o c o o CM 3 <0 . C E CO -2 CL CU o> «J v_ d> >• < [Win] asoon/o euusejd a6ejaA\f i- m CO CM h o o o 0 0 o CM I o to CO CO CO (luu/nrf) unnsui euuseid CO CD C TJ CO CD CD > o TJ CD CD CD i _ CD > co _co CD > _ CD CO — "55 .g i i c -W CO — CD ro co CD jQ CO TJ TJ C C O TJ " CD • N TJ g CD -tS CO 03 CO I S I s 1 § CO "^3 > 03 O CD CD -Q O O CO ~ CO E CO Q. 5 O • co c g "co 03 i I O) IP. c a. ^ TJ 0) CD a> •• c CD OJ CD T3 CD C -Q W C c .2 2 E a) 4-> 1— TO ! r 2 8 E l ^< c re w > CD X - > O JD o g . CO * E CO CO a> ^ it. a. 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Relat ive to control, p lasma creatinine w a s higher in the DT and D groups, and was reduced in the D P group. In addit ion, D P had significantly reduced p lasma triglyceride levels. P l a s m a cholesterol and G O T were not different in diabetic groups relative to C . Al though urea and G P T levels were significantly greater in D, these dif ferences were abol ished in DT and D P . Vanad ium levels in DT were: p lasma (nmol/ml): 7.8 ± 1.0, (nmol/g): liver: 30.6 ± 3.7, musc le : 4.5 ± 0.2, pancreas: 8.0 ± 1.0, bone: 415.0 ± 40.4 and kidney: 129.4 ± 6.9. T h e s e levels are similar to reported va lues in diabetic rats after one year of chronic treatment (Dai et a l , 1994). P l a s m a and t issue vanad ium levels were not different between D T - H C and D T - L C despi te a higher calculated vanad ium intake in D T - H C . 6.3.6. Effects of vanadium and pair-feeding on pancreatic insul in content. Although there was a profound reduction (<15% of control: 1.6 ± 0.1 mU/g) in the pancreat ic insulin content (PIC) in diabetic rats at 5 weeks , it w a s higher in D P and D T relative to D by ~2 and 4-fold, respectively (Fig. 6.6A, inset). The correlation between residual insulin store and fed g lycemia in D (r=-0.93, p = 0.0003) w a s distinct from D P (r = -0.88, p = 0.0007), whereas no correlation was found in DT (p = 0.087) (F ig. 6.6A). D T - L C rats which had required low vanadyl concentrat ions to ach ieve normoglycemia had a P IC > 160 mU/g , consistently greater than rats which required higher concentrat ions (DT-HC) . Signif icant correlations were found between A U C j and P I C (Fig. 6 .6B). Th is relationship for D P (r = 0.71, p = 0.02) w a s shifted slightly to the right of D (r=0.80, p=0.006). In DT rats which had a P I C higher than the D P range (>100 mU/g) , there was a strong correlation (r = 0.94, p = 0.002) which was shifted further to the right. Thus , the A U C j / P I C ratio was 2.2 (DP) and 5.1 (DT) t imes that of D. Table 15. P lasma Parameters at Termination C D DT D P (n=4) (n=10) (n=10) (n=10) G l u c o s e (mM) 6.9 26.8* 7.8+ 19.8*+ (0.1) (1.3) (0.3) (1.8) Insulin (LiU/ml) 39.0 21 .3* 17.0* 20 .8* (0.8) (2.9) (2.1) (2.7) Tr iglycer ides (mM) 1.20 1.37 1.25 0.44*+ (0.19) (0.32) (0.15) (0.05) Cholestero l (mM) 1.32 1.66 1.54 1.37 (0.15) (0.12) (0.05) (0.08) A lbumin (uM) 521.6 456.4 556.4+ 520.2 (30.4) (21.7) (20.3) (23.2) Creat in ine (uM) 25.6 34 .1* 33.6* 13.6*+ (4.1) (6.7) (3.1) (2.8) Urea (mM) 10.7 12.4* 11.9 9.8+ (0.3) (0.7) (0.6) (0.5) G O T (U/l) 102.5 114.6 93.9 99.2 (8.2) (6.2) (5.4) (6.3) G P T (U/l) 41.4 74.8* 52.8+ 51.4+ (2.3) (8.7) (4.9) (6.6) Data is mean (± S E M ) * p < 0.05 vs . C , + p < 0.05 vs . 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CD -Jw • - ^ DQ CD T— ^ 6 CD o O (0 CO •— g <0 CD a> CD o o c c CD 5 c " a cu £ (j) CD a> E .Q CD -I - CD <2 g Q_ Q co" > m o o v Q. + CD X 5 . -co O T J X • Q Q" 0> T 3 C CD CO > m o d v o CD . a CD ^ ^ • C -— 'm CD CM o CO c o o ^ 3 * CO c i i ! o CD CD Di c o O CO CO CD CO £ a> o 3 O LL CD CD i _ O c CD CL * - L CO •O TJ C CD CO N c < 2 147 6.4 DISCUSSION It was previously suggested that the ant ihyperglycemic effects of vanad ium in STZ-d iabet ic rats can be attributed entirely to its auxil iary effects in reducing food intake (Malabu et a l , 1994; Domingo et a l , 1994). Resul ts of the present study do not support this hypothesis. First, despite an increased normoglycemic response with higher V O S 0 4 concentrat ions, no further reduction in food intake w a s s e e n . In this context, more severe ly diabetic animals appear to require higher concentrat ions of vanad ium and otherwise do not deve lop normoglycemia even after prolonged (5 month) treatment. A l though reduced food intake resulted in a higher pancreat ic insulin store and lower g lycemia, these changes were modest, and occurred only in a subgroup of (4 out of 10) D P animals. Moreover, the reduction in g lycemia in s o m e pair-fed rats may not reflect a true improvement in g lucose homeostas is , as D P - R rats remained g lucose-intolerant. In support, Blondel et al (1989) reported that unlike vanad ium treatment, pair-feeding of diabet ic rats reduced basa l g lycemia but did not e n h a n c e insul in sensitivity at submaximal hyperinsul inemia. In this study, D P and D shared strong correlat ions between g lucose and insulin levels in the fed state, which exc luded DT. Thus , al though food restriction appears to have s o m e benef ic ial effects, the mechanism(s) of g lucose- lowering by vanadium is distinct from, and hence cannot be attributed to, that of a reduction in food intake per se , at least in the present condi t ions. A number of factors may contribute to the dispari t ies be tween this and previous studies. The presence of g lucose- into lerance in D P suggests that slight reduct ions in hyperglycemia may simply reflect the feeding state of the an imals . Despi te a more c losely monitored feeding schedu le of pair-fed an imals in this study, g lucose levels actually represent 14-hour fasted levels s ince D P rats c o n s u m e d their food within 1 hour. Indeed, at 5 minutes after feeding, g lycemia was not iceably higher (>27 mM). In the previous studies (Malabu et a l , 1994; Domingo et a l , 1994), both vanad ium and pair-feeding appeared to produce parallel reduct ions in fed g lycemia . 148 However, one of the laboratories a lso reported (McKibbin et a l , 1992) that food-restricted diabetic rats ate quickly. A s bleeding w a s done routinely at 0900 h, pair-fed rats were likely fasted for at least 22 hours, having been fed at 0900 h the previous day. Interestingly, when D P rats were fasted for - 2 2 hrs prior to an O G T T , p lasma g lucose w a s lower ( -12 mM), and w a s similar to D which had been fasted for 16 hrs. Another confounding factor which could explain the var iances between this and previous studies (Malabu et a l , 1994; Domingo et a l , 1994) may involve di f ferences in severity of the diabetic state. Indeed, STZ- induced hyperglycemia w a s high (>25 mM) in both previous studies relative to the present study (<20 mM). In addit ion, whereas untreated diabetic rats in the previous studies showed a complete a b s e n c e of weight gain, body weight in D was less than control only at 4-5 weeks . In this study, a gradual decl ine in p lasma insulin and steady rise in hyperglycemia over 5 w e e k s in D suggests a more progressive diabetic state. O n the other hand , a profound weight loss in the vanadium-treated and pair-fed rats (Malabu et a l , 1994) suggests the p resence of a malnour ished and catabol ic state (Rao and Menon , 1993). W h e r e a s D P rats in this study had low triglycerides and creatinine, s igns of protein malnutrition and cachec t ic states, p lasma albumin and urea nitrogen levels were unaffected. Prev iously , vanad ium treatment has been reported to result in d iarrhea, dehydrat ion, and severe weight loss. In this study, it was observed that more severe ly diabetic rats, which had less pancreat ic insulin reserve at termination, were also prone to deve lop diarrhea and severe weight loss prior to achieving stable normoglycemia on higher concentrat ions of V O S 0 4 . Notably, these symptoms fol lowed a discernible rise in vanady l intake (50-90 ml/day) for 1-2 days prior to diarrhea, and were reversed by administer ing water for 24 hours. Hence , the reported diarrhea and lack of response to vanad ium in the drinking water (Malabu et a l , 1994) is consistent with the notion of severe ly diabet ic rats consuming high amounts of vanadium in solution. Factors such as body weight (Masiel lo et a l , 1979), and source of S T Z could have contributed to these var iances . 149 A lowered insulin secretory activity at the onset of d iabetes has been shown to preserve f i-cel l function and insulin content (Shah et a l , 1989). Thus , a similar mechan ism of vanad ium in protecting fi-cells and reversing the diabet ic state could result from its effects on reducing insulin secret ion following the induction of S T Z -diabetes. It w a s previously demonstrated that while pretreatment with vanad ium a lone does not alter the diabetic state induced by S T Z , continuing treatment for 2 w e e k s following S T Z results in a partial reversal of the diabetic state after withdrawal from vanad ium (Chapter 5). S ince the glucose- lowering effect of vanad ium treatment is not immediate, and may require severa l days , vanadium was administered for one week prior to the onset of STZ-d iabe tes . The fi-cytotoxic effect of S T Z is dose -dependen t and high doses of S T Z (>60 mg/kg) can severely deplete pancreat ic insulin content to <5% by 24 hours (Junod et a l , 1969). At lower doses , however, the initial d a m a g e to f i-cel ls is not as extensive, although pancreat ic insulin can gradual ly diminish to <5% by 28 days . The high circulating insulin in untreated diabetic rats for 2 w e e k s fol lowing S T Z suggests the presence of viable fi-cells which survived STZ- i nduced cytotoxicity, whereas the subsequent decl ine in insulin levels over 3 weeks is consistent with gradual exhaust ion of insulin stores, which was minimal (<5%) at 5 weeks . V a n a d i u m treatment, which induced a greater preservat ion of residual insulin stores at 5 w e e k s than pair-feeding, a lso reduced circulating insulin to a greater extent than food restriction both prior to (at normoglycemia) and at 3 days after (at hyperglycemia) S T Z . Interestingly, at 5 weeks , the insulin response to oral g lucose w a s low in D T despi te maximal stimulatory g lucose levels (>15 mM) at 20-30 minutes, and in spite of profound improvements in pancreat ic insulin at this time. The insulin response to i.v. g lucose has been reported to correlate highly with both pancreat ic insulin content and fi-cell m a s s in STZ-d iabet ic baboons (McCul loch et a l , 1991). In this study, strong correlat ions were found between insulin response (AUCj) and pancreat ic insulin content in the diabetic rats. This relationship in DT w a s shifted to the right of D and D P , 150 suggest ing a higher threshold for glucose-st imulated insulin response with vanad ium treatment. The modest shift in D P suggests that a reduced food intake may inf luence insulin secret ion. Indeed, food restriction lowered insulin secret ion in control and diabetic rats (Chu et a l , 1991; R a o , 1991). A more profound shift in DT rats sugges ts an exist ing fi-cell dysfunct ion, a direct or indirect inhibition by vanad ium or an enhanced insulin sensitivity. S ince chronic and glucose-st imulated insulin levels are increased in diabetic rats after withdrawal from vanadium treatment, it is likely that the preserved f i-cell mass is functional, but that a full secretory capaci ty is manifested only upon removal of vanad ium. Thus , apart from an indirect component (perhaps a reduced intestinal secret ion of incretins such as G I P or G L P - 1 ) , a direct inhibition of g lucose-stimulated insulin re lease reported at low vanadate concentrat ions (1-50 pM) in vitro (Voss et a l , 1992), could a lso occur in vivo. Converse ly , the insulinotropic effects of vanad ium have been shown at 0.1-1 m M (Fagin et a l , 1987; Zhang et a l , 1991), levels greatly exceed ing those currently detected in p lasma and pancreas (<10 uM). A 2-fold greater pancreat ic insulin content was assoc ia ted with a 2.2-fold shift in A U C / P I C in D P . Similarly, A U C / P I C w a s increased by 5.1-fold only in a subset of 7/10 DT rats, which together had a pancreat ic insulin content (156 ± 9 mU/g) 5.5 t imes higher than D. T h e s e results suggest that protection of residual insulin stores may be l inked to a suppress ion of persistent glucose-st imulated insulin re lease by vanad ium and to a lesser extent, by food-restriction. Al though hyperglycemia per se can inhibit insulin gene transcription in f i-cells (Olson et a l , 1993), hyperglycemia w a s equal ly severe in D and D P throughout most of the study. Hence , the increased insulin store in D P likely resulted from reduced insulin secret ion rather than relief from hyperg lycemia. The correlation between fed g lucose and insulin levels demonst ra tes a modulatory effect of p lasma insulin on g lycemia. The observat ion that a separate correlation of DT appeared consistently below the correlation shared by D and D P suggests an ability of vanadium-treated rats to maintain g lucose homeostas is at a lower 151 setpoint of circulating insulin. These results support the notion that vanad ium can increase insulin sensitivity and lower insulin demand in insulin-deficient an imals in the chronic fed state. Furthermore, despite a similar insulin response (AUCj) in the D P - R and D T - L C animals , A U C g in the vanadium-treated group w a s signif icantly lower than the pair-fed group. These observat ions suggest that vanad ium treatment may have effects on either enhancing insulin sensitivity or preventing the progression towards insulin resistance in the diabetic animals. Finally, the in vivo ef fect iveness of vanad ium w a s found to be dependent on the underlying diabetic state, as reflected in pretreatment circulating insulin and g lucose levels. Th is study further descr ibes a critical level of pancreat ic insulin store ( -160 mU/g), below which higher concentrat ions were required to ach ieve stable normoglycemia. Notably, 3 DT rats had a pancreat ic insulin content < 100 mU/g , levels which could not support normoglycemia in either D or D P rats. Hence , al though the effect iveness of vanad ium treatment is initially dependent on residual insulin stores, normoglycemia ach ieved with high vanad ium concentrat ions is not limited by insulin stores as low as 2 .5% of control. In summary, although the moderately beneficial effects in pair-fed diabet ic rats suggests that a reduction in food intake contributes to the overal l effects of vanad ium treatment, the degree of g lucose- lowering by food restriction is c losely l inked to p lasma and pancreat ic insulin levels, and distinct from that induced by vanad ium treatment. Moreover , al though the relative requirement for vanad ium is dependent on the level of insulin store, normoglycemia is ach ieved at extremely low insulin reserves, wh ich suggests that vanad ium may be of some use in newly d iagnosed IDDM. Furthermore, these results suggest that vanadium had an addit ional inhibitory effect on g lucose-st imulated insulin secret ion, as reflected in low insulin levels pos t -STZ and a profound shift in A U C j / P I C . It is not known whether the suppress ion of insulin re lease by vanad ium is via a direct or indirect mechan ism, but it appears to have benef ic ial c o n s e q u e n c e s in the chronic preservation of residual insulin stores. 