An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.

In type 1 diabetes (T1D), there is a specific destruction of the insulin secreting pancreatic ␤ cell. Although the exact molecular mechanisms underlying ␤ cell destruction are not known, sera from T1D patients have been shown to promote Ca 2؉ -induced apoptosis. We now demonstrate that apolipoprotein CIII (apoCIII) is increased in serum from T1D patients and that this serum factor both induces increased cytoplasmic free intracellular Ca 2؉ R esearch over the last 30 years has established that type 1 diabetes (T1D) is an autoimmune disease, but the mechanisms͞events that trigger the initiation and progression of the disease are still not identified. Genetic, immunological, and environmental factors are involved in the pathogenesis of T1D and most likely the events involved differ between different patients. Voltage-gated L-type Ca 2ϩ channels have an important physiological role in pancreatic ␤ cell signal transduction (1). These channels constitute an essential link between transient changes in membrane potential and insulin release. Changes in cytoplasmic free intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) in the ␤ cell are associated with the activation of a spectrum of intracellular signals and are strictly regulated because prolonged high [Ca 2ϩ ] i is harmful to the cells. Sera from newly diagnosed T1D patients have been shown to increase the activity of voltage-gated L-type Ca 2ϩ channels in ␤ cells, resulting in increased [Ca 2ϩ ] i upon depolarization and ␤ cell apoptosis, effects that can be prevented by Ca 2ϩ channel blockers (2). The key question has been what factor in T1D serum that is responsible for the changes in [Ca 2ϩ ] i . In the present study, we have revealed the identity of a key factor in T1D sera that increases [Ca 2ϩ ] i as well as promotes apoptosis and found it to correspond to apolipoprotein CIII (apoCIII). The fact that not all sera from T1D patients affected [Ca 2ϩ ] i indicates that T1D is not caused by a single factor like apoCIII, which is in agreement with clinical observations, suggesting that different factors can act in concert with the autoimmune abnormalities resulting in ␤ cell destruction. MethodsMedia. The basal medium used both for isolation of cells and for experiments was a Hepes buffer (pH 7.4), containing: 125 mM NaCl, 5.9 mM KCl, 1.3 mM CaCl 2 , 1.2 mM MgCl 2 , and 25 mM Hepes. BSA was added to the medium at a concentration of 1 mg͞ml. For cell culture, RPMI medium 1640 was supplemented with 100 g͞ml streptomycin, 100 units of penicillin, and 10% FCS, normal human, or diabetic serum.Preparation of Cells. Adult mice from a local colony (3) were starved overnight. Pancreatic islets were isolated by a collagenase technique, and cell suspensions were prepared as described (4, 5). Cells were seeded onto glass coverslips and cultured at 37°C in a humidified atmosphere of 5% CO 2 in air.Preparation and Purification of Sera. Sera from T1D patients and control subjects were collected, identically sterile-processed, and stored frozen at Ϫ20°C until used. The sera we...

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The insulinotropic activity of the novel imidazoline compound BL11282 was investigated. Intravenous administration of BL11282 (0.3 mg ⅐ kg -1 ⅐ min -1 ) to anesthetized rats did not change blood glucose and insulin levels under basal conditions, but produced a higher increase in blood insulin levels and a faster glucose removal from the blood after glucose infusion. Similarly, in isolated Wistar rat pancreatic islets, 0.1-100 mol/l BL11282 potently stimulated glucose-induced insulin secretion but did not modulate basal insulin secretion. Unlike previously described imidazolines, BL11282 did not block ATP-dependent K ؉ channels. Furthermore, the compound stimulated insulin secretion in islets depolarized with high concentrations of KCl or permeabilized with electric shock. Insulinotropic activity of BL11282 was dependent on activity of protein kinases A and C. In pancreatic islets from spontaneously diabetic GK rats, the imidazoline compound restored the impaired insulin response to glucose. In conclusion, the imidazoline BL11282 constitutes a new class of insulinotropic compounds that exerts an exclusive glucose-dependent insulinotropic activity in pancreatic islets by stimulating insulin exocytosis. Diabetes 50:797-802, 2001 C ompounds with imidazoline structure are potent stimulators of insulin secretion in pancreatic ␤-cells (1). This effect is thought to be due to the blockade of ATP-dependent K ϩ channel (K ATP ) activity and the increase in cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] i ) (2). Recently, we have shown that the imidazoline compound RX871024, in addition to blocking K ATP channels and increasing [Ca 2ϩ ] i , exerts its insulinotropic effect by enhancing insulin exocytosis at stages distal to the increase in [Ca 2ϩ ] i (3-5). This direct stimulation of exocytosis was only observed at elevated glucose concentrations and required activation of protein kinases A and C.Sulfonylurea drugs, which are currently used in the treatment of type 2 diabetes, block K ATP channels in ␤-cells and stimulate insulin secretion at both low and high glucose concentrations (6). The strong insulinotropic effect of sulfonylureas observed at low glucose concentrations often provokes pronounced hypoglycemia in patients treated with these drugs (7). Therefore, agents that do not stimulate basal insulin secretion but augment glucoseinduced insulin secretion by modulating Ca 2ϩ -stimulated insulin exocytosis could provide a better therapeutic alternative to sulfonylureas.We now demonstrate that an imidazoline compound, BL11282 (Fig. 1A, insert), represents a new class of insulinotropic compounds that do not stimulate basal insulin secretion and lack the effect on K ATP channel activity, but markedly potentiate glucose-induced insulin secretion. RESEARCH DESIGN AND METHODS BL11282 and LY333531 were obtained from Eli Lilly (Indianapolis, IN).Rp-cAMPS was from Calbiochem (San Diego, CA). All other reagents were from Sigma (St. Louis, MO). Intravenous glucose tolerance test. The intravenous glucose tolerance test ...

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