High concentrations of glucose are considered to be toxic for the pancreatic ␤-cell. However, the mechanisms underlying ␤-cell dysfunction and resulting cell death are not fully characterized. In the present study we have demonstrated that incubation of pancreatic islets and ␤-cells from ob/ob mice and Wistar rats with glucose induced a process of apoptotic ␤-cell death, as shown by DNA laddering, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, and by using DNA-staining dye HOECHST 33342. The obtained results show that the percentage of apoptotic cells was dependent on glucose concentration, being minimal at 11 mM glucose. At a concentration of 100 M, aurintricarboxylic acid, an inhibitor of endonuclease activity, almost completely inhibited apoptosis triggered by 17 mM glucose. We have also shown that long term incubation with 100 M sulfonylurea, tolbutamide, triggered apoptosis in pancreatic ␤-cells. The process of ␤-cell death induced by high glucose concentration and tolbutamide were Ca 2؉ -dependent, because introduction to the culture medium of 50 M D-600 or 200 M diazoxide, which blocked glucose-and tolbutamide-induced [Ca 2؉ ] i increase, inhibited apoptosis. Thus, this study shows for the first time that high glucose concentrations and tolbutamide induce apoptosis in pancreatic ␤-cells, and that this process is Ca 2؉ -dependent.Glucose is the principal regulator of pancreatic ␤-cell function. Metabolism of glucose in the pancreatic ␤-cell leads to generation of ATP, closure of ATP-regulated K ϩ channels (K ATP channels), plasma membrane depolarization, opening of voltage-dependent Ca 2ϩ channels, increase in cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] i ) 1 and thereby insulin release (1). Long term exposure to a hyperglycemic environment leads to ␤-cell dysfunction (2-6), so-called glucose toxicity (7-9), and glucose desensitization (10, 11). The persistent hyperglycemia, which is a feature of diabetes mellitus, may also cause eye, nerve, and kidney microvascular complications.Despite the convincing evidence for glucose toxicity in pancreatic ␤-cells, resulting in cell death, the underlying mechanisms have not yet been identified. Cell death could be categorized as either necrotic (accidental) or apoptotic (programmed). Apoptosis is a mechanism of self-destruction, and a variety of stimuli and processes have been shown to induce apoptosis. Among those are DNA damage, growth factor deprivation, environmental toxins, heat shock, viral infections, and immune response (12).The aim of the present study was to investigate whether glucose and the antidiabetic sulfonylurea drug, tolbutamide, can trigger apoptosis of pancreatic ␤-cells. EXPERIMENTAL PROCEDURESMaterials-RPMI 1640 medium and fetal calf serum were obtained from Life Technologies, Inc. IL-1␤, tolbutamide, bovine serum albumin, aurintricarboxylic acid (ATA), diazoxide, palmitate, and oleate were purchased from Sigma. The fluorescent dyes fluorescein isothiocyanate (FITC), propidium iodide (PI), HOECHST 33342 (HO 342), and fura-2 ace...

A novel imidazoline compound, RX871024, was used to investigate the mechanisms by which imidazoline derivatives promote insulin secretion in rat pancreatic beta-cells and HIT T15 cells. RX871024 stimulated insulin release from rat pancreatic beta-cells and HIT T15 cells in a glucose-dependent way. This effect was not related to alpha2-adrenergic, I1-, and I2-imidazoline receptors. RX871024 promoted insulin release by at least two modes of action. One included an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), subsequent to blocking of ATP-dependent K+ channels, membrane depolarization, and activation of voltage-dependent Ca2+ channels. The other, a more distal effect of imidazoline, affected the exocytotic machinery and was unrelated to changes in membrane potential and [Ca2+]i. The mechanism of RX871024-induced insulin release was dependent on protein kinases A and C. The sensitizing effect of a low dose of RX871024 on glucose-induced insulin secretion suggests that imidazoline compounds of this kind may constitute the basis for development of a new class of oral hypoglycemic agents.

The objective of this study was to compare effects of RX-871024, a compound with imidazoline structure, and the sulfonylurea glibenclamide, representatives of two groups of ATP-dependent potassium channel (KATP) blockers, on insulin secretion and cytoplasmic free calcium concentration ([Ca2+]i). Furthermore, we studied the interaction of the compounds on these two parameters. The experiments were performed in the perfused rat pancreas, isolated rat pancreatic islets, and dispersed β-cells. At maximal effective concentrations of the compounds, RX-871024 had a more pronounced insulinotropic effect than glibenclamide, but the increase in [Ca2+]iwas similar. Glibenclamide enhanced the insulinotropic effect of suboptimal concentrations of RX-871024 at 3.3 and 16.7 mM glucose. Notably, glibenclamide and RX-871024 also stimulated insulin secretion under Ca2+-clamped conditions, i.e., during plasma membrane depolarization with KCl and glucose or in permeabilized islets. The magnitudes of insulin stimulation under the latter types of conditions were similar for both compounds. It is concluded that RX-871024 and the sulfonylurea glibenclamide promote insulin secretion by two mechanisms, namely closure of KATP channels and a direct stimulation of exocytosis. At a similar increase in [Ca2+]i, the maximal stimulatory effect of RX-871024 on insulin secretion was stronger than that of glibenclamide, implying that RX-871024 also affects insulin secretion by a signal transduction pathway that is not activated by glibenclamide.