152 Chapter 7 THE E F F E C T OF VANADIUM TREATMENT ON IN VIVO GLUT4 T R A N S L O C A T I O N IN ADIPOSE TISSUE IN STZ-DIABETIC RATS 7.1. INTRODUCTION Recent studies have demonstrated the role of structurally related facilitative g lucose transporters in the t issue-speci f ic uptake of g lucose (Klip et a l , 1992, G o u l d and Ho lman, 1993; Mueckler , 1993). G L U T 4 , one of the 6 isoforms of g lucose transporters is expressed only in peripheral t issues that respond to insulin: skeletal and card iac musc le and ad ipose t issue, and hence has also been referred to as the insul in-regulatable g lucose transporter. Found almost exclusively in intracellular s torage ves ic les at basa l condit ions, the G L U T 4 isoform is t ranslocated to the p lasma membrane upon exposure of the cell to insulin, an event which is fol lowed by enhanced transport of g lucose into the cell (Cushman and Wardza la , 1980; S u z u k i and K o n o , 1980). A l though initially reported in rat adipocytes, a similar mechan ism has been demonstrated in skeletal muscle (Klip et a l , 1987) and heart (Slot et a l , 1991). In musc le and ad ipose t issue, g lucose transport appears to be rate-limiting for g lucose utilization at low physiological g lucose and insulin levels and in d iabetes (Ziel et a l , 1988). In adipocytes, skeletal muscle and heart, G L U T 1 (responsible for basa l g lucose transport) is present in quantit ies ~10-fold less than G L U T 4 and similarly t rans locates in response to insul in, albeit at a lower level (Zorzano et a l , 1989). T h u s , in rat ad ipocytes, insul in- induced redistribution of G L U T 4 rather than of G L U T 1 more c losely paral lels the increase in transport activity (Holman et a l , 1990). The rate and extent of insul in-mediated g lucose utilization is critically dependent on cellular G L U T 4 levels, their ability to translocate to the cell surface, and intrinsic activity. Indeed, total G L U T 4 protein in nondiabet ic human skeletal muscle was found to correlate with the rate of whole-body g lucose d isposal (Koranyi et a l , 1991). 153 In STZ-d iabet ic rats, whole body insulin-stimulated g lucose uptake is d e c r e a s e d , coinciding with the reduced cellular content of G L U T 4 in ad ipose t issue (Berger et a l , 1989), skeletal musc le (Garvey et a l , 1989) and heart (Camps et al , 1992). Moreover , the reduction of G L U T 4 content in adipocytes in STZ-d iabet ic rats was accompan ied by an enhanced phosphorylat ion state (Begum and Draznin, 1992), sugges ted to diminish the ability of insulin to stimulate the intrinsic activity of G L U T 4 (Reusch et a l , 1993). Insulin treatment of diabetic rats restored cellular express ion of g lucose transporters and enhanced transport activity in isolated adipocytes (Karnieli et a l , 1987). The glucose- lower ing mechan ism of vanad ium in chronic STZ-d iabe tes has been sugges ted to include an enhanced basa l g lucose uptake (Meyerovi tch et a l , 1987). Vanad ium treatment a lso prevented insulin resistance at the level of peripheral g lucose d isposa l in STZ-d iabet ic (Blondel et a l , 1989) and partially pancreatectomized (Rossett i et a l , 1990) rats. S ince g lucose tolerance was shown to be improved in vanad ium-treated diabetic rats without concomitant increases in insulin secret ion, it w a s hypothesized that vanadium may enhance G L U T 4 translocat ion in insul in-sensit ive t issues at low physiological insulin. Indeed, studies have supported that vanadate activated G L U T 4 translocation in vitro in isolated rat adipocytes (Paquet et a l , 1992). Furthermore, vanad ium treatment restored skeletal musc le G L U T 4 protein and m R N A content in STZ-d iabet ic rats (Strout et a l , 1990), therefore, it was specu la ted that the post-insulin receptor effect of vanadate could involve induction of g lucose transporter gene express ion. In contrast, vanadate treatment of STZ-d iabet ic rats, despi te normalizing p lasma g lucose levels, did not restore cellular G L U T 4 content in ad ipocytes (Begum and Draznin, 1992). Never theless, G L U T 4 content in subcel lu lar fract ions w a s not determined in the vanadium-treated animals, and in vivo t ranslocat ion of G L U T 4 in response to insulin and/or g lucose has not been examined. Therefore, in the present study, the effect of chronic vanadium treatment on in vivo G L U T 4 translocat ion in ad ipose t issue in STZ-d iabet ic rats was investigated. 154 7.2 MATERIALS AND METHODS 7.2.1. Treatment and maintenance of animals Male Wistar rats (200-250 g) were obtained from Char les River and maintained on standard laboratory chow. The rats were divided into control and diabet ic groups. S T Z (55 mg/kg i.v.) was administered to the diabetic groups, while control received vehic le (NaC l , 154 mmol/ l , pH 7.2). At 3 days after administration of S T Z , d iabetes w a s conf i rmed by p lasma g lucose levels (> 13 mM) in the fed state. Contro l rats were divided into untreated (C, n=8) and treated (CT, n=8) groups while diabet ic rats were divided into untreated (D, n=12) and treated (DT, n=12) groups. A n O G T T w a s performed on day 5 pos t -STZ in overnight-fasted rats. The diabet ic groups (untreated vs . treated) were a s s e s s e d to have a similar range in body weight, nonfasted g lucose levels and integrated g lucose response ( A U C g ) during the O G T T . At 7 days pos t -STZ , V O S 0 4 was administered at a concentrat ion of 0.75 mg/ml in the drinking water to the D T group, and increased to 1.00 mg/ml by week 2. A t week 3, the concentrat ion of vanady l w a s increased to 1.25 mg/ml, and further raised by 0.25 mg/ml every two weeks to a max imum of 1.75 mg/ml. P l a s m a g lucose, insulin, body weight, and daily food and fluid intake were monitored. The study was terminated at 10 weeks pos t -STZ , and pancreat ic insulin extraction was carried out as previously descr ibed. 7.2.2. Oral and intravenous g lucose tolerance tests (OGTT/IVGTT) The oral g lucose tolerance test w a s performed as previously descr ibed in overnight-fasted rats. S ince the pre-treatment O G T T demonstrated a high correlat ion between A U C g and g lucose levels at 60 min (r=0.99, F ig . 7 .2C), the level of g lucose to lerance in overnight fasted rats was determined on a weekly bas is by measur ing p lasma g lucose at 60 min after a 1 g/kg oral g lucose dose . At 9 weeks , an intravenous g lucose tolerance test ( IVGTT) was performed in overnight-fasted rats anaesthet ized with sod ium pentobarbital (30 mg/kg i.p.). Rats were administered g lucose (0.5 g/kg) 155 into the tail ve in. B lood samples were col lected prior to (time 0) and at 5, 15, 30 and 60 min following the g lucose dose . P l a s m a was ana lyzed for insulin and g lucose as previously descr ibed. The g lucose d isappearance rate constant ( K g ) w a s calculated as the s lope of the least squares regression line relating the log-transformed g lucose concentrat ion to time from 5 to 30 minutes after the g lucose bolus during the IVGTT. Kg is expressed as percent per minute (%/min). 7.2.3. Subcel lular fractionation of adipose t issue At 10 weeks , overnight fasted rats were anaesthet ized with sod ium pentobarbital (30 mg/kg, i.p.) and administered g lucose (0.5 mg/kg, i.v.). At 5 minutes, an imals were bled from the tail vein for p lasma insulin levels, and at 7.5 minutes (previously determined to be the time of maximal ad ipose t issue P M G L U T 4 content in response to i.v. g lucose) , rats were killed by guillotine and ad ipose t issue removed for subcel lu lar fractionation. The fractionation procedure is modif ied from the methodology used for isolated adipocytes (McKee l and Jarett, 1970; S impson et a l , 1983), and is outl ined in Figure 7.1. Four epididymal fat pads (2 rats) were used per preparat ion. T i ssue homogenizat ion and subsequent handling ef f ract ions was performed in H E S buffer (20 m M Hepes , 1 m M E G T A , 255 m M sucrose; pH 7.4) at 4°C. Fat pads were r insed in 10 vo lumes of H E S to remove blood, minced in 10 ml H E S buffer, and homogen ized v ia polytron homogenizer (2 x 1 s bursts, position 8). Ad ipose t issue homogena tes were centri fuged at 3,000 rpm (1,000 xg, J A - 1 7 rotor) for 5 minutes to separate fat and cel l debr is. The infranatant containing crude membrane (CM) was removed using a 10 ml syr inge and 16 G needle and centrifuged at 11,000 rpm (17,500 xg, J A - 1 7 rotor) for 30 minutes. The supernatant (A) was further centri fuged at 21 ,000 rpm (50,000 xg, Ty 65 rotor) for 20 minutes and the resulting pellet (B) w a s resuspended and centr i fuged at 17,000 rpm (50,000 xg, J A - 1 7 rotor) for 60 minutes to obtain high density microsomal (HDM) membranes . The pellet (A) from the 11,000 rpm centrifugation step w a s w a s h e d 156 twice by resuspens ion in 10 ml H E S and centrifugation at 11,000 rpm (17,500 xg, J A - 1 7 rotor) for 15 minutes. The pellet was resuspended in 4 ml H E S and layered on top of a d iscont inuous sucrose gradient made up of 4 ml each of 0.8 M and 1.12 M suc rose and centr i fuged at 35,000 rpm (150,000 xg , SW-41 rotor) for 40 minutes. P l a s m a membrane (PM) w a s col lected from the 0.8/1.12 M interface and centr i fuged in 10 ml H E S at 17,000 rpm (50,000 xg, J A - 1 7 rotor) for 60 minutes. The supernatant (B) on top of pellet (B) was centrifuged at 60,000 rpm (200,000 xg, Ty 65 rotor) for 60 minutes. The pellet containing low density microsomes (LDM) was further purified by the method of J a m e s et al (1987) to obtain the G L U T 4 enr iched fraction. The pellet w a s resuspended in 4 ml of buffer containing 0.1 M sucrose, 2 m M M g S 0 4 and 1 m M E G T A (pH 7.4), appl ied on a discont inuous gradient containing 0.4 M and 1.5 M suc rose and centrifuged at 35,000 rpm (150,000 xg, SW-41 rotor) for 40 minutes. The purified L D M fraction was col lected at the 0.4/1.5 M interface and centr i fuged at 60 ,000 rpm (200,000 xg, Ty 65 rotor) for 60 minutes. Al l final pellets were resuspended in 200 pi of H E S buffer, f rozen in liquid nitrogen, and stored at -70°C for G L U T 4 analys is . 7.2.4. Membrane marker enzyme assays E n z y m e marker analys is was performed on fresh subcel lu lar fractions. A marker for endop lasmic reticulum, NADH-cy tochrome c reductase, was measured v ia the spectrophotometr ic method of Strobel and Dignam (1978). Briefly, ox id ized cytochrome c (from bovine heart, C -3131 , S igma) was diluted to 5 mg/ml in 0.2 M potass ium phosphate buffer (pH 7.5) and ft-NADH (disodium salt, S igma) d isso lved to a f inal concentrat ion of 3 mg/ml in distilled water. Both substrate and cofactor were kept at 4° C . In a g lass microcuvette, 0.85 ml of 0.2 M phosphate buffer, 50 pi of cy tochrome c, 20 pi of 10 m M K C N , 10 pi of 2 mg/ml antimycin A (Sigma), and 20 pi of samp le (20 pg) were added , mixed by inversion and preincubated for 2 minutes at 25°C in a thermostatted cell compartment. Fol lowing an initial absorbance reading ( N A D H -157 Fat Pad (Minced in 10 ml HES) Polytron ( 2 x 1 sec bursts) 3,000 rpm [5 min) Infranatant Crude Membrane (CM) 11,000 rpm (30 min) Supernatant (A) 21,500 rpm (20 min) Pellet (A) 11,000 rpm (15 min) Pellet Supernatant (6) Pellet (B) 60,000 rpm (60 min) 17,000 rpm (60 min) 11,000 rpm (15 min) Pellet Pellet 35,000 rpm (40 min) (S/0.4/1.5) Interface 60,000 (60 min) Pellet Low Density Microsomes (LDM) Pellet 35,000 rpm High Density Microsomes (40 min) (HDM) (S/0.8/1.12) Interface 17,000 rpm (60 min) Pellet Plasma Membrane (PM) Pellet Figure 7.1 rat fat pad Modified protocol for subcel lu lar fractionation of whole epididymal S e e methods sect ion for detai ls. (S = sample) 158 independent rate) at 550 nm for 30 seconds , the enzymat ic reaction w a s started by adding and rapidly mixing 50 pi of N A D H into the reaction mixture and the change in absorbance at 550 nm (characterizing formation of reduced (ferrous) cy tochrome c) w a s monitored for 30 seconds . E n z y m e activity per mg protein was calculated from the initial rate (0-20 seconds) , subtracted by the NADH- independen t rate. Ouaba in -sensit ive N a + / K + - A T P a s e activity was measured as descr ibed by Gangu ly et al (1983). 