No abstract

We investigated the effect of 24 h of exposure to various glucose concentrations on insulin secretion by isolated rat pancreatic islets and purified rat beta-cells. Compared with islets cultured with standard medium (5.5 mM glucose), islets cultured with 16.7 mM glucose showed a higher basal insulin release (means +/- SE, 3.0 +/- 0.5 vs. 0.7 +/- 0.2%, n = 8, P less than .005) and reduced glucose-stimulated insulin secretion (2.4 +/- 0.3 vs. 5.8 +/- 0.4%, n = 8, P less than .005). Similar results were also obtained with purified beta-cells. The effect of high glucose was time dependent (present after 12 h, maximal after 24 h) and reversible: when islets cultured with high glucose were transferred to standard medium, normal responsiveness to glucose was restored within 8 h and normal basal release within 24 h. Mannitol, 3-O-methylglucose, and 2-deoxyglucose were not able to mimic the effects of glucose. Islets or purified beta-cells cultured in the presence of high glucose had a normal response when stimulated with glyburide, dibutyryl cyclic AMP, and isobutylmethylxanthine. Tunicamycin, an inhibitor of N-terminal glycosylation, prevented glucose-induced desensitization when added during 24 h of islet culture with 16.7 mM glucose. Swainsonine, another agent that influences glycosylation, had a similar effect. Our study indicates 1) that 24 h of exposure to high glucose induces a specific and reversible impairment of insulin secretion in response to glucose, 2) that this is a direct effect of glucose on beta-cells, and 3) that islet glucose metabolism and glycosylation processes may play a critical role in determining glucose desensitization.

We studied the internalization of [125I]insulin into circulating human monocytes, a cell type widely used for insulin binding studies. The internalization of [125I]insulin was assessed by both an acid extraction technique, which removes surface-bound insulin but not intracellular insulin, and by a trypsinization technique, which removes cell surface-bound hormone. After 5 h of incubation at 22 C, over 40% of the total cell-associated [125I]insulin was internalized into monocytes of normal subjects. This internalization was temperature dependent; the fraction of internalized hormone was progressively decreased when the incubation temperature was reduced from 37 to 4 C. Treatment of monocytes with increasing concentrations of 2,4-dinitrophenol also decreased [125I]insulin internalization, whereas dansylcadaverine, an inhibitor of transglutaminase, had no effect. Analysis by gel filtration of the internalized labeled hormone after 4 h of incubation at 22 C indicated that 50-60% of the label was degraded insulin, but detectable intact insulin was still present. Internalization of insulin was then studied in monocytes from eight obese patients (161% of ideal body weight) with type II diabetes mellitus. After 4 h of incubation at 22 C, the specific total monocyte-associated [125I]insulin was decreased compared to that in cells from 7 normal subjects [6.02 +/- 0.38% (+/- SE) vs. 3.91 +/- 0.31% of the total; P less than 0.001]. Moreover, the percentage of hormone that was internalized was also decreased from 41.4 +/- 1.2% of the total to 28.9 +/- 1.8% (P less than 0.001). In 20 nondiabetic obese subjects, specific cell-associated [125I]insulin was reduced to 3.9 +/- 0.3% (P less than 0.001). However, compared to that in normal subjects, the percentage of hormone that was internalized was not decreased (39.7 +/- 3.51% of the total). The present findings indicate that human circulating monocytes internalize [125I]insulin; this process is temperature and energy dependent; and monocytes from obese type II diabetic patients have a significantly decreased ability to internalize insulin. This decreased internalization may play a role in the cellular resistance to insulin that occurs in these patients.

We investigated the effect of 24 h exposure to 100 nmol/l glibenclamide on insulin secretion in isolated rat pancreatic islets. The insulin content was similar in control islets and in islets preincubated with 100 nmol/l glibenclamide for 24 h. In islets preexposed to glibenclamide: 1) the subsequent response to a maximal glibenclamide stimulatory concentration (10 mumol/l, 1 h at 37 C) was greatly reduced in comparison to control islets (0.69 +/- 0.20% vs 2.16 +/- 0.41%; mean +/- SE; n = 14; p less than 0.001); 2) the response to 100 mumol/l tolbutamide stimulation was also reduced (0.55 +/- 0.15% vs 2.38 +/- 0.44%; n = 8; p less than 0.001); 3) the response to 16.7 mmo/l glucose, both in the presence or in the absence of 1 mmol/l IBMX, a phosphodiesterase inhibitor, was also diminished by about 50% (1.79 +/- 0.39% vs. 3.22 +/- 0.42%; n = 14, p less than 0.001). In glibenclamide pretreated islets, blunted responses to stimuli were confirmed also by dynamic studies using a perifusion system. The effect of glibenclamide preincubation was fully reversible: when islets cultured in the presence of glibenclamide were transferred to a glibenclamide-free medium for further 24 h, insulin release in response to glibenclamide stimulation returned to control values. We conclude that prolonged exposure of rat pancreatic islets to glibenclamide induces a reversible desensitization to a variety of metabolic stimuli. The inhibition by prolonged glibenclamide exposure of a common pathway in the mechanism of insulin release is one possible explanation for these results.