Interacting Effects of Sulfonylureas and Glucose on Cyclic AMP Metabolism and Insulin Release in Pancreatic Islets of the Rat

Interacting Effects of Sulfonylureas and Glucose on Cyclic AMP Metabolism and Insulin Release in Pancreatic Islets of the Rat V. Grill, E. Cerasi J Clin Invest. 1978;61(5):1346-1354. https://doi.org/10.1172/JCI109052. Research Article The effects of tolbutamide and glibenclamide on the metabolism of cyclic AMP were investigated in pancreatic islets of the rat. Changes in cyclic AMP were assessed by measuring [3H]cyclic AMP after labeling of the islets with [2-3H]adenine. In the presence of a nonstimulatory concentration of glucose (3.3 mM), both sulfonylureas caused a rapid increase in islet [3H]cyclic AMP, which declined within 5 (tolbutamide) or 10 min (glibenclamide). In the absence of glucose, the glibenclamide effect was shortened, but the initial (1 min) response of [3H]-cyclic AMP was unaffected. Glucose could be substituted with d-glyceraldehyde but not pyruvate for prolongation of the glibenclamide response. The effect of glucose withdrawal on the glibenclamide response was reproduced by the addition of d-mannoheptulose to glucose containing media. The [3H]cyclic AMP response to glibenclamide was influenced by prior exposure of the islets to glucose, a 30-min preincubation with 27.7 mM glucose, enhancing the response to the sulfonylurea over a subsequent 5-min stimulation period. Sulfonylureas exerted their effects at low but not at high glucose concentrations, i.e., shifted the glucose dose-response curve to the left both for [3H]cyclic AMP accumulation and insulin release. On the other hand, increasing concentrations of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, progressively augmented the effects of the drugs. Omission of Ca++ from the incubation media inhibited both the glucose and the sulfonylurea 3[H]-cyclic […] Find the latest version: https://jci.me/109052/pdf Interacting Effects of Sulfonylureas and Glucose on Cyclic AMP Metabolism and Insulin Release in Pancreatic Islets of the Rat V. GRILL and E. CERASI, Department of Endocrinology, Karolinska Hospital, S-104 01 Stockholm 60, Sweden A B S T R A C T The effects of tolbutamide and gliben- hibited both the glucose and the sulfonylurea [3H]- clamide on the metabolism of cyclic AMP were in- cyclic AMP and insulin responses. Epinephrine (1 uM) vestigated in pancreatic islets of the rat. Changes in partially inhibited the [3H]cyclic AMP response to cyclic AMP were assessed by measuring [3H]cyclic both glucose and sulfonylurea, whereas insulin release AMP after labeling of the islets with [2-3H]adenine. was completely abolished. In the presence of a nonstimulatory concentration of It is concluded that the sulfonylurea effects on islet glucose (3.3 mM), both sulfonylureas caused a rapid cyclic AMP are intimately related to those of glucose. increase in islet [3H]cyclic AMP, which declined It is suggested that sulfonylureas exert a major part within 5 (tolbutamide) or 10 min (glibenclamide). In of their action by facilitating the effect of glucose on the absence of glucose, the glibenclamide effect was the beta-cell adenylate cyclase; the increased cyclic shortened, but the initial (1 min) response of [3H]- AMP level, in its turn, enhances the secretion rate of cyclic AMP was unaffected. Glucose could be sub- insulin. stituted with D-glyceraldehyde but not pyruvate for prolongation of the glibenclamide response. The effect INTRODUCTION of glucose withdrawal on the glibenclamide response was reproduced by the addition of D-mannoheptulose The mode of action of sulfonylureas, well-known to glucose containing media. insulin secreting agents (1), is not well understood. The [3H]cyclic AMP response to glibenclamide was However, in recent years evidence has been ac- influenced by prior exposure of the islets to glucose, cumulating suggesting that the adenylate cyclase- a 30-min preincubation with 27.7 mM glucose, en- cyclic AMP' system of the islets is involved in the hancing the response to the sulfonylurea over a subse- transmission of the drug effect. In islet homogenates, quent 5-min stimulation period. tolbutamide stimulates adenylate cyclase to a moderate Sulfonylureas exerted their effects at low but not at extent (2) and inhibits phosphodiesterase enzyme ac- high glucose concentrations, i.e., shifted the glucose tivity (3-5). Recently, Charles and co-workers have dose-response curve to the left both for [3H]cyclic reported that tolbutamide increases the cyclic AMP AMP accumulation and insulin release. On the level of perifused islets simultaneously with insulin other hand, increasing concentrations of the phos- release (6). phodiesterase inhibitor, 3-isobutyl-l-methylxanthine, Insulin secretion induced by sulfonylureas is de- progressively augmented the effects of the drugs. pendent upon the prevailing glucose concentration. Omission of Ca++ from the incubation media in- Thus sulfonylurea induces a shift to the left in the glucose dose-response curve for insulin release (1, 7). Part of this work was previously reported at the symposium The nature of interaction between these drugs and glu- "Diabetes Research Today," held in Capri, Italy, 13-15 April cose is unknown. 