7.2.5. GLUT4 analysis In order to more quantitatively detect subcel lu lar membrane G L U T 4 content, a new competit ive E L I S A method was des igned for this study. Lyophi l ized G L U T 4 C -terminus pept ide ( C T E L E Y L G P E N D , Eas t A c r e s Bio logica ls , Southbr idge, M A ) w a s initially made up to 125 pg/ml in deionized distilled water (pH 8.0) and stored in al iquots at -70°C. The coating antigen was prepared by diluting the peptide solution 1:10 in 0.1 M sod ium carbonate-bicarbonate buffer (pH 9.5) and adding 50 ul/well of di luted peptide to high-binding Maxisorp 96-well E L I S A plates (Nunc, Denmark) . Coa ted plates were baked overnight ( -12 hours) at 50°C in a dry oven , whereas all subsequent incubation s teps of the coated E L I S A plate were performed at 37°C in a shak ing , covered water bath. The coated plates were washed 3 t imes in w a s h buffer (0 .1% Tween in modified phosphate-buffered sal ine (KBS) : 0.138 M N a C l , 0.01 M K H 2 P 0 4 , 0.01 M N a 2 H P 0 4 , 0 .05% Tween) and b locked for 1 hour with 10% goat se rum in K B S . Compet ing antigen (subcel lular membrane fractions) was solubi l ized in 1% Triton X -100. 60 pi w a s added to the first well in each row of untreated V-wel l plates (Evergreen Scient i f ic, L o s Ange les , C A ) , and serial ly diluted (1:2) in 30 pi of dilution buffer ( 1 % Triton X -100 in K B S ) . Monoc lona l an t i -GLUT4 antibody (1F8, Eas t A c r e s Bio logicals) was diluted 1:3000 in antibody dilution buffer (0.5% B S A ( E L I S A grade, S igma) , 1% S k i m milk in K B S ) and 30 pl/well added to the compet ing ant igen mixture 5 minutes prior to transferring to the b locked, pept ide-coated plates. Fol lowing b locking, the 159 coated plates were washed 4 times, and 50 pi of antigen/antibody mixture was carefully pipetted to each well. Following incubation for 6 hours, plates were washed 3 times and further incubated for 2 hours with 100 pi of peroxidase-linked sheep anti-mouse Ig F(ab')2 fragment (Amersham, Arlington Heights, IL), diluted 1:1000 in antibody dilution buffer. After washing 3 times, 150 pi of substrate solution (2 mM o-phenylenediamene (Sigma) and 0.01% H 2 0 2 in 0.1 M citrate buffer, pH 5.0) was added and incubated for 30 minutes. The enzyme reaction was stopped with 40 pi of 8 N H 2 S 0 4 . Absorbance was measured at 490 nm using a microplate reader (Biotek Instruments, Model EL309). Curve-fitting and calculation of data was performed via Fig. P Scientific Processor (Fig. P Software Corp., Durham, NC). 7.2.6. Protein assay Membrane protein was assayed by the fluorescamine method (Bohlen et al, 1973) adapted for use to a fluorescence plate reader (Lorenzen and Kennedy, 1993). To overcome the interference of high detergent concentrations on the protein assay, appropriate volumes (0.5-6 pi) of protein standard (1.5 mg/ml y-globulin) and Triton-solubilized membranes were pipetted into a microplate and made up to 100 pi with 1% Triton. At time 0, 50 pi of 0.2 N Boric Acid was added to each well and mixed for 1 minute. At 7 minutes, 75 pi of 0.3 mg/ml fluorescamine (Sigma) in acetone was added and mixed for 10 sec. Fluorescence was read at 18 minutes by a multiwell plate reader (Millipore Cytofluor 2350, excitation X = 360 ± 20 nm, emission X = 460 ± 30 nm). 7.2.7. Statistical analysis Two-way analysis of variance (ANOVA) was used followed by the Fisher's LSD test. Linear and nonlinear regression analysis was performed using the Fig.P Scientific Processor. Data are expressed as mean ± S.E.M. unless otherwise specified, p < 0.05 was considered significant. 160 7.3. RESULTS 7.3.1. General characteristics of diabetic animals prior to vanadium treatment The 55 mg/kg dose of S T Z induces a variable degree in the severity of d iabetes, hence, it was important to establ ish that diabetic animals, prior to being ass igned to the untreated and treated groups, exhibit similar characterist ics of the diabetic state. At 5-7 days after S T Z , fed p lasma g lucose and insulin levels were measured , and a n O G T T was performed in all the animals. Indeed, diabetic animals during the week fol lowing S T Z showed a wide range in the levels of fed p lasma g lucose (17.8 - 31.7 mM) and oral g lucose tolerance ( A U C g = 740.6 - 1139 mM-60 min). The diabetic an imals were subdiv ided such that both D (n=12) and DT (n=12) groups had initial levels of g lucose to lerance (Fig. 7.2), body weight (Fig. 7.3A), and fed p lasma g lucose (Fig. 7.4A) and insulin (Fig. 7.4B) which were not significantly different at week 1, prior to treatment. 7.3.2. Effects of vanadium on body weight, food and fluid intake At 1 week pos t -STZ, vanadyl treatment was started at 0.75 mg/ml and increased gradual ly over 10 weeks to 1.75 mg/ml in C T and DT. Vanad ium treatment lowered body weight gain in both C T and DT animals, and this w a s lowest in DT (4 g/week) at the highest concentrat ion (Fig. 7.3A). At 10 weeks , body weight in C T w a s not significantly different from D and was lowest in the DT group. Throughout the study, food intake in the D group was 3 0 % greater than all other groups and w a s significantly lowered by vanad ium treatment to levels which were not different from control at 10 weeks (Fig. 7.3B). Fluid intake w a s significantly higher (by 75%) in D relative to C , whereas vanad ium treatment lowered fluid intake in the DT and C T groups to 5 0 % that of C (Fig. 7.3C). Rais ing the V O S 0 4 concentrat ion did not affect food or fluid intake over an prolonged period. Thus , food intake in C T and DT was not signif icantly different from control at 10 weeks (at 1.75 mg/ml) and at week 1 (at 0.75 mg/ml). Fluid intake was similarly unaffected by raising V O S 0 4 concentrat ions. 161 o o CO ID CM O CM i n I m o o m o in CM o o o o m o o m o m CM c E o O (Win) ,09 ie a s o o n / £ > euiseid m o co o m o o CO o CM L- O c J 00 LO CM O CM I LO I o LO ~I o o oo o CO o o CM o fiuj/nrl) uijnsui euiseid • .2 -4—' . CO O g> 1 8 C CO o § o o "§ » I S = 5 • ^ O LO r- O | . E d 1 8 o (0 o Q. (0 > CD _ CO O o to * - o Q . 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V O S 0 4 concentrat ions administered are as indicated above the x-axis. 163 7.3.3. Effects of vanadium on glycemia and g lucose tolerance Glycem ia w a s normal ized with vanad ium at concentrat ions of 0.75 to 1.25 mg/ml (< 9.0 mM) in all DT animals, and was unaffected in the C T group (Fig. 7.4A). In the D group, there was a trend towards lowering of average p lasma g lucose levels over severa l weeks , a result of spontaneous reversion towards normoglycemia in 6 out of 12 animals (D-R), starting from 3 weeks pos t -STZ administration (Fig. 7.5A). The relatively mild diabetic state induced in the D group was also evident in the fed p lasma insulin levels, which was only lower in D relative to C at weeks 6 to 10 (Fig. 7.4B). V a n a d i u m treatment reduced p lasma insulin to the s a m e level in C T and DT groups, which at 10 weeks was 4 0 % of C . W h e n separated into subgroups, p lasma insulin levels in the "reverted" (D-R) untreated diabetic group were higher than D T or non-reverted D - N R groups during weeks 6-10 (Fig 7.5B), and were not significantly different from controls. S i n c e A U C g correlated c losely with g lycemia at 60 minutes (r = 0.99) (F ig. 7 .2C), the degree of g lucose tolerance was determined via weekly readings at 60 minutes post-oral g lucose load in fasted animals. DT rats demonstrated an improved g lucose tolerance from 2 weeks pos t -STZ which was significant from D (Fig. 7 .4C). D-R rats a lso demonstrated an improved g lucose tolerance at a similar t ime as DT, which occurred prior to the onset of normoglycemia in these animals (Fig. 7 .5C). Despi te an attempt to correct g lucose tolerance in the DT group beyond that of the untreated D-R group by further raising the V O S 0 4 concentrat ion to 1.75 mg/ml, DT maintained a slight g lucose intolerance relative to control, and was similar to D-R at week 8. To el iminate any effects of vanadium on the oral absorpt ion of g lucose (Madsen et a l , 1993), an IVGTT was performed at week 9 (Fig. 7.6). Fast ing g lucose and A U C g were significantly lower in D T and D-R relative to D-NR, and not significantly different from control (Fig. 7 .6A,C) . Peak p lasma insulin at 5 minutes and A U C j in C T were lower than C (Fig 7 .6B.C) . A l though the peak insulin levels were simi lar be tween the diabet ic groups, A U C j was reduced in D -NR relative to D-R. 164 3 5 A 0.75 1.00 1.25 1.50 1.75 £ U I — I — I — I — I — I — I — I — I — I — I 0 1 2 3 4 5 6 7 8 9 10 Weeks Post-STZ Figure 7 .4 . Effects of vanadium treatment on fed plasma g lucose and insul in levels and on oral g lucose tolerance in STZ-diabetic rats P l a s m a g lucose (A), insulin (B) and g lucose tolerance a s est imated by p lasma g lucose at 60 minutes following a 1 g/kg oral g lucose dose (C) in control (C, • ) , control-treated (CT, • ) , diabet ic (D, O ) , and diabetic-treated (DT, • ) groups. (* p < 0.05 vs . C ; + p < 0.05 vs . D). V O S 0 4 concentrat ions administered are as indicated above the x-axis. 165 2 CD to O O E </> CU 30 - i 25 -20 -15 -10 -5 0 J =3 =3. 3 to . C E to 0L 60 n 40 H 20 H B 6 o CO CO to o o 3 E to 25 20 15 10 -5 -0 -0.75\1.00\ 1.25 1.50 1.75 I 1 1 1 1 1 1 1 1 1 I 0 1 2 3 4 5 6 7 8 9 10 Weeks Post-STZ Figure 7.5. Spontaneous reversion in STZ-diabetic rats: chronic fed g lycemia, p lasma insul in and oral g lucose tolerance levels P l a s m a g lucose (A), insulin (B) and g lucose tolerance as est imated by g lycemia at 60 minutes fol lowing a 1 g/kg oral g lucose dose (C) in diabetic "reverted" (D-R, 0), d iabet ic "non-reverted" (D-NR, • ) , and diabetic-treated (DT, • ) groups. (* p < 0.05 vs . C) . V O S 0 4 concentrat ions administered are as indicated above the x-ax is . 166 * * o o o o LT) o o o o o I o o o o t o o o CO o o o C M o o o - 1 o (uiui "onv (WUJ 09-iw/nrf) !onv o CO O ID ID O c J .EE m CM o CM i n o m - 1 o o o o o o CM O <X> CD "<* o o CM (Viiu) asoonjQ euusejd (iuu/nri) ujjnsuj evuseid c to O CD CO O 5 > o O B I P 2 + • c o o TJ CD -+-» CO .CD CD CO o TJ CD CO o O cn > o> •-CD CD t ^ ID O > g ^ o w & CD CD <-> a> c o — C _co 1 ^ +•>„ .. CD CQ CO O C O ~ 3 3 — CO O) c CO ' 3 -a O c c CD * J CD ^ £ co • o • co 5 " 5 ) ^ a) 2 -o i - E a) 3 OT m u. a . j= • o a £ 73 P CD O to V O Q . •v * o ' U—» CD >, •S a5 .2 > 73 CD C CL CD CO ^ CD • r r co Z c 6 8. -— co = CD 73 CD •e o > 2 4— ' ^ c .E - C CO ~ c O CD CD O > O CD 3 i _ — = CD O 73 CD cu -£ -Q CD .2 CD 73 CD O 'CL CD 73 73 C CD 167 Kg was not significantly different between C and C T , whereas K g w a s markedly reduced in D -NR to 2 0 % of C . K g w a s 2.5-fold greater in D-R and DT; however , it w a s still - 4 0 % less than C (Fig. 7.7A). K g in the various groups was significantly correlated to the integrated insulin area from 5-30 minutes (Al) (r = 0.69, p < 0.0001) (Fig. 7.7 B). 7.3.4. Characterization of adipose t issue subcel lular fractionation The enzyme marker activities characterist ic of endop lasmic reticulum ( E R ) and p lasma membrane were measured in the membrane fractions prepared as descr ibed (n=6). The activity of the E R marker, cytochrome c reductase, was consistent ly higher in the H D M fraction (1.48 ± 0.03 pmol/min/mg) than in P M and L D M (0.26 ± 0.02 and 0.83 ± 0.05 pmol/min/mg, respectively). Ouabain-sensi t ive N a + / K + - A T P a s e activity detected in the L D M and H D M fractions was <10% and - 5 0 % of P M , respect ively. Thus , the relative contamination of p lasma membrane with E R , and of low densi ty m ic rosomes with P M was similar to that previously reported for isolated ad ipocytes (S impson et a l , 1983). However, the H D M fraction appeared to have a higher contaminat ion with P M than the 12% reported in the previous method (Weber et a l , 1988). Th is could be attributed to a greater cel lular disruption in initial homogenizat ion of ad ipose t issue via a polytron homogenizer unlike the gentler homogenizat ion method via a Tef lon pestle grinder for isolated adipocytes. Indeed, inc reased c ross -contaminat ion of E R in the P M fraction (to 40%) and increased P M - G L U T 4 content w a s found with longer homogenizat ion t imes (> 2 sec) . The recovery of protein from 2 rats w a s 0.26 ± 0.02, 0.46 ± 0.02 and 0.22 ± 0.01 mg for the P M , H D M and L D M fract ions, respectively. The comparat ively higher recovery for the H D M fraction (15% of homogenate) from ad ipose t issue relative to isolated adipocytes (3%) could a lso be attributed to the net loss of P M to the H D M fraction. There is no known marker for L D M other than G L U T 4 , which was enr iched in this fraction at least 30-fold, 10-fold and - 2 -fold the levels found in C M , P M and H D M , respectively. 168 Figure 7.7. Relationship between glucose disappearance rate and integrated insul in release over 30 minutes (A) G l u c o s e d isappearance rate ( K g ) and (B) correlation between K g and integrated insulin response (Al) from 5-30 minutes in control (C, • ) , control-treated (CT, • ) , "non-reverted" diabetic (D-NR, • ) , "reverted" diabetic (D-R,0), and diabetic-treated (DT, • ) groups (r = 0.69, p < 0.0001). (* p < 0.05 vs . C ; + p < 0.05 vs . D). . 