Glucose Utilization in Islets of Hyperglycemic Rat Models With Impaired Glucose-Induced Insulin Secretion zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR R.M. Colella, J.M. May, S. Bonner-Weir, J.L. Leahy, and G.C. Weir Under most experimental conditions islet glucose metabolism is well-correlated with short-term glucose-induced insulin secretion. Two hyperglycemic rat models (neonatal streptozotocin and glucose infusion) have been previously found to have markedly impaired insulin responses to glucose. and the glucose utilization of islets isolated from these models was therefore studied to see if reduced glucose metabolism might be related to the secretory abnormalities. It was found that glucose utilization in the islets of the two models was similar or higher than in comparable control islets. These data suggest that the secretory defect of these models, which is presumably induced by chronic hyperglycemia, is at a step in the secretion process distal to glucose metabolism. D 1987 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA by Grune & Stratton, Inc. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA U NDER most circumstances, short-term glucose- induced insulin secretion is thought to be tightly linked to the rate of islet glucose metabolism, whether measured by glucose oxidation to CO, or by conversion to water.“’ The mechanisms through which fuel metabolism stimulates the terminal steps of insulin release are, however, poorly understood. The process is particularly important because acute glucose-induced insulin secretion is markedly reduced in diabetic man, both in type II (non-insulin- dependent) diabetes mellitus and in the early stages of type I (insulin-dependent) diabetes mellitus.4-6 Our laboratory has been testing the hypothesis that B-cells that are chronically exposed to a severe, or even modest degree of hyperglycemia, lose their ability to respond to a sudden increase of glucose concentration.6 This finding has been obtained in three rat models with hyperglycemia: those treated as neonates with streptozotocin (NSZ),‘~* those given partial pancreatecto- mies (Px)p and those receiving glucose infusions.” The current experiments were designed to determine whether glucose metabolism is altered in isolated islets obtained from the neonatal streptozotocin model and the glucose infusion model. MATERIALS AND METHODS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Injection of Newborn zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Rats W ith Streptozotocin (NSZ) Male Sprague-Dawley rats (Flow Labs, Dublin, VA) were injected at two days of age with 90 mg/kg streptozotocin (a gift from Dr W. Dulin, Upjohn Co, Kalamazoo, MI) as described previously.’ Controls from the same litter were injected with citrate buffer alone. Two days after the injection plasma glucose levels were determined from blood obtained by cardiac puncture. Only streptozotocin animals with a plasma glucose greater than 275 mg/dL were kept for further study. Islets were obtained from NSZ and control rats which were 8 to 12 weeks of age. The plasma glucose values of the NSZ rats were 180 to 250 mg/dL. An indwelling intravenous catheter was placed in male Sprague- Dawley rats weighing 220 to 300 g using a standard technique. The external jugular vein was exposed and cannulated with a 3-cm piece of soft sialastic tubing which was attached to a IO-cm length of 0.23 x 0.38 in polyethylene tubing. This end was tunneled under- neath the skin and externalized between the shoulders. After one to two days, it was flushed with saline containing a 3 U/mL of heparin sulphate and then a solution containing 50% glucose in 0.45% NaCl was infused at 2 mL/h for 48 hours using a Harvard syringe pump M efabolism, Vol 36, No 4 (April), 1987: pp 335-337 (Harvard Apparatus, Millis, MA). The infusion caused the plasma glucose to rise to approximately 320 mg/dL.“’ The rats were unrestrained and had free access to food and water during the whole 48-hour period. M easurement of Glucose Utilization Rates in Isolated Islets Islets were isolated using the collagenase digestion technique of Lacy and Kostianovsky.” The islets were washed with a Krebs- Ringer bicarbonate buffer (KRB) containing 10 mmol/L Hepcs (2-(N-2-hydroxyethylpiperazin)-N’-yl) ethanesulphonic acid (Grand Island Biological Co, Grand Island, NY), 5 mmol/L NaHCO,, 5 mg/mL bovine serum albumin (BSA, fraction V). and 1 mmol/L glucose. Groups of 30 islets were transferred to 9 x I5 mm siliconized glass tubes. Glucose utilization rates were measured by following the conversion of ‘H-5-glucose to ‘H,O as described by Ashcroft et al.” To each tube was added 125 rL of KRB with Hepes containing 2 pCi/mL ‘H-5- glucose (New England Nuclear, Boston) and the appropriate concentration of cold glucose. The KRB with Hepes had been equilibrated with air prior to the incubation. The tubes were placed inside 20 mL glass scintillation vial and incubated without shaking for one hour at 37OC. At the end of the incubation period, two 50 PL aliquots were taken to measure ‘Hz0 production. The duplicate 50 IIL aliquots were then placed in 9 x I5 mm tubes containing 5 @L of 1 mol/L HCl. These tubes were gently placed in 20 mL glass scintillation vials containing 0.5 mL distilled H,O and incubated overnight at 37°C for ‘H,O equilibration. Tissue blanks consisted of 9 x 15 mm tubes containing medium treated in the same way only without islets. In addition, 50 FL of tritrated water standard was also incubated to allow correction for incomplete equilibration during the diffusion step. After the overnight incuba- tion period, the inner tube was transferred from the scintillation vial to another scintillation vial containing 0.5 mL distilled H,O. Ten milliliters of Beckman Ready-Solv EP was then added to all vials, which were then counted. After correction for the incomplete equilibration of the ‘Hz0 formed and for the ‘H,O in the blanks, the From the Department of Internal Medicine, M edical College of Virginia, Richmond, VA. Supported by a grant from the US National Institutes zyxwvutsrqponmlkjihg of Health, AM-20349. Presented in part at the 44th Annual Meeting of the American Diabetes Association, 1984. Address reprint requests to G.C. W eir. MD. Joslin Diabetes Center, One Joslin PI, Boston, MA 02215. 0 I987 by Grune & Stratton, Inc. 0026-0495/87/3604-0006$03.00/O 335