Glucose-induced insulin secretion from hyperglycemic 90% pancreatectomized rats is markedly impaired, possibly because of loss of ␤ cell differentiation. Association of these changes with ␤ cell hypertrophy, increased mRNA levels of the transcription factor c-Myc, and their complete normalization by phlorizin treatment suggested a link between chronic hyperglycemia, increased c-Myc expression, and altered ␤ cell function.In this study, we tested the effect of hyperglycemia on rat pancreatic islet c-Myc expression both in vivo and in vitro. Elevation of plasma glucose for 1-4 days (glucose infusion/clamp) was followed by parallel increases in islet mRNA levels (relative to TATA-binding protein) of c-Myc and two of its target genes, ornithine decarboxylase and lactate dehydrogenase A. Similar changes were observed in vitro upon stimulation of cultured islets or purified ␤ cells with 20 and 30 mmol⅐liter ؊1 glucose for 18 h. These effects of high glucose were reproduced by high potassium-induced depolarization or dibutyrylcAMP and were inhibited by agents decreasing cytosolic Ca 2؉ or cAMP concentrations. In conclusion, the expression of the early response gene c-Myc in rat pancreatic ␤ cells is stimulated by high glucose in a Ca 2؉ -dependent manner and by cAMP. c-Myc could therefore participate to the regulation of ␤ cell growth, apoptosis, and differentiation under physiological or pathophysiological conditions.Besides being the major stimulus of insulin secretion, glucose exerts pleiotropic effects in pancreatic ␤ cells, including stimulation of protein synthesis (1) and cell proliferation and cell growth (2). Stimulus-secretion coupling is largely understood (3), but how glucose exerts its other effects is unclear. Moreover, the influence of glucose is not always beneficial, because chronic hyperglycemia impairs ␤ cell gene expression, function and survival (4 -6). Several mechanisms have been proposed to explain this so called "glucose toxicity," including ␤ cell overstimulation, increased oxidative stress, protein glycation, and islet amyloid pancreatic polypeptide deposition. We have recently shown that 2-4 weeks after a 90% pancreatectomy, chronic hyperglycemia induces a loss of ␤ cell differentiation that could account for the marked alteration of glucoseinduced insulin secretion in this animal model of diabetes (7,8). Thus, the expression of several transcription factors involved in ␤ cell differentiation (transcription factor Pdx1, hepatocyte nuclear factors, etc.) and of a panel of genes involved in glucose recognition and stimulation of secretion (insulin, glucose transporter 2, mitochondrial glycerol-P dehydrogenase, etc.) was decreased in islets from hyperglycemic 90% pancrea-

Two sarcoendoplasmic reticulum Ca(2+)-ATPases, SERCA3 and SERCA2b, are expressed in pancreatic islets. Immunocytochemistry showed that SERCA3 is restricted to beta-cells in the mouse pancreas. Control and SERCA3-deficient mice were used to evaluate the role of SERCA3 in beta-cell cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) regulation, insulin secretion, and glucose homeostasis. Basal [Ca(2+)](c) was not increased by SERCA3 ablation. Stimulation with glucose induced a transient drop in basal [Ca(2+)](c) that was suppressed by inhibition of all SERCAs with thapsigargin (TG) but unaffected by selective SERCA3 ablation. Ca(2+) mobilization by acetylcholine was normal in SERCA3-deficient beta-cells. In contrast, [Ca(2+)](c) oscillations resulting from intermittent glucose-stimulated Ca(2+) influx and [Ca(2+)](c) transients induced by pulses of high K(+) were similarly affected by SERCA3 ablation or TG pretreatment of control islets; their amplitude was increased and their slow descending phase suppressed. This suggests that, during the decay of each oscillation, the endoplasmic reticulum releases Ca(2+) that was pumped by SERCA3 during the upstroke phase. SERCA3 ablation increased the insulin response of islets to 15 mmol/l glucose. However, basal and postprandial plasma glucose and insulin concentrations in SERCA3-deficient mice were normal. In conclusion, SERCA2b, but not SERCA3, is involved in basal [Ca(2+)](c) regulation in beta-cells. SERCA3 becomes operative when [Ca(2+)](c) rises and is required for normal [Ca(2+)](c) oscillations in response to glucose. However, a lack of SERCA3 is insufficient in itself to alter glucose homeostasis or impair insulin secretion in mice.