169 7.3.5. Quantitation of GLUT4 in subcel lular fractions by competitive ELISA Figure 7.8 depicts typical curves generated for the competi t ive E L I S A des igned in this study for the detection of G L U T 4 in subcel lular membrane fractions. The epi tope specif ical ly recognized by the monoclonal antibody (1F8) is thought to be the carboxy terminal end (James et a l , 1988). This was confirmed by proportionately greater binding of ant ibody to increased amounts of synthetic C-terminus peptide ant igen coat, and by the competit ion of antibody-binding with synthetic peptide (Fig. 7.8). However , it was further determined that at least in the a s s a y condit ions used , the relative affinity of ant ibody for native G L U T 4 protein (in membrane fractions) w a s 10 3 - fo ld higher than that for the synthetic peptide, thus precluding use of the peptide a s a s tandard for the absolute quantitation of G L U T 4 in subcel lular membranes . Never the less, the E L I S A method would still al low for determination of relative amounts of G L U T 4 protein with greater accuracy as compared to Western Blot, and with more specif icity relative to cytochalas in B binding, which does not dist inguish between different isoforms of g lucose transporters. Furthermore, the E L I S A method w a s highly reproducible, having inter- and intra-assay coefficients of variation of < 10%. Thus , the relative amounts of G L U T 4 per mg protein in solubi l ized subcel lular membrane fractions could be determined against a s ingle internal standard (e.g. crude membrane) . B e c a u s e of the marked di f ferences in enrichment of G L U T 4 in the var ious ad ipose t issue subcel lu lar fractions, the ideal amount of protein required to run dupl icate E L I S A a s s a y s w a s for P M : 150 pg, L D M : 30 pg, H D M : 60 pg, and C M : 300 pg. From an initial set of exper iments (n=2), it w a s ascerta ined that G L U T 4 content in the p lasma membrane fraction increased by - 5 0 % in vivo in response to an i.v. g lucose (0.5 mg/kg) cha l lenge in overnight fasted control rats (Fig. 7.8, inset). Thus , i.v. g lucose-st imulated P M G L U T 4 content was found to be maximal at 7.5 minutes and returned to basa l levels at longer t ime points (data not shown). A slightly higher degree of t ranslocat ion w a s found at 5 minutes following 10 U/kg i.v. insulin (data not shown). 170 o in —1 t~ o u> (maiojd Bui/siiun a/ije/aa; joejuoo W/1T5 r -LO LO d q d q d o q d tut/ 06f7 ie aoueqjosqy c o ro c E CD 0) Q c CD ** c o u c CD E CD *§ E O C L T J o x: CD E < to CD C CO 1_ XI E CD E CO E CO _co Q. CD Z> CO CO . CD CD CO CO o o .9-CL O -a ET 3 co j CO TJ O > g 3 CO E LO d c CO CT c o CD U X! 3 0) _ -O CO a E (0 w a> o _ o c 8 s CO Q . W CD * - Q. 0) CO CO 3 O C ~ E to a) = 6 0) CD O cn c c CD CD O C O CO "<t CO H c ~3 =^ - CD co a) "3 c E . CO < CO — 1 _ LO o TJ O c o CO CM • £ ' CD LO E •2 o ° +- c CO CD CO o _ ^ -1—' I c =1 o LU CD > CD a E o o 00 1^  £ 3 CO Q . E o o CO > o TJ 2 Q) Q . "co i -co o >+- M— co 3 CD c re XI Ii c o re 92 E ^ to CO •S ® Q. O c re co E -e "5 ° m CO E < CD > O E o CO CD 1_ CO o X CO TJ tf) i £ co I « o £ CD I § J= CT 171 7.3.6. Effect of chronic vanadium treatment on maximal GLUT4 translocation in vivo in response to an i.v. g lucose load in control and STZ-diabetic rats At 10 weeks , overnight fasted animals were administered g lucose 0.5 mg/kg into the tail ve in, bled from the tail vein at 5 minutes, and ad ipose t issue removed at 7.5 minutes. Total cel lular G L U T 4 content (in crude membrane) was not signif icantly different among the var ious groups (Fig. 7.9A). G L U T 4 content in p lasma membrane w a s significantly lower in C T (by 20%) and in the diabetic groups (by 40%) relative to C . However , P M G L U T 4 content did not differ between the diabetic groups (Fig. 7.9B). L D M G L U T 4 content was not significantly different between D -NR and C , and w a s higher in control-treated (by 60%) and in D-R and D T groups (by 40%) relative to the untreated control and D -NR groups, respectively (Fig. 7.9D). The degree of change in H D M G L U T 4 content in the various groups was similar to L D M (Fig. 7 .9C). 7.3.7. Relationship between residual insulin store, g lycemia, A U C g and GLUT4 Pancreat ic insulin content in the various groups were 18% (D-R), 1 1 % (DT) and 3 % (D-NR) of control (Fig. 7.1 OA). To examine the effect of the severity of the diabet ic state perse on the degree of g lycemic control, relat ionships between fed g lycemia , and A U C g with residual insulin content (Fig. 7 .10B.C) were determined, and found to be similar to those shown previously (Chapter 6). It appeared that the D-R and D T an imals which had relatively greater pancreat ic insulin stores a lso p o s s e s s e d a higher G L U T 4 content in the L D M and H D M fractions; thus, a relationship between subcel lu lar G L U T 4 and insulin content was confirmed (Fig. 7.11). There w a s a signif icant correlat ion between residual insulin store and G L U T 4 levels in intracellular stores, L D M (r = 0.87, p = 0.0002) (Fig. 7.11A), and H D M (r = 0.67, p < 0.02) fractions, al though not in P M (p > 0.05) (data not shown). Instead, P M G L U T 4 content w a s found to be highly correlated with ambient p lasma insulin levels prior to removal of ad ipose t issue (r = 0.85, p < 0.0001) (Fig. 7.11B). 172 fuiaiOJd Bui/siiun akiiBiayj IUBIUOO Pimo nd (ma)OJd Bui/siiun e /^je/ey; loejuoo pxnis nan (uiBiojd Bw/siwn OApeieyl j ua juoo trimD *VO (uiBiojd Bui/stiup BAiieiey) »UBIUOO trims *VOH 6 2 to -2^  8 ' g 3 CD B ? > S> — Ic c ^ « O CO II to 2 CU _ Q "3 E • i ! 5 1 r— co 4-1 CO to . J S O ^ l l O P IT ° CO C L 2 co CD Q co -—^  8 -° 3 3 °> $ > V •— o CO CO E T3 LO o co co CO , c "5 o o _to 3 0 C CO CD - Q S E Xi 3 (0 CU 3 to to CD E CD T J i — O "co CD _ Q CO T J T J CD •c CD > CD ".t3 - — • CO tu S " to g co a. c a: 2 Q o ^ o a) - Q co S- 5 a -o TJ .2 re TJ CO ' T J _ CD c 2 n i t § 3 •4-" _ J o g I- CO 3 c _ l — O c CD •4—' CD o £ ? i l O CD > CD M 2 t £ o co E =c Q c i n CD o "? d | "Jo v T J C c o CO CO o > 173 c C o O 400 300 200 H 100 H 18% 3% 11% D-NR D-R DT 5 E o u 3 5 CO E «J 30 25 20 15 10 5 0 B o° o 8 2000 | 1500 o | 1000 o -1 1 1 1 0 200 400 600 500 H r = - 0 . 8 5 - i 1 1 1 0 200 400 600 Pancreatic Insulin Content (mU/g) Figure 7.10. Relationship between residual pancreatic insul in content, fed p lasma g lucose levels and A U C g (A) Pancreat ic insulin store in STZ-d iabet ic animals, showing "non-reverted" diabet ic (D-NR) , "reverted" diabetic (D-R) and diabetic-treated (DT) groups, and correlat ion with (B) fed p lasma g lucose levels and (C) g lucose response ( A U C g ) during an I V G T T in: diabet ic (D, O), and diabetic-treated (DT, • ) rats. 174 Plasma Insulin at 5' (yU/ml) Figure 7.11. Relationships between residual insul in store with LDM GLUT4 content, and between plasma insulin levels and PM GLUT4 content (A) Correlat ion between pancreat ic insulin store in diabetic an imals with G L U T 4 content in L D M (r = 0.87, p = 0.0002) (B) Correlat ion between P M G L U T 4 content and p lasma insulin levels at 2.5 min prior to sacrif ice (r = 0.85, p < 0.0001), showing: control (C, • ) , control-treated (CT, • ) , diabetic (D, O ) , and diabetic-treated (DT, • ) rats. 175 7.4. DISCUSSION The current study examined a possible mechan ism for the improved g lucose to lerance observed in STZ-d iabet ic rats following chronic vanad ium treatment. Notably, the diabet ic state induced in this study w a s mild; i.e. fed insulin levels were similar to control until 6 weeks , and 5 0 % of untreated diabetic rats reverted spontaneous ly to normoglycemia. There were sufficient dif ferences within the untreated diabet ic group to subclass i fy them either as "reverters" (D-R) or "non-reverters" (D-NR) . D-R rats had improved fed normoglycemia, A U C g and K g relative to D -NR rats. M e a n pancreat ic insulin content of D-R w a s -6- fo ld that of D -NR (<4%) and w a s reflected in an improved ability of D-R rats to secrete insulin in the fed state and in acute response to g lucose . The high rate of reversion may be due to the particular lot of S T Z used , s ince the s a m e S T Z dose in previous studies produced stable d iabetes in >90% of the an imals (Chapter 6,7). Spon taneous recovery has been reported following low d o s e s of S T Z , 30-40 mg/kg (Junod et a l , 1969). Insulin content in untreated D-R rats w a s greater than that observed in the vanadium-treated group, which may have been a c o n s e q u e n c e of hypertrophy and hyperplasia of uninjured f i-cells in response to high g lucose (Bonner-Wei r et a l , 1989), a phenomenon also reflected in an apparent beneficial effect of hyperglycemia in rats with a mild ft-cell injury (Thibault et a l , 1992). It is poss ib le that s o m e of the antidiabetic act ions of vanad ium may be m a s k e d by beneficial effects secondary to an improved pancreat ic insulin content per se. However , if vanad ium had insulin-like or insul in-enhancing activity in vivo, one would expect that g lucose tolerance in DT would be improved beyond an intermediate level attained by a spontaneous recovery of insulin stores a lone. Interestingly, the g lycemic status of vanadium-treated rats was virtually indist inguishable from rats wh ich had spontaneous ly recovered from diabetes; i.e. no significant di f ferences were detected between D-R and DT according to fed and fasted g lycemia, A U C g and K g levels. Importantly, raising V O S 0 4 concentrat ions beyond that which normal ized fed g lycemia 176 did not further improve the ability of DT animals to acutely d ispose of a g lucose load. Thus , al though K g in D T was 2.5-fold greater than D-NR, it was not different from D-R, and remained at 6 0 % of control. Similarly, Blondel et al (1989) showed that vanad ium did not normal ize K g in STZ-d iabet ic rats despi te enhanc ing insulin sensit ivity at the level of hepatic g lucose production and peripheral g lucose utilization during a hyper insul inemic c lamp. Thus , it appears that correct ion of insulin res is tance at high susta ined insulin levels may not manifest in an improved g lucose to lerance in a setting of low insulin. Whi le the rate-limiting step(s) for g lucose d isposa l at low insulin levels are thought to include g lucose transport (Ziel et a l , 1988) and g lucose phosphorylat ion (Manches ter et a l , 1994), at higher insulin levels, intracellular g lucose metabol ic pathways become important (Kubo and Foley, 1986). Recent ly , g lucose process ing during an I V G T T w a s found to involve g lucose oxidation with no apparent c h a n g e in musc le g lucose storage, unlike during an insulin c lamp wherein g lycogen synthes is b e c o m e s a signif icant mechan ism of g lucose d isposa l (Henr iksen et a l , 1996). Thus , the enhanced insulin sensitivity in vanadium-treated diabetic an imals as measured during high, susta ined insulin levels, relative to the more limited effects fol lowing a n acute, dynamic rise in insulin (during an IVGTT) could be attributed to the preferential effects of vanad ium on enhanc ing nonoxidat ive g lucose d isposa l (Rossett i et a l , 1990). Notably, al though vanadium-treated animals demonstrated a 7.8-fold increase in pancreat ic insulin stores, and fed insulin levels which were not different from control , the reversal of insulin resistance was not cons idered to be assoc ia ted to an improved f i-cel l function (Blondel et a l , 1989). In this study, A U C g levels in the diabet ic rats were assoc ia ted to the residual insulin stores, as previously shown (Chapter 6). S i n c e this relat ionship w a s a lso s e e n in DT, it appears that at least s o m e effects of vanad ium on g lucose tolerance may be linked to an improved residual insulin store. The observat ion that A U C g remained slightly above control in D-R and D T is consistent with the notion that an impaired g lucose tolerance is manifested when the residual insulin store is 177 reduced to <26% of control (Kramp and Burr, 1981). B e c a u s e a reduction in fed glycemia in the DT group occurred prior to that seen with spontaneous recovery of the diabetic animals, it appears that vanadium could have affected fed g lucose levels by enhanc ing basal g lucose transport (Meyerovitch et a l , 1987) or by inhibiting g lucose output (Bruck et a l , 1991). In support of direct, short-term effects, infusion of B M O V acutely lowered g lycemia in STZ-d iabet ic rats within 30 minutes (Yuen et a l , 1995). After an i.v. g lucose load, over 70-80% of administered g lucose is taken up by peripheral t issues, primarily muscle (DeFronzo et a l , 1983). Both insul in-dependent g lucose uptake (combined effects of insulin sensitivity and incremental insulin response to g lucose) and g lucose effect iveness were found to be strong determinants of the g lucose d isappearance rate, K g (Kahn et al , 1994). In this study, incremental insulin re lease correlated well with K g , support ing the role of insul in-mediated effects. O n the other hand, g lucose uptake via mass act ion, although not dependent on ambient insulin levels (Del Prato et a l , 1995), w a s found to be dec reased in IDDM (F inegood et a l , 1990) and in STZ-d iabe tes (Tobin and F inegood, 1993). Thus , the observat ion that K g in DT animals did not exceed that of D-R suggests that vanad ium, despi te normal iz ing chronic g lycemia, may have limited effects on acute insulin- or g lucose-media ted g lucose transport in vivo in STZ-d iabet ic animals. However, K g reflects not only the rate of peripheral g lucose uptake, but also the inhibition of hepatic g lucose output (Steele et a l , 1968; A d e r et al , 1985). Thus , specif ic changes in insul in-mediated g lucose transport is difficult to extract from