1976, and published in Symposia Medica Hoechst 12, 223- We and others have obtained evidence that glucose 240 (1976), Friedrich-Karl Schattauer-Verlag GmbH, Stuttgart, increases cyclic AMP concomittantly with insulin West Germany. constitutes an Dr. Cerasi's present address is the Department of Endo- secretion, and that this action important crinology and Metabolism, the Hebrew University Hadassah Medical School, Jerusalem, Israel. 1Abbreviations used in this paper: cyclic AMP, cyclic aden- Received for publication 25 July 1977 and in revised form osine -3', 5'-monophosphate; IBMX, 3-isobutyl-1-methyl- 8 November 1977. xanthine; KHB, Krebs-Henseleit bicarbonate buffer. 1346 J. Clin. Invest. C The American Society for Clinical Investigation, Inc., 0021-9738178/0501-1346 $1.00 factor in the regulation of insulin release (8). Against pork insulin (obtained from the Radiochemical Centre, this background it seemed important to investigate the Amersham, Buckinghamshire, England) was used. Purified rat insulin (kindly supplied by Dr. J. Schlichtkrull, Novo Re- relationship, if any, between sulfonylurea- and glu- search Institute, Bagsvaerd, Denmark) served as standard. cose-induced changes in beta-cell cyclic AMP metab- Presentation of results. In view of the considerable inter- olism. To this end, different aspects of a glucose- experimental variation, quantitative comparisons have been sulfonylurea interaction were investigated: (a), the restricted to variables measured within the same experi- time-course of sulfonylurea effect in the presence or ments. Standard statistical procedures were used, all levels of significance being calculated from paired differences. For absence of glucose, (b), the effects of preincubation clarity, results are expressed, when deemed appropriate, with glucose on the subsequent sulfonylurea response, after the subtraction of control values. and (c), the effects of sulfonylurea over a range of glucose concentrations. In addition, the effects of dif- RESULTS ferent conditions inhibiting insulin release on the glucose- and sulfonylurea-induced responses were com- Dose dependency of sulfonylurea-induced insulin pared. An indirect method for assessing changes in release. The effects of 50-400 ,ug/ml of tolbutamide, cyclic AMP metabolism was utilized, namely the con- and 0.5-8.0 ,ug/ml of glibenclamide were tested to- version of [3H]ATP to [3H]cyclic AMP in prelabeled gether with 3.3 mM glucose in incubations of 30 min islets, a procedure whose validity has been confirmed (Table I). A maximal response was obtained with 200 (9). Two sulfonylureas were chosen: a classical one gg/ml tolbutamide and 2 ug/ml glibenclamide. These (tolbutamide), and one representative of the "second concentrations were chosen for further study. Under generation" of these drugs (glibenclamide). our experimental conditions, glibenclamide was a somewhat more potent insulin secretagogue than tolbutamide at their respective maximal doses. METHODS Time course of the sulfonylurea effect on [3H]cyclic Preparation of islets, labeling with [3H]adenine, and AMP in the presence of a nonstimulatory glucose incubation of islets. Male Sprague-Dawley rats weighing concentration. In the presence of 3.3 mM glucose, 100-150 g were fed ad libitum until decapitation. Pan- both tolbutamide and glibenclamide rapidly increased creatic islets were isolated by the collagenase method of Lacy and Kostianovsky (10). The collagenase employed was from Worthington Biochemical Corp., Freehold, N. J. Krebs TABLE I Henseleit-bicarbonate buffer (KHB) with 0.2% bovine serum Insulin and [3H ]Cyclic AMP Responses to Sulfonylurca albumin, 10 mM Hepes, and, when not otherwise indicated, in 30-min Incubations 3.3 mM glucose was used throughout the experiments. Islets were pulse labeled with 100 ,uCi/ml of [2-3H]adenine Immuno- No. (25 Ci/mmol, New England Nuclear, Dreieichenhain, W. reactive of Germany) during a 60-min preincubation period as previously Substance tested [3H]cAMP insulin expt. described (11). Islets were then transferred in batches of 15 either to small incubation tubes with a final incubation ±gIml dpm/islet dpm/islet M(U/islet volume of 1 ml or, in some experiments, to "baskets." These tissue medium were prepared by cutting small microcentrifugation plastic 6.9+0.6 1.3±0.1 1.6+0.1 7 tubes with an upper inner diameter of -4 mm (Beckman Instruments, Munich, W. Germany). The hollow cylinders Tolbutamide thus obtained were capped with nylon gauze (70 threads/cm2 50 9.0±+1.8 3.8±+1.1 4.4+ 1.4* of gauze, 36% open space). The baskets with the islets were 200 6.2±1.0 3.8±0.8* 6.6± 1.8* placed in small incubation tubes in a final volume of 0.5 400 6.8± 1.5 3.6±0.8* 6.6+0.8* ml. In all experiments incubations were carried out at 37°C together with the agents to be tested, and 0.1 mM of 3- 6.6+0.7 1.9±0.6 1.1+0.4 4 isobutyl-l-methylxanthine (IBMX) unless otherwise indi- Glibenclamide cated.

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