Endocrine cells are continually regulating the balance between hormone biosynthesis, secretion, and intracellular degradation to ensure that cellular hormone stores are maintained at optimal levels. In pancreatic beta-cells, intracellular insulin stores in beta-granules are mostly upheld by efficiently up-regulating proinsulin biosynthesis at the translational level to rapidly replenish the insulin lost via exocytosis. Under normal circumstances, intracellular degradation of insulin plays a relatively minor janitorial role in retiring aged beta-granules, apparently via crinophagy. However, this mechanism alone is not sufficient to maintain optimal insulin storage in beta-cells when insulin secretion is dysfunctional. Here, we show that despite an abnormal imbalance of glucose/glucagon-like peptide 1 regulated insulin production over secretion in Rab3A(-/-) mice compared with control animals, insulin storage levels were maintained due to increased intracellular beta-granule degradation. Electron microscopy analysis indicated that this was mediated by a significant 12-fold up-regulation of multigranular degradation vacuoles in Rab3A(-/-) mouse islet beta-cells (P

Insulin secretory dysfunction of the pancreatic ␤-cell in type-2 diabetes is thought to be due to defective nutrient sensing and/or deficiencies in the mechanism of insulin exocytosis. Previous studies have indicated that the GTP-binding protein, Rab3A, plays a mechanistic role in insulin exocytosis. Here, we report that Rab3A ؊/؊ mice develop fasting hyperglycemia and upon a glucose challenge show significant glucose intolerance coupled to ablated first-phase insulin release and consequential insufficient insulin secretion in vivo, without insulin resistance. The in vivo insulin secretory response to arginine was similar in Rab3A ؊/؊ mice as Rab3A ؉/؉ control animals, indicating a phenotype reminiscent of insulin secretory dysfunction found in type-2 diabetes. However, when a second arginine dose was given 10 min after, there was a negligible insulin secretory response in Rab3A ؊/؊ mice, compared with that in Rab3A ؉/؉ animals, that was markedly increased above that to the first arginine stimulus. There was no difference in ␤-cell mass or insulin production between Rab3A ؊/؊ and Rab3A ؉/؉ mice. However, in isolated islets, secretagogue-induced insulin release (by glucose, GLP-1, glyburide, or fatty acid) was ϳ60 -70% lower in Rab3A

1. We examined whether pituitary adenylate cyclase-activating polypeptide with 38 or 27 residues (PACAP_38 or PACAP_27) serves as an intra-islet regulator of glucose-induced insulin secretion in rats. PACAP antiserum specific for PACAP_38 and PACAP_27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca¥ concentration ([Ca¥]é) in single â-cells, monitored by dual-wavelength fura_2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription-polymerase chain reaction (RT_PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2. High glucose-stimulated insulin release from isolated islets was attenuated by PACAP antiserum but not by non-immune sera. 3. The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca¥]é in â_cells. PACAP antiserum also neutralized the [Ca¥]é-increasing action of synthetic PACAP_38 and PACAP_27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4. Both Med and synthetic PACAP increased [Ca¥]é in â_cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from â_cells, increased [Ca¥]é in â_cells at both basal and stimulatory glucose concentrations. 5. Expression of PACAP mRNA and biosynthesis of PACAP_38 were detected in islets and a â_cell line, MIN6. 6. Immunoreactivity for PACAP-selective type-I receptor was observed in islets. 7.[Ca¥]é measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose-unresponsive â_cells, many of which were recruited by PACAP_38 into [Ca¥]é responses. 8. These results indicate that PACAP_38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine andÏor paracrine manner, potentiates and arouses â-cell responses to glucose, thereby amplifying glucose-induced insulin secretion in islets.

ABSTRACT. Oxygen-free radicals are thought to be a major cause of b-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca 2+ concentration ([Ca 2+ ]i) and the activity of ATP-sensitive potassium (K + ATP) channels in isolated rat pancreatic b-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca 2+ ]i from 114.3±15.4 nM to 531.1±71.9 nM (n=6) and also increased frequency of K + ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca 2+ ]i, the transient inhibition of K + ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca 2+ , 0.1 mM H2O2 increased [Ca 2+ ]i from 88.8±7.2 nM to 134.6±8.3 nM. Magnitude of [Ca 2+ ]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ pump (45.8±4.9 nM vs 15.0±4.8 nM). Small increase in [Ca 2+ ]i in response to an increase of external Ca 2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5±122.7 nM). We concluded that H2O2 not only activates K + ATP channels in association with metabolic inhibition, but also increases partly the Ca 2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic b-cells.