K g va lues a lone. Present ly , the effect of vanad ium on G L U T 4 translocation in vivo in an insulin-sensit ive t issue w a s examined . P l a s m a membrane G L U T 4 content in ad ipose t issue following a g lucose chal lenge w a s determined to reflect the extent of g lucose transport in an imals with a reduced insulin secretory capacity. The 5 0 % rise in P M G L U T 4 following i.v. g lucose in control an imals is similar to that reported in skeletal muscle after an oral g lucose load (Napol i et a l , 1995). G lucose was administered i.v. to eliminate the possibi l i ty of an 178 inhibitory effect of vanadium on oral absorpt ion of g lucose (Madsen et a l , 1993). S i nce skeletal musc le m a s s represents 3 6 - 4 0 % of body weight, it is a lso the major site of g lucose d isposa l , account ing for - 7 0 % of the load, whereas ad ipose t issue and heart account for <10% (Kel ley et a l , 1988). Never the less, ad ipose t issue is highly responsive to insulin and displays similar cellular mechan isms as skeletal musc le for the acute glucose-transport response to insulin (Kahn and C u s h m a n , 1985). Moreover , the subcel lu lar fractionation procedure in adipocytes is better character ized relative to skeletal musc le (Zorzano et a l , 1996). Al though pentobarbital anes thes ia reduces g lucose utilization in postural musc les such as so leus by ~4-5-fold, other t i ssues such a s white ad ipose t issue and d iaphragm are unaffected (Pen icaud et a l , 1987). The strong correlation between P M G L U T 4 content and p lasma insulin supports the notion that G L U T 4 translocation is highly sensit ive to, and dependent on , ambient insulin levels. The inclusion of vanadium-treated animals in this correlation contradicts the notion that vanad ium can enhance insulin signal ing in vivo at the level of G L U T 4 transporter translocation in ad ipose t issue. Previously, vanad ium was shown to activate g lucose transport a lone and enhance the effects of insulin on g lucose uptake in isolated rat adipocytes (Dubyak and Kleinzel ler, 1980; Er iksson et a l , 1992), an effect sugges ted to be due to activated translocation of G L U T 4 to the cel l sur face (Paquet et al , 1992), and/or an enhanced intrinsic activity as shown in sarco lemmal ves ic les (Okumura and S h i m a z u , 1992). Notably, these effects were demonstrated at 10~ 4 -10~ 3 M, levels unattainable in p lasma in vanadium-treated animals. Prev iously , p lasma vanad ium in STZ-d iabet ic animals with similar treatment was < 1 0 - 5 M (Chapter 7). Moreover , pervanadate but not vanadate at 20 p M enhanced basa l or insul in-st imulated g lucose transport in isolated rat adipocytes (Sh isheva and Shechter , 1993b). G l u c o s e transport in rat skeletal muscle in vitro was similarly demonstrated at 1 0 - 4 - 1 0 - 3 M (C lausen et a l , 1981; Clark et a l , 1985). Hence , the lack of a n insul in-enhancing effect in vivo may be due to higher (>100-fold) vanadium levels used in vitro. 179 Hyperg lycemic D -NR rats showed a 30 -40% reduction in the intracellular G L U T 4 pool ( L D M / H D M ) , consistent with f indings in ad ipose (Karniel i et a l , 1987, Berger et a l , 1989) and skeletal musc le (Klip et a l , 1990) of STZ-d iabet ic rats. G L U T 4 m R N A and protein in ad ipose t issue are tightly regulated by insul inemia (Sivitz et a l , 1989) rather than g lycemia (Burcel in et a l , 1993). Previously, Strout et al (1990) reported an increased skeletal muscle G L U T 4 protein and m R N A content in STZ-d iabe t i c rats treated with vanad ium, suggest ing that vanadium may increase G L U T 4 express ion at a pretranslational level. Interestingly, the amount of intracellular G L U T 4 in ad ipose t issue in the diabetic animals was highly correlated with residual pancreat ic insulin content and not with chronic p lasma insulin levels. A l though this appears to be a contradictory f inding, acute changes in circulating insulin, such as following a g lucose cha l lenge, may not reflect in the fed p lasma insulin levels measured at a single time point. However , despi te a reduced intracellular G L U T 4 pool in D -NR relative to D-R and D-T, G L U T 4 levels in p lasma membrane were not different among the diabetic groups in response to i.v. g lucose. Never the less, P M G L U T 4 does not necessar i ly reflect transporter activity, as phosphorylat ion of G L U T 4 , which is enhanced during STZ-d iabe tes , may a lso inhibit its intrinsic activity (Begum and Draznin, 1992). In summary, these results demonstrate the relative ineffect iveness of vanad ium in enhanc ing K g and ad ipose t issue G L U T 4 translocat ion to a level beyond that ach ieved with spontaneous recovery from diabetes. Thus , vanad ium may have limited effects on acute insulin-stimulated G L U T 4 translocation in vivo. Instead, it may inf luence the overal l course of the diabetic state by preventing the progression towards insulin resistance, possibly by increasing basa l g lucose transport and/or reducing hepatic g lucose output, in concert with circulating insulin. The results of this study a lso suggest that some of the insulin-like effects of vanad ium in STZ-d iabe tes , such as improvements in g lucose tolerance and in the maintenance of ad ipose t issue intracellular G L U T 4 pool, may be secondary to the preservat ion of f i-cell insulin stores. 180 CONCLUSIONS AND FUTURE DIRECTIONS The mechanism(s) of the antidiabetic effects of vanad ium in vivo in STZ-d iabe t i c an imals was examined by conduct ing a ser ies of treatment studies. A l though vanad ium has been demonstrated to have many insulin-mimetic effects in vitro, ones which play a signif icant role in vivo remain undetermined. Severa l studies which examined the in vivo effects of chronic vanadium treatment employ the STZ-d iabet ic rat as a model of Type 1 d iabetes, and maintain the diabetic animals for chronic per iods of t ime (weeks-months). However, most of these studies fail to recognize severa l key aspec ts of the STZ-d iabe t i c state which could influence the interpretation of results. Thus , one important e lement which is usually over looked, and is a source of complexi ty with this model , is the exis tence of residual pancreat ic insulin stores in the diabet ic an imals which albeit smal l , are sufficient for survival. This phenomenon in itself d is t inguishes this model from Type 1 diabetes in humans, and may instead reflect the condit ion of a poorly controlled diabetic. As ide from this, it is clear from the studies in this thes is , that the diabetic state induced by S T Z can be immensely var ied within a single study, and between studies even under well-controlled and reproduced condit ions. Furthermore, al though most (85-95%) insulin-secreting cel ls are effectively depleted by S T Z , residual f i-cel ls which survive continue to be regulated by vanad ium and/or g lucose which can potentially inf luence fi-cell insulin stores and secretory function in the long-term. Thus , the effect of vanad ium in preserving even smal l amounts of insulin store, can potentially reverse the diabet ic state, resulting in normoglycemia and the induction of severa l " insulin-mimetic" effects in vivo. Indeed, the express ion of severa l proteins key to g lucose and lipid metabol ism (i.e. G L U T 4 , g lucok inase, pyruvate k inase, fatty ac id synthase, acetyl C o A carboxy lase, P E P C K , etc.) are tightly regulated by insulin and/or g lucose, and may be significantly affected by modest changes in circulating insulin (and hence, g lucose) . 181 Having noted this, it was the aim of this thesis to determine, through a ser ies of treatment studies, the relative role(s) for vanad ium, both at the level of the fi-cell and in peripheral t issues, in inducing an overall amelioration of the diabet ic state. Thus , the following general conc lus ions can be made: 1. B e c a u s e normoglycemia and correction of abnormal ad ipose t issue function were observed in similar proportions of diabetic animals even after treatment w a s initiated 10 or 17 days pos t -STZ, it appears that the eff icacy of vanady l treatment at 3 days after induction of d iabetes is unrelated to a protective effect of vanad ium from the acute STZ- induced fi-cytotoxicity. Instead, the observat ion that a diabet ic subgroup which had responded to normoglycemia had some residual insulin secretory function suggested that the chronic preservation of f i-cel ls may contribute to an effective response to administered vanad ium. 2. A n organic complex of vanadyl (naglivan) was tested as a more lipophilic, more orally bioavai lable form of vanad ium. Nagl ivan, administered once daily to STZ-d iabe t i c rats, lowered exogenous insulin requirement and preserved card iac function, and had an oral potency 7.6 t imes greater than V O S 0 4 . The absence of d iarrhea with nagl ivan suggests that it could be a more therapeutically desirable form of vanady l . 3. It w a s found that unresponsive diabetic animals become normoglycemic when g iven higher concentrat ions/doses of vanad ium ( V O S 0 4 or nagl ivan). S i n c e the animals requiring higher amounts of vanadium were more severe ly diabet ic prior to start of treatment than those which responded to lower amounts, residual circulating insulin appears to be important in achieving a normoglycemic effect to a given d o s e of vanad ium. This finding suggests that vanad ium and insulin work in an interdependent and complementary manner in vivo. 182 4. Eug lycemia was observed up to 20-30 weeks after withdrawal from vanad ium treatment in diabet ic an imals which demonstrated an improved residual insulin secretory capaci ty. It was determined that while the susta ined normoglycemia fol lowing withdrawal from treatment may be related to an enhanced sensitivity in the short-term, it may depend more critically on improved fi-cell secretory function in the long-term. 5. Vanad ium treatment might directly inhibit the fi-cytotoxic effects of S T Z by lowering insulin demand and fi-cell secretory activity. However, it w a s found that 1-week pretreatment with vanad ium did not prevent the STZ- induced fi-cell cytotoxicity. Alternately, short-term treatment after S T Z induced a partial preservat ion of f i -cel ls ( - 12% of control) which was sufficient for chronic reversal of d iabetes. Thus , smal l changes in pancreat ic insulin content can have profound c o n s e q u e n c e s on g lucose homeostas is both in the chronic fed state, and following an acute g lucose cha l lenge. 6. A l though the ant ihyperglycemic effects of vanad ium in STZ-d iabe t i c rats could be partly ascr ibed to its anorexigenic effects, the contribution Of a reduced food intake to the hypoglycemic and fi-cell protective effects of vanad ium is minor. Moreover , the mechan i sm of glucose- lower ing by food restriction is l inked to p lasma and pancreat ic insulin levels and distinct from that of vanad ium. The protection of residual insulin stores by vanad ium may be secondary to a higher threshold for insulin re lease. 7. A l though it was hypothesized that vanad ium may improve g lucose to lerance in diabetic an imals by enhancing insul in-mediated g lucose transport, vanad ium did not appear to enhance the acute insulin-stimulated G L U T 4 translocat ion in ad ipose t issue in vivo. Moreover , the improvement in g lucose tolerance and main tenance of intracellular G L U T 4 pool in ad ipose t issue with vanad ium treatment appears to be secondary to a preservation of residual fi-cell insulin stores perse. 183 Al though chronic vanad ium treatment can induce an overal l amel iorat ion of the diabet ic state, it appears that some of its "insulin-l ike" effects, especia l ly after chronic withdrawal from treatment, are secondary to the preservat ion of fi-cell insulin stores per se. S i n c e vanad ium has limited effects on enhanc ing insul in-st imulated g lucose d isposa l and transporter translocation at least in ad ipose t issue in vivo, these observat ions suggest that in vitro effects do not necessar i ly translate to mechan isms in vivo, especia l ly when these results are demonstrated at concentrat ions greater than the 10-20 p M levels which are ach ieved in vivo. This becomes an issue of greater concern when one real izes the vast number of in vitro s tudies in the literature (see Introduction). Alternately, the suggest ion that the in vivo ant ihyperglycemic effects of vanad ium may be completely secondary to its anorexigenic effects may have ar isen from poorly controlled pair-feeding studies, s ince carefully conducted, the contribution of food intake to the glucose- lower ing and fi-cell protective effects of vanad ium is minimal. A consistent observat ion is that vanad ium induces chronic normoglycemia at low circulating insulin level and residual insulin stores. Hence , al though the insulin reserve determines an initially effective vanadium dose , normoglycemia can be observed at an insulin content - 2 . 5 % of control. This suggests an important role of vanad ium in perhaps enhanc ing basa l g lucose transport through G L U T 1 or G L U T 2 (Meyerovi tch et a l , 1987), or by inhibiting hepat ic g lucose output, a s demonstrated at low vanad ium levels (10" 7 M) (Bruck et a l , 1991). The chronic maintenance of basa l normoglycemia would then prevent the onset of insulin resistance and defects in gene express ion , al though its stimulatory effects on g lycogen synthase appear to be speci f ic (Cohen et al , 1995). The select ive mechan isms of vanadium in vivo a lso support the notion that vanad ium may not affect the insulin-signaling pathway global ly, but may be directed towards certain intracellular events which could even be distinct from those of insulin (Sh isheva and Shechter , 1993a). 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Diabetes 45 (Suppl 1): S 7 0 - S 8 1 , 1996. 214 Appendix 1 A SENSITIVE RADIOIMMUNOASSAY OPTIMIZED FOR R E P R O D U C I B L E M E A S U R E M E N T OF RAT P L A S M A INSULIN A1.1. INTRODUCTION P l a s m a insulin levels reported in the literature have been noted to be somewhat variable and lacking in uniformity (Clark and Ha les , 1994; Robb ins et a l , 1996). In a col laborative study involving severa l laboratories, significant variability w a s found for identical p lasma samples when ana lyzed by different kits (Cotes et a l ,1969). In addit ion, the "within-kit" variability in p lasma insulin levels recorded by severa l laboratories was also high ( % C V > 15%) (Shishiba et a l , 1982). More recently, a task force was ass igned by the Amer ican Diabetes Assoc ia t ion to a s s e s s the comparabi l i ty of p lasma insulin measurements from different laboratories (Robbins et a l , 1996). Th is extensive study reported that different laboratories produced widely d isparate results for identical serum and p lasma samples , demonstrat ing an overal l lack of acceptabi l i ty for population compar isons. Most seriously, use of the s a m e a s s a y kit in different laboratories did not a lways yield similar results and even providing a single reference insulin standard to the various laboratories did not improve the variat ions in results. U s e of ant ibodies with a narrow specificity for insulin did not consistent ly yield lower va lues than nonspeci f ic assays which had high cross-reactivity with proinsulin and proinsulin intermediates. Both charcoa l precipitation and double ant ibody techn iques yielded similar performance in terms of linearity over a ser ies of dilutions and recovery of sp iked insulin standards in fasting serum, suggest ing that matrix effects of full vo lume serum may affect a s s a y performance. The task force conc luded that s ince there w a s no highly accurate, precise and reliable procedure to detect the amount of insulin in a biological matrix, that internal performance of a s s a y s be maintained within acceptab le limits. 215 Dextran-coated charcoal has been used extensively for separat ion of free from ant ibody-bound hormone in rad io immunoassays (Herbert et a l . , 1965). However , use of the charcoal separat ion technique has been severely limited s ince other p lasma proteins such as albumin compete for binding to charcoal (Albano et a l , 1972). T h e s e problems with charcoal lead to erroneously low insulin va lues, but other techn iques are also subject to errors including false high va lues, when using the double ant ibody method (Meek et a l , 1970, Silbert and Saw in , 1975). O n e proposed modif ication w a s to minimize the variability in protein content between standard buffer and p lasma by the addit ion of insulin-free p lasma to the standard curve (Albano et a l , 1972, Och i et a l , 1973). However , unacceptable variability and undetectable insulin levels in fasted rats remained following the addition of charcoal-extracted p lasma ( C E P ) to the standard curve, s ince the effects on adsorpt ion of free insulin to charcoal w a s different between C E P and untreated p lasma (Frayn, 1976). More appropriate alternatives for insulin-free p lasma have subsequent ly been proposed, including p lasma obta ined from pancreatectomized animals, or p lasma treated by immunoadsorpt ion (Odel l , 1980). Prior to this study, attempts to find a reproducible RIA for insulin in rat p lasma using both the charcoal adsorpt ion and double antibody (RIA kits) techniques of separat ion, there was unacceptable interassay variability. Moreover , as previously reported with the charcoa l adsorpt ion technique (Frayn, 1976), we found that insulin w a s undetected in p lasma from fasted and insul inopenic rats, a phenomenon which persisted even after adding C E P to the standard curve. Thus , severa l trials were conducted to ach ieve a more sensit ive and reproducible a s s a y for insulin in p lasma. This study descr ibes a correction of experimental data by using the a s s a y of C E P a lone as a distinct "zero insulin" level for the analysis of p lasma samp les . This approach was found to reduce interassay variability and al lows for detect ion of very low physio logical insulin levels. Thus , this modif ication provides a s imple way of correct ing for p lasma effects when included in the charcoal separat ion technique of rad io immunoassay. 216 A1.2. MATERIALS AND METHODS A1.2.1. Materials The rad io immunoassay method descr ibed in this study is a modif ication of procedures of Pede rson and Brown (1979), and Novo B io labs (Copenhagen , Denmark) . Freeze-dr ied rat insulin standard, and lyophil ized m o n o - 1 2 5 l - ( T y r - A 1 9 ) -human insulin ( -30 mCi /mg) and freeze-dr ied anti-porcine insulin gu inea pig serum (M 8309, 1:300) were purchased from Novo R e s e a r c h Institute. The undiluted serum has a binding capaci ty of 3 U insulin/ml of serum (Novo). Bov ine serum albumin (RIA grade) w a s obtained from S igma (St. Louis , MO. ) , Dextran-T70 purchased from Pharmac ia Biotech (Piscataway, New Jersey, NY. ) , and activated charcoa l (Norit carbon decolor iz ing) obtained from B D H . 1 2 5 | . j n s u | j n a n c | insulin ant iserum were diluted to 4-6 ng/ml and 1:60, respectively with 0 .1% B S A and 0 .025% thimerosal in phosphate buffer (0.04 M, pH 7.5) as per specif icat ions (Novo Nordisk). T h e s e final, working dilutions were stored as al iquots at -20°C. Severa l al iquots of rat insul in s tandards were prepared in RIA buffer suppl ied by ICN Biomedica ls (Costa M e s a , C A . ) and stored at -20°C. Charcoa l extracted p lasma was prepared from nonhemolyzed frozen human p lasma. Briefly, p lasma was mixed with charcoal (1g/100 ml) at 4°C for 90 minutes and centrifuged at 10,000 rpm for 30 minutes. The supernatant w a s filtered through Shark Sk in® analyt ical paper (Schle icher & Schue l l , Inc., N H , U S A ) us ing a vacuum flask until p lasma was clear of charcoal particles, and al iquots kept at -20°C. Charcoa l separat ion buffer (0.5% dextran T-70, 5 % activated charcoal in 0.04 M phosphate buffer) was prepared by mixing charcoal in buffer containing d isso lved dextran. The charcoal suspens ion was stirred overnight and kept stirring for at least 2 hours at 4°C prior to use. Un less otherwise noted, rat p lasma samp les used as internal s tandards were pooled from several fasted ( F A S T ) , diabetic (DIA) or control ( C O N ) rats, which represented low, middle and high insulin concentrat ion va lues. Al iquots were f lash-frozen in liquid nitrogen and stored at -20°C. 217 A1.2.2. RIA Procedure Working reagents were thawed immediately prior to use. A s s a y tubes were kept on ice at all t imes. O n day 1, 775 pi of RIA a s s a y buffer (5% C E P in 0.04 M phosphate buffer) and 25 pi of either standard buffer (blank), or s tandards (range: 0.5-20 ng/ml or 7.1-284 pU/ml), internal standards, C E P , and p lasma samp les were pipetted to the tubes. Subsequent ly , 100 pi of ant ibody w a s added to all tubes except for total counts. The tubes were vortex-mixed and kept overnight at 4°C. O n day 2, 1 2 5 l - i n s u l i n (100 pi, >4000 cpm, final dilution 6.0 - 8.1 pU/ml) was added to all tubes, vortex-mixed and incubated for an addit ional 24 hours at 4°C. O n day 3, 200 pi of charcoal separat ion buffer was added to each tube (except total counts) fol lowed by vortex-mixing. After incubation for 30 minutes, the tubes were centri fuged at 4 ,200 rpm at 4°C for 30 minutes, the supernatant aspirated and charcoal precipitate counted. E a c h a s s a y (equivalent to one centrifugation run) included one standard curve, a set of internal s tandards, C E P and 60 samples . Al l samples /s tandards were a s s a y e d in triplicate. Data was ana lyzed via Scienti f ic F ig . P rocesso r (F ig .P Software Corporat ion, B I O S O F T , Durham, N C ) . The nonlinear equation for asymmetr ic s igmoid w a s used to generate standard curves and to ana lyze the data. The Students ' T-test w a s used to determine significant dif ferences and p < 0.05 was cons idered significant. W h e r e a s the radioactivity counted in the charcoa l pellet is charcoa l -bound 1 2 5 l - i n s u l i n or C P M (free), ant ibody-bound counts (supernatant) are obtained by subtracting C P M (free) from total (T) counts, yielding the following C P M (bound) va lues : for insulin-free buffer or zero standard (Bo), for insulin s tandards and samp les (B), and for C E P (Bp). Thus , the ant ibody-bound fraction for s tandards and samp les are expressed as % B / B o , and for C E P as % B p / B o . S ince the % B p / B o for the a s s a y is used as a distinct "zero insulin" level for p lasma samples , the original % B / B o va lues are corrected via the following formula: % B / B o 4 - % B p / B o = % B / B o ' , and the corrected % B / B o ' va lues are evaluated using the standard curve to determine p lasma insul in. 218 A1.3. R E S U L T S A1.3.1. Effects of C E P on the standard curve The effect of correcting for p lasma effects on the determination of insulin by adding C E P to the standard curve was examined. In Figure A 1 . 1 , 25 (A) and 50 (B) pi of C E P were added to equal vo lumes of standard, whereas the vo lumes of all other reagents were unchanged. The insets show the difference in C P M (bound) between the C E P - s t a n d a r d and the normal standard curves. The addit ion of C E P resulted in a significant increase (above zero) in C P M (bound) in some but not all of the standard concentrat ions. This resulted in skewed linearity in the logit-log correlat ions (F ig. A 1 . 1 C - D ) . Thus , the linear fit was found to be highest (r=-0.997) using a standard vo lume of 25 pi without added C E P (Fig. A 1 . 1 C ) , whereas the addit ion of 25-50 pi C E P altered the s lopes and reduced the correlation coefficients of the l inear plots. A similar d isp lacement pattern was obtained when high concentrat ions of a lbumin (6% w/v) were present in the standard buffer (data not shown). A1.3.2. Correcting for C E P (%Bp/Bo) binding A typical example of 3 a s s a y s done in sequence is depicted in Tab le 16. P l a s m a samp les obtained from fasted animals ( F A S T ) consistent ly had a % B / B o which w a s greater than 100%, thus appear ing beyond the lower limits of the standard curve, and resulting in non-detectabil ity. It was thought that insulin-free p lasma (in the form of C E P ) could represent the true "zero insulin" level for p lasma samp les , in p lace of the original zero binding (Bo) which is obtained from the a s s a y of insulin-free standard buffer. This hypothesis was supported by the observat ion that the % B p / B o (resulting from the a s s a y of C E P alone) was consistently detected as >100%, (range: 100-115%, s e e F ig . A1 .5B) . In turn, the % B / B o va lues of p lasma samp les obtained from fasted or severe ly insul inopenic animals, which also exceeded 100%, were found not to exceed the % B p / B o within the s a m e assay . Thus , when internal s tandards were normal ized 219 N. at o> d d l l n n o • r 00 h CM to <o o> 00 o> o> d d O • 3 O) o o o o -1 1 CO C\J o CM I I CO oo •>— I o 1 - CM I I • o o I- o L O =5 =1. 3 C CC T3 C 3 O CL O < o o O O C I (M o o '; ° m o o o o m CM o o o o 1— o —r~ o 1 o 1 1 o o o 1 b o 1 o 1 1 o o o o o o o o o o o o in o LO o LO LO o LO o LO CM CM i — 1— CM CM *— CD CO -C > O D_ 2 co LU CD CD Q N | t e g — CD H CO CD O v CD CD o T J C Z3 O X I T J C CO Z i o x: CO T J i_ co T J C > -2 i » o J i T 3 Z> 1- CO CO c T J ~ "co CO X CO I (0 CD c re £ » o C — D_ £ O c c ° — CO CD C CD CO St ^ = CD T J O •£ o CD ^ •— Z i - CO D_ c ^ W +- "S O «2 | X I co o C O CD CO Q . co w 3 — (0 CQ P " T J 0. C QJ CO f LTT Zt C M o LO T J CD , „ — o .!2 CD Z l ' T J O X co T J C CO m CN <+- p ° CO Uj to - i t < I CD £ = ^ CO Q_ i l O S. CD "g "E £ w ® c < CD CD £ CO U T5 "2 i_ CO O T J * - CD C CO k— C O o CO CD 2: Z i o T J c CD £ D . CO CO * 1 g 0) t O x: o ^ C Q CD T J (0 RO CO CD CD Z l o Z i C J a> (A C T J C O - CO - ° — ' T J ° - c £b L U CO Q 220 against the "zero insulin" level for p lasma by dividing the % B / B o by the respect ive % B p / B o for each assay , the corrected % B / B o ' va lues subsequent ly fell within the standard concentrat ion range, and higher insulin levels were detected for all the samp les . Thus , using the uncorrected % B / B o va lues, insulin detected in DIA w a s low and variable, with an interassay coefficient of variation (%CV) of 3 0 % (Table 16). After re-evaluating the corrected % B / B o ' va lues, the % C V for DIA was reduced to 3.4%, and did not exceed 6.0% in all internal standards. Furthermore, use of the correction factor transformed previously undetectable fasting ( F A S T ) insulin levels into consistent and reproducible va lues within the expected range. Hence , using this s imple formula for the correction of % B / B o va lues for p lasma samp les routinely increased the detectabil i ty of low p lasma insulin, and diminished intra- and interassay variability. F igures A1 .2 (A-B) are Figures A1.1 (A-B) reevaluated as % B / B o curves. In the a b s e n c e of C E P , the working range of the normal standard curves w a s observed to be between 7 to 200 pU/ml with standard vo lumes of 25 (A) and 50 (B) pi. W h e n both 25 and 50 pi p lasma samples were ana lyzed on the respect ive normal standard curves, fasting insulin levels were undetectable, even with higher sample vo lumes of 100 pi were assayed (data not shown). Furthermore, the % B p / B o (from the a s s a y of C E P alone) w a s >100% and also increased with higher vo lumes, from 106% (25 pi) to 112% (50 pi) and 120% (100 pi). After dividing the sample % B / B o by the % B p / B o of the s a m e assay , to yield the corrected % B / B o ' , subsequent reevaluat ion of the % B / B o ' on the normal standard curve resulted in p lasma insulin levels which were reproducible and within the range of detect ion. On the other hand, using the C E P - s t a n d a r d curve, p lasma insulin levels in the low ( F A S T ) and high ( C O N ) range consistent ly fell beyond the detect ion range. Thus , al though the % B p / B o va lues approached 1 0 0 % using the C E P - s t a n d a r d curve, using the original % B / B o va lues and correcting for p lasma effects by the addit ion of equal vo lumes of insulin-free p lasma ( C E P ) to the standard curve did not improve the detectability of insulin in p lasma. 