In the present study, we examined the ability of adenosine 3′,5′-cyclic monophosphate (cAMP) to reduce elevated levels of cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic β-cells. [Ca2+]iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single β-cells by fura 2 and autofluorescence microfluorometry. Sustained [Ca2+]ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activator forskolin (10 μM), and an incretin glucagon-like peptide-1-(7–36) amide (10−9 M), as well as by glucose (16.7 mM). The [Ca2+]i-reducing effects of cAMP were greater at elevated glucose (8.3–16.7 mM) than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA), H-89, counteracted [Ca2+]i-reducing effects of cAMP but not those of glucose. Okadaic acid, a phosphatase inhibitor, at 10–100 nM also reduced sustained [Ca2+]ielevation in a concentration-dependent manner. Glucose, but not DBcAMP, increased NAD(P)H in β-cells. [Ca2+]i-reducing effects of cAMP were inhibited by 0.3 μM thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+ pump. In contrast, [Ca2+]i-reducing effects of cAMP were not altered by ryanodine, an ER Ca2+-release inhibitor, Na+-free conditions, or diazoxide, an ATP-sensitive K+ channel opener. In conclusion, the cAMP-PKA pathway reduces [Ca2+]ielevation by sequestering Ca2+ in thapsigargin-sensitive stores. This process does not involve, but is potentiated by, activation of β-cell metabolism. Together with the known [Ca2+]i-increasing action of cAMP, our results reveal dual regulation of β-cell [Ca2+]iby the cAMP-signaling pathway and by a physiological incretin.

1. Calcium signalling by acetylcholine (ACh) in single rat pancreatic /3-cells was studied. The cytosolic free Ca2+ concentration ([Ca2+]

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cAMP and the insulinotropic peptides that raise cAMP glucose-dependently increase the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells, which is tightly linked to the potentiation of glucose-induced insulin release. We examined whether cAMP increases [Ca2+]i in specific cooperation only with glucose or also with other insulin secretagogues that act through different mechanisms. [Ca2+]i in single rat pancreatic beta-cells was measured by dual-wavelength fura-2 microfluorometry. In the presence of a stimulatory concentration of glucose (8.3 mmol/l) and the moderate elevation in [Ca2+]i induced by it, forskolin, an activator of adenylyl cyclase, or dibutyryl cAMP produced a marked additional increase in [Ca2+]i but was ineffective at the basal 2.8 mmol/l glucose. These cAMP-elevating agents also potentiated the effect of tolbutamide on [Ca2+]i. The cAMP-induced increase in [Ca2+]i was completely and selectively inhibited by a blocker of cAMP-dependent protein kinase A (PKA), and by nitrendipine, a blocker of the L-type Ca2+ channel. However, in the presence of high KCl and the [Ca2+]i elevation induced by it, a rise in cAMP failed to further increase [Ca2+]i, whereas BAY K8644, an agonist of L-type Ca2+ channels, evoked an additional increase in [Ca2+]i. Under low Na+ conditions, the [Ca2+]i response to cAMP was observed in the majority of the cells. In the cells in which glucose at 4.5-5 mmol/l was inadequate to increase [Ca2+]i, the glucose together with a rise in cAMP often increased [Ca2+]i. Likewise, tolbutamide and a rise in cAMP acted in concert to increase [Ca2+]i. Thus, cAMP left-shifted the concentration-[Ca2+]i response relationship for glucose and tolbutamide. In conclusion, the cAMP-PKA pathway acts in selective synergism with glucose and tolbutamide to initiate [Ca2+]i signals in pancreatic beta- cells. cAMP appears to regulate beta-cell sensitivity to glucose and tolbutamide. In contrast, cAMP fails to cooperate with high KCl to increase [Ca2+]i. It is suggested that cAMP acts mainly on a site that is more proximal but functionally linked to the L-type Ca2+ channel, thereby finally increasing Ca2+ influx through this channel.