221 o CO CO c o *3 u a> t_ i_ o o 1-Q) re T J C (0 CD a> .a to _a> a E re to (0 E to re a T J re _o CO o JO a> > CD 3 to c CD 0) A re I— CO CD O CN in o oo o 03 CO l< Q_ LU o o co a. 00 O c o O o CO CD o c o O 3 . o c o O o CO CO o eg CO Tj o e o o 5 CD O) m CO o oo CN TJ- a> o CT) TJ-CD d CD T -1- CT) CO od ^r c> ^ • 0 ) 0 O CO Q ^ CD Z LO ^j- co LO ^ N CO m o o _ CJ O ^ "O O Q LL CJ c o o o DO CO *01 o c o a o DO CO 0s o c o u o CO CO Tj o o Z> 3 . Z) CD LO d cN co CM CD CT) CT) CT) CO OJ d co t o i d d LO t o o CT) TJ- CO od T - d S CM t -CD CO s d in Tf CT) CT) o O *r; T J o .2 S O Q LL o CO do o co CO O DO CO o CO CO s5 E o L— <+-c o "-4—» CD i— "c CD O c o ( J CJ c o o a) CD CJ CD CD T J O C 2 2 2 o I — CO 51 Q O O a. LU O o s <o o ^ 9 CM CM >- CO co" cd CO o •r- CM I + =3. =1 LO LO CM CM o • CM * ~ CO Q ^ LO CM CO LO 2 5 . =S. O O LO LO o • •- o o o m 3 co C co QC o o o CO o CO o o CM o o o oo o CO o o CM CO D_ o E LU — 2 o < M— CD o 3. co (2 LO C | CM § •a » CD CO >» CD CO 3 CO — CO cp CO > CO c CL CO E . E $ g E co co pr-o > to n CT CD C co CO © 0 1 1 2 CO E T J 52 co CO xj M o ^ CO CO o "O _ C ZL CO ^ OT CO Q. CO I- 9> CO £ < 1 3 c ^ *= T J CO co CO T J T J C C TO CO CO ^ < LTL S LU Q O "CD ~ XJ T J CO C T J TO O p O o LO T J c CO . C ««_ CL LU O 8| I 8 O 7= 5 C M TJ TJ CO LU CM < CD CO CD o "2 CO T J c CO A-» CO o m CO =» CD T J ^ — ^ CD cp co 3 > =2 "co C CO > 3 CD o co CO C CD ^ *= co a . _ 3 CO CO CD CD ^ = 0 0 I— LO O 223 A1.3.3. Effect of increased charcoal -binding It has been noted that p lasma proteins can preferentially d isp lace antibody-bound insulin from charcoal adsorpt ion sites. However, increasing the availabil ity of charcoal not only results in complete adsorpt ion of free hormone but a lso increases the adsorpt ion of the ant ibody-bound fraction (Odell et a l . , 1980). To determine whether the method for correction of p lasma effects can be similarly appl ied when ant ibody-bound insulin is being d isp laced, an excess ive amount of charcoal (25 mg/tube) w a s compared to the original amount (10 mg/tube). A n increased amount of charcoa l resulted in a reduced C P M (bound) fraction throughout the curve (Fig. A 1 . 3 A ) . However , when replotted as % B / B o curves, both standard curves were super imposed (Fig. A1 .3B ) . Al though the % B p / B o was higher with greater amounts of charcoa l , correction with the respect ive % B p / B o to obtain % B / B o ' resulted in p lasma insulin va lues which appeared within the expected range of the normal method. A1.3.4. Recovery Experiment To detect whether possib le variations in the recovery of insulin exist in the p resence of 25 pi of p lasma over the range of the standard curve, p lasma samp les (in which insulin levels were averaged over severa l assays ) were added to the standard curve. This experiment was done in p lace of analyz ing the recovery of s tandards in the p resence of C E P , s ince when standards were assayed in the p resence of equal vo lumes of C E P , the distinct shifts in the C E P - s t a n d a r d curves demonstrate both inadequate and variable recovery of insulin throughout the curve (Fig. A1.1) . Instead, by adding 25 pi of untreated p lasma which had relatively low insulin concentrat ions to the entire range of the standard curve, the effects of untreated p lasma on the recovery of total insulin, which is additive from both p lasma and standard, could be determined. The degree of recovery in the presence of p lasma was thus compared to total recovery obtained when a known insulin standard (in buffer) was added to the standard curve. 2 2 4 o o o o (ct O CD -c o O ^ 3 3 . o 8 o » § i •«* * < CO CO r: r; <o ^ o o ^ <q Q U> CM ^ d o S % O Q UL o Q . =3 5 . o CD w O CD k. o 8 oo o> CO IO io p o> c\i <=> CS| 00 S 1^  K <*> O) o> ^ <*) (O « ci d <o m ! : CM O «~ CO ^ O) O) - , I— O S £ O Q LL o o o o =3 2 3 3 c m r o o o o in o CM CM O O in o o o o o in i o o o o CO o CO o o CM I o (punoq) NdO og/8% c o re c E v_ 0) T J " D C re o £ 3 O "S re T J C re to c o a a> *5 c co — c o 4 3 2 re a a> co "re c re o o i_ re s: u to 4-" c o E re CD o c 9 a> in •D - C C M— CD O o a. o oo CN T J r-g ai o ct : a) CD t co 8 CD CD • a c£ C CD JS - D CO C CD 3 5 c -• - 9 4-> 'l— CD CL .15J CO CD o CD T J c TO "co CO o go co CD > CD CO c CL CD -t—* CO TO c CD T J c CD C < £ X f «1 1st IP CO • — c T™ re Q < E " (j) tO CD L a ^ " 5 . E .2* o U. O O CD .Q c .2 ^ 2 iS CD C CL — CD C CO £ "CD ° 8 » i- CD CD C x: CD " CO T J CD T J C CO -«—' CO 225 Figure A1 .4 depicts the addition of 25 pi each of diabetic rat p lasma (20.8 pU/ml insulin, A) , fasting rat p lasma (6.9 pU/ml insulin, B) and rat insulin standard in buffer (7.1 pU/ml insulin, C ) to the standard curve. The % B / B o curves were subsequent ly plotted against the expected total (standard + fixed standard/plasma) insulin concentrat ions. W h e n predetermined insulin in p lasma w a s added to the standard curve, there w a s a distinct shift of the % B / B o curve (A-B) upwards, which resulted in a consistent ly low recovery of total insulin throughout the curve (between 38-89%), particularly in the low concentrat ion range (D-E). However , after correction of e a c h point by the % B p / B o for the assay , the resultant plot of % B / B o ' vs . total insulin concentrat ion w a s found to super impose the normal curve. Accordingly, using the modified % B / B o ' , there w a s >90% recovery of total insulin (p lasma + standard) in the p resence of 25 pi of untreated p lasma throughout the entire range of standards. O n the other hand, addit ion of rat insulin standard (in buffer) did not d isp lace the normal standard curve (C), and the recovery of total insulin w a s consistent ly >95% (F). A1.3.5. Reproducibil ity of the assay Reproducibi l i ty of the RIA procedure w a s tested in 29 a s s a y s over a period of 15 days (Fig. A1.5) . F rozen aliquots of rat insulin standard, antibody and radiolabeled insulin were thawed and pooled for two separate a s s a y s per day. Al iquots of pooled rat p lasma samp les were used as internal s tandards (Fig. A1 .5A) . % B p / B o over severa l days ranged between 100-112% and varied by as much as 10 percentage units between a s s a y s performed on the s a m e day (Fig. A1 .5B) . Reevaluat ing the internal s tandards using the corrected % B / B o ' brought insulin levels to the s a m e range, with a n average % C V (intra-assay) of 4 . 3 % (range: 0.2-15.4%) for control and 7 . 1 % (range: 0.1-20.5%) for diabetic standards. The average % C V (interassay, within-day) w a s 3 .4% (range 0.2-9.9%) for control and 3 .8% (range: 0.1-7.7%) for diabet ic s tandards. Overal l interassay % C V over 29 a s s a y s was 6.6% for control and 7 . 1 % for diabet ic 226 %B/Bo 100 -I A 80 -60 -40 -20 -Insulin (fjU/ml) Observed 100 -80 -60 -40 -20 -100 -I C . .• 80 60 40 20 •o I 1 1 1 1 10 100 1000 Insulin (fjU/ml) 150 -I D 1 0 0 H 50 0 150 100 H 50 H 0 —' o CT -I E .o cm F .o 0 -1 cm 150 H 100 H 50 H I 1 1 1 1 0 50 100 150 200 Insulin (fjU/ml) Expected Figure A1.4. Effects of untreated rat plasma on recovery of total insul in % B / B o standard curves (dotted line) to which 25 ul of diabetic rat p lasma (A), fasted rat p lasma (B) and rat insulin standard in buffer (C) was added prior to ( • , % B / B o ) and after ( O , %B/Bo ' ) correcting for % B p / B o (105%). D-F show observed total insulin from A - C plotted against expected total insulin. The observed insulin levels calculated from % B / B o (• ) and % B / B o ' ( O ) are shown with the theoretical 100% recovery (dotted line). 227 . C E 51 220 ~i 200 180 -160 -140 -40 20 -t 0 • • O- • * • - A - £ . . A - A . - A - i . - A - t » A - 4 " * - i -- T -2 4 8 10 - 1 — 12 i — 14 16 DAY of ASSAY O Q. Q3 115 110 H 105 H 100 95 B o o ^ 0 -o • o o • • o o o o • o 6 1^  8 - 1 — 10 -~1— 12 - 1 — 14 i 16 DAY of ASSAY Figure A1.5 Reproducibi l ity test of radioimmunoassay over 15 days (A) Resul ts for internal standards: control ( • , • ) and diabetic ( A , A ) rat p lasma for a s s a y s 1 ( " . A ) and 2 ( D .A ) performed sequential ly on the s a m e day, and recorded over 15 days . (B) % B p / B o for a s s a y s 1 (•) and 2 ( O ) were used to correct % B / B o va lues of p lasma samp les from the respect ive a s s a y s over the s a m e time per iod. Dotted l ines represent the average. 228 standards. The average (± S.D.) va lues for control and diabetic p lasma insulin over the s a m e number of a s s a y s were 170.5 ± 1 1 . 3 and 20.8 ± 1.5 pU/ml , respectively. A1.3.6. Effects of site of plasma sampling The effect of obtaining p lasma from two common methods of blood col lect ion in rats on the measurement of insulin was tested (Fig. A1.6) . Wistar rats were administered either sal ine ( C O N , n =4), or made diabetic with 50 mg/kg (DIA(50), n=4) and 75 mg/kg (DIA(75), n=4) i.v. streptozotocin (STZ) . At 3 days after S T Z , rats were given an i.p. injection of pentobarbital (60 mg/kg). B lood was drawn from the tail vein (by nicking the end of the tail and collecting in heparinized capil lary tubes), and from the chest cavity (after cutting the chest open below the d iaphragm and removing the heart) into hepar in ized test tubes. Insulin measured from tail vein p lasma of DIA(50) rats were similar to control at 3 days pos t -STZ. However, DIA(75) an imals were clearly insul inopenic at this time point. Insulin levels of C O N in p lasma col lected from the chest cavity was significantly (3 t imes) higher than levels measured from tail vein p lasma. O n the other hand, the site of p lasma sampl ing did not signif icantly affect insulin levels in diabetic rats. This is despi te similar insulin levels (obtained from tail vein) between the C O N and DIA(50) groups. Thus , with chest cavity p lasma, both diabetic groups appear to be quite hypoinsul inemic relative to control, whereas using tail vein p lasma, insulin levels are only significantly lower in the DIA(75) group. T h e protein concentrat ions were significantly lower for p lasma obtained from the chest cavity in control rats (tail vein: 37.4 ± 0.9 vs. chest cavity: 33.5 ± 0.5 mg/ml, p < 0.05). However , protein concentrat ions in chest cavity p lasma between control and diabet ic rats were not significantly different from one another. 229 CD co h-O q> O LO Q o LO Q O O o in CNJ o o CM o LO o o o (iw/nri) ui/nsui euiseid T J CD i _ £ to c ' E T J CO CO "co o CD X I CO T J T J c CO II c <= O a " CO _ c g 0 o c C J .2 CO * J (0 CO c E— T J i _ 0 CD "o v co "O E W C D CO CO C «*-= o a co 1 ® CO CO CO CD II • - J = I— CD 2 E CO o co -e > » CO CO CD T J - C "co co T J > 8 1 H= CD 0 i -co © CO i t T J 3 ^ > TJ- CO n " c +-CO ^ - C CD L O SZ N - O < £ . o C D *J= 1 = E co L O -C= r-~ CD T J > S !s > ^ CD i - c to +-(0 i -a £ o " § a> . E if E (0 CD M - T J O co el LU c £ I 3 CO CO CO o L O E O P O co — > < =§ ^ c 3 g . i C CO V Q. + O T J L O CD o CO CO S CD CO o T J -4—' CO N h-O CO CO CD > co -4—' CO > L O LU Q_ C/D DQ o v 230 A1.4. DISCUSSION It has been reported that p lasma proteins can compete for adsorpt ion sites in charcoal and interfere with the final separat ion of free from ant ibody-bound insul in. Thus , the free 1 2 5 - i n s u l i n binding to charcoal is reduced, which artificially ra ises the C P M (bound) fraction, erroneously lowering p lasma insulin levels. The addit ion of hormone-free p lasma to standard tubes has been proposed to equal ize the var iances in protein content between p lasma samp les and standards (A lbano et a l . , 1972). However , the addit ion of C E P from either rats (Frayn, 1976) or humans (current study) appears to be inadequate for removing these dissimilarit ies. Indeed, adding C E P to the standard curve resulted in a variable degree of d isp lacement throughout the curve, and internal s tandards C O N and F A S T remained beyond the working range of the curve. Th is finding supports the proposal that charcoal extraction can remove severa l highly charged smal l molecules which influence binding of charcoal to hormone (Odel l , 1980), unlike the reported lack of changes with charcoal extraction (Albano et a l . , 1972). Initially, the impetus for acquir ing an insulin RIA to reproducibly measu re insulin in p lasma was not satisfactorily fulfilled with the use of commerc ia l insulin RIA kits, most of which employed double-ant ibody precipitation methods. Indeed, se rum has also been shown to interfere with the double ant ibody technique, thus prompting the suggest ion of adding hormone-depleted serum to the standard curve (Silbert and Saw in , 1975). Importantly, an unacceptable kit-to-kit variability w a s found, which would require pooling a considerable number of samp les from one study to be a s s a y e d at a single t ime, which was not feasible. More recently, E L I S A methods have been introduced which reproducibly measure insulin in pancreat ic extracts or culture med ium (Bank, 1988, Webs te r et al . , 1990). However, one E L I S A method in which serum samp les were tested was reported to be incompatible with serum, which act ively d is lodged ant ibodies from the plastic surface (MacDona ld and Gap insk i , 1989). 231 Using the charcoal adsorpt ion technique, it w a s hypothesized that the a s s a y of insulin-free p lasma in the form of C E P , rather than insulin-free buffer, could represent a distinct "zero insulin" level for p lasma samples . This hypothesis is supported by severa l observat ions. Firstly, the % B p / B o obtained when C E P alone was a s s a y e d w a s a lways > 100%, not unlike p lasma taken from overnight-fasted animals . Moreover , the interassay variability in % B p / B o values was similar to that of % B / B o of p lasma samp les . Thirdly, the % B / B o va lues for all p lasma samples , particularly fasted samp les , were consistent ly found not to exceed the % B p / B o value obtained for the s a m e assay . Thus , when the % B / B o va lues of p lasma samp les were corrected against the % B p / B o for each assay , low (diabetic and fasting) insulin levels were a lways posit ive, and interassay variability was diminished. That C E P can be used as a "zero insul in" level for p lasma but is inadequate for correction when added to other levels of insulin can be expla ined by the observat ion that the increase in C P M (bound) is consistent with the zero , but occurs to a variable extent with the other insulin standards. Theoret ical ly, the interassay variability can be thought to be reduced by obtaining large batches of rat insulin standard, antibody and radiolabeled insulin, which are then stored as aliquots of final working dilutions. However, despi te removing this potential source of variability, reproducibility remained poor between a s s a y s performed even on the s a m e day. The specif ic factor(s) contributing to the observed cons iderab le interassay variability is unknown. It was hypothesized that certain aspec ts of the final separat ion step may be a potential source of variability. S ince the centrifugation step limited the number of tubes to be processed simul taneously to 220, batches of tubes were precipitated sequential ly, with incubation and centrifugation t imes kept constant. S ince the % B p / B o value was found to differ as high as 1 0 % between a s s a y s done on the s a m e day, whereas % B p / B o appeared to be relatively resistant to changes in charcoa l ; i.e. doubl ing the amount of charcoal increased % B p / B o va lue by only 5%, the actual contribution of var iances within the charcoal stock suspens ion itself is unlikely. 232 Thus , it appears that the adsorpt ion characterist ics of dextran-coated charcoa l may be sensit ive to some other variable(s), perhaps involving even minute changes in incubation t imes, particularly when p lasma samp les are being handled. In this regard, it was reported that the interassay variability in kit-determined p lasma insulin levels were markedly reduced ( % C V < 10%) when the standards were prepared in serum rendered free of insulin via immunoadsorpt ion (Shish iba et a l . , 1982). It has been noted that immunoassays often make erroneous assumpt ions about the ex is tence of parallel curves between serially diluted p lasma and s tandards (Plikaytis et a l . , 1994). Thus , it was important to test paral lel ism of the curve in the p resence of p lasma, and the extent of insulin detect ion in p lasma throughout the standard curve. S ince the addition of C E P resulted in a nonparal lel d isp lacement of the standard curve, it was considered an unsuitable medium for serial ly diluting p lasma. Therefore, the recovery of total insulin when untreated p lasma w a s added to insulin s tandards w a s tested. Unlike C E P , addition of untreated p lasma resulted in parallel upward-shifted curves which led to a consistently low recovery of total insulin in the p resence of p lasma. W h e n corrected % B / B o ' va lues were plotted, the modif ied curve was found to super impose the original standard curve, thus indicating linearity between progressing va lues a s s a y e d in 25 pi p lasma vo lumes. Accord ingly , there w a s a marked improvement in the recovery of insulin, which was within 10% of the predicted total. Hence , these results val idate the use of % B p / B o as a specif ic "zero insul in" level for the correction of p lasma effects throughout the range of the standard curve. Alternatively, addit ion of an insulin standard did not d isp lace the standard curve, and recovery w a s > 9 5 % . This finding further supports the notion that d isp lacement from charcoa l binding sites is specif ical ly related to factors present in p lasma, and is absent when assay ing for insulin in extracts or buffer which contain comparat ively smal l amounts of protein. Indeed, protein concentrat ions of p lasma were found to range between 35 - 40 mg/ml as compared to - 0 . 7 mg/ml for insulin standards. 233 There has been a notable lack of cons is tency in p lasma insulin va lues reported in the literature (Clark and Hales , 1994; Robb ins et a l , 1996). S i nce there are severa l methods for blood collection from animals, it was quest ioned whether the source of p lasma sampl ing could also affect the detection of insulin. It appeared that p lasma from chest cavity in control but not diabetic rats showed dramatical ly inc reased insulin concentrat ions relative to p lasma obtained from the tail ve in. T h e s e e levated insulin levels in chest cavity p lasma for control animals were a lso found using the double-ant ibody method of RIA (Yuen V . , personal communicat ion). A l though protein levels in chest cavity p lasma were found to be significantly lower than tail vein p lasma from control rats, protein concentrat ions from chest cavity p lasma were not signif icantly different between control and diabetic rats. Thus , one poss ib le explanat ion cou ld be that the mixture of chest cavity blood with peritoneal fluid enr iched with insulin from surrounding pancreat ic islets contributed significantly to the marked elevat ion in p lasma insulin levels. The a b s e n c e of e levated insulin levels in p lasma obtained from chest cavity blood in the DIA(50) group, despite similar tail vein p lasma insulin levels to control, is consistent with the dramatic reduction in pancreat ic insulin stores in these rats. Diabet ic an imals administered lower d iabetogenic d o s e s of S T Z (45-55 mg/kg i.v.) have been shown to have reduced pancreat ic insulin content to - 1 0 % of control by 24 hours, but do not demonstrate significant hypoinsul inemia for 1-2 weeks (Junod et a l . , 1969). T h e s e results a lso suggest that one potential source of variability in insulin levels reported in the literature, at least among control rats, could be related to the site of the p lasma sampl ing. It is a lso apparent that a different conc lus ion could be made with regard to the state of hypoinsul inemia of the diabet ic rats, depend ing on the source of p lasma. B e c a u s e of the large volume of p lasma (>30 ml) required for internal s tandards, p lasma was col lected and pooled from chest cavity blood from severa l an imals , which explains the relatively high insulin levels detected in the control internal s tandards ( -170 pU/ml). 234 The sensitivity of the a s s a y to low insulin can be attributed to the relatively low concentrat ions of both insulin ant iserum and radiolabeled insulin in the react ion medium (Poznansk i and Poznansk i , 1969). Overnight preincubation of unlabeled insulin with ant ibody was also crucial in maintaining the high sensitivity of the assay . The use of smal l p lasma vo lumes for insulin a s s a y s has been invaluable in long-term treatment studies (Chapter 4) for several reasons. Firstly, less time is spent on bleeding rats, thus reducing temporal variabilit ies when a large number of an imals need to be bled at the same time. In addit ion, one avoids the removal of large quantit ies of blood which can potentially induce the re lease of stress hormones which can , in turn, affect other exper imental parameters (Rao, 1992). Thirdly, the feasibil i ty of performing an experiment in which repetitive sampl ing is required from severa l an imals is enhanced , for instance while performing a g lucose tolerance test. A s i d e from increased reproducibil ity, performance of this modified RIA method a lso al lows for the a s s a y of samp les in triplicate, which further enhances the reliability of the results. Final ly, severa l suggest ions have been made towards optimizing the charcoal -separat ion technique for the measurement of insulin. For instance, uncoated charcoal has been suggested to be more efficient and provide more adsorpt ion si tes for free hormone especia l ly in the p resence of high protein concentrat ions s u c h a s in serum (Binoux and Odel l , 1973). O n the other hand, it has been sugges ted that an optimal amount of dextran-coated charcoal which binds more free and less bound hormone be tested prior to establ ishing the final condit ions for the RIA (Odel l , 1980). T h e s e results demonstrate the relative resistance of the a s s a y towards extreme changes in the number of charcoal adsorpt ion sites, provided that C E P be a s s a y e d s imul taneously and used to correct p lasma sample % B / B o va lues. Thus , establ ishing a s imple, sensit ive and reproducible a s s a y for p lasma insulin is easi ly ach ieved , and need not require tedious preparations beyond optimizing the final dilutions of commerc ia l ly avai lable ant iserum and labeled hormone. 235 A1.5. R E F E R E N C E S Albano J D M , Ek ins R P , Maritz G , Turner R C : A sensit ive, precise rad io immunoassay of serum insulin relying on charcoal separat ion of bound and free moiet ies. Acta Endocrinol 70: 487-509, 1972. Bank HL : A quantitative enzyme- l inked immunosorbent a s s a y for rat insulin. J Immunol 9: 135-158, 1988. Binoux M A , Odel l W D : U s e of dextran-coated charcoal to separate ant ibody-bound from free hormone: a critique. J Clin Endocrinol Metab 36: 303-310, 1973. Clark P M S , Ha les C N : How to measure p lasma insulin. Diab Metab Rev 10: 79-90, 1994. Co tes P M , Musset t M V , Berryman I, Ek ins R, G lover S , Ha les N, Hunter W M , Lowy C , Nevi l le R W J , S a m o l s E, Woodward P M : Col laborat ive study of est imates by rad io immunoassay of insulin concentrat ions in p lasma samp les examined in groups of five or six laboratories. J Endocrinol 45 : 557-569, 1969. Frayn K N : Effects of insulin-free p lasma on the charcoal -separat ion method for rad io immunoassay of insulin. Horm Metab Res 8 : 1 0 2 - 1 0 5 , 1 9 7 6 . Herbert V , Lau K S , Gottl ieb C W , Ble icher S J : Coa ted charcoal immunoassay of insulin. JCIinEndocr 25 : 1375-1384, 1965. Junod A , Lambert A E , Stauffacher W, Reno ld A E : Diabetogenic act ion of streptozotocin: Relat ionship of dose to metabol ic response. J Clin Invest 48 : 2129-2139, 1969. MacDona ld M J , Gap insk i J P : A rapid E L I S A for measur ing insulin in a large number of research samples . Metabolism 38: 450-452, 1989. M e e k J C , S toskopf M M , Bol inger R E : Optimizat ion of rad io immunoassay for human growth hormone by the charcoal-dextran technique. Clin Chem 16: 845-848, 1970. Och i Y , Katsuhiko S , Hach iya T, Yosh imura M , Miyazak i T: Dextran-coated charcoa l technique to make the hormone-free serum as a diluent for standard curve of rad io immunoassay. Endocrinol Japon 20: 1-7, 1973. Odel l W D : U s e of charcoal to separate ant ibody complexes from free l igand in rad io immunoassay. Meth Enzymol 70: 274-279, 1980. 236 Pede rson R A and Brown J C : Effect of cholecystokinin, secret in, and gastr ic inhibitory polypept ide on insulin re lease from the isolated perfused rat pancreas . Can J Pharmacol Physiol 57: 1233-1237, 1979. Pl ikaytis B D , Holder P F , Pa is L B , Mas lanka S E , Ghees l ing LL , Car lone G M : Determination of paral lel ism and nonparal lel ism in b ioassay dilution curves. J Clin Microbiol 22: 2441-2447, 1994. Poznansk i N, Poznansk i W J (1969) Laboratory appl icat ion of the dextran-coated charcoal rad io immunoassay of insulin. Clin Chem 15: 908-918. R a o R H : C h a n g e s in insulin sensitivity from stress during repetitive sampl ing in anaesthet ized rats. Am J Physiol 262: R1033-1039 , 1992. Robb ins D C , Ande rsen L, Bowsher R, C h a n c e R, D inesen B, Frank B, G inger ich R, Goldste in D, Widemeyer H M , Haffner S , Ha les C N , Jarett L, Po lonsky K, Porte D, Sky ler J , Webb G , Gal lagher K: Report of the amer ican d iabetes assoc ia t ion 's task force on standardizat ion of the insulin assay . Diabetes 4 5 : 242-256, 1996. Sh ish iba Y , Takino H, Takag i A , Sato S , Irie M: The large "kit-to-kit" variation in insulin rad io immunoassay is mainly due to difference in standard concentrat ion. Clin Chem 28: 2443-2444, 1982. Silbert C K , Saw in C T : Double-ant ibody rad io immunoassay of serum insulin: effect of use of hormone-depleted human serum. Clin Chem 21 : 1520-1522, 1975. Webs te r HV , Bone A J , Webs te r K A , Wilkin T J : Compar i son of an enzyme- l inked immunosorbent a s s a y (EL ISA) with a rad io immunoassay (RIA) for the measurement of rat insulin. J Immunol Meth 134: 95-100, 1990. 

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