Buffered Ca(2+) diffusion in the cytosol of neuroendocrine cells is a plausible explanation for the slowness and latency in the secretion of hormones. We have developed a Monte Carlo simulation to treat the problem of 3-D diffusion and kinetic reactions of ions and buffers. The 3-D diffusion is modeled as a random walk process that follows the path of each ion and buffer molecule, combined locally with a stochastic treatment of the first-order kinetic reactions involved. Such modeling is able to predict [Ca(2+)] and buffer concentration time courses regardless of how low the calcium influx is, and it is therefore a convenient method for dealing with physiological calcium currents and concentrations. We study the effects of the diffusional and kinetic parameters of the model on the concentration time courses as well as on the local equilibrium of buffers with calcium. An in-mobile and fast endogenous buffer as described by, Biophys. J. 72:674-690) was able to reach local equilibrium with calcium; however, the exogenous buffers considered are displaced drastically from equilibrium at the start of the calcium pulse, particularly below the pores. The versatility of the method also allows the effect of different arrangements of calcium channels on submembrane gradients to be studied, including random distribution of calcium channels and channel clusters. The simulation shows how the particular distribution of channels or clusters can be of relevance for secretion in the case where the distribution of release granules is correlated with the channels or clusters.

The mutual enhancement of insulin release by glucose and amino acids is not clearly understood. In this study, the effects on electrical activity and insulin release of a mixture of amino acids and glucose at concentrations found in fed (aaFD) and fasted (aaFT) animals were determined using freshly isolated mouse islets. Islets perifused with aaFD mixture showed an oscillatory pattern of electrical activity at lower glucose concentrations (5 mmol/l) than in islets perifused with the aaFT mixture and with glucose (G) alone (10 mmol/l). The concentration/response curve for the fraction of time spent by the membrane potential in the active phase in aaFD-stimulated islets was found to be significantly shifted to the left and had a smaller slope than that for glucose-stimulated islets. Insulin release followed the same pattern. This resulted in a concentration/response curve for glucose that was closer to that recorded "in vivo". We have also found that four amino acids (leucine, isoleucine, alanine and arginine) are largely responsible for the observed effects and that there is a non-linear enhancement of insulin release as a consequence of the combined effect of amino acids and glucose. This effect was more pronounced in the second phase of insulin release and was dependent on intracellular Ca2+. These findings indicate that amino acids account for most of the left-ward shift in the concentration/response curve for glucose and that a reduction in the threshold for the glucose-induced oscillatory electrical activity response and in the generation of Ca2+ spikes accounts for the triggering of insulin release at lower glucose concentrations. Nevertheless, the effects on insulin release at high glucose concentrations cannot be explained solely by the increase in glucose-induced electrical activity.

Increased ␤-cell sensitivity to glucose precedes the loss of glucose-induced insulin secretion in diabetic animals. Changes at the level of ␤-cell glucose sensor have been described in these situations, but it is not clear whether they fully account for the increased insulin secretion. Using a euglycemic-normolipidemic 60% pancreatectomized (60%-Px) mouse model, we have studied the ionic mechanisms responsible for increased ␤-cell glucose sensitivity. Two weeks after Px (Px14 group), Px mice maintained normoglycemia with a reduced ␤c e l l mass (0.88 ± 0.18 mg) compared with control mice (1.41 ± 0.21 mg). At this stage, the dose-response curve for glucose-induced insulin release showed a signific a n t displacement to the left (P < 0.001). Islets from the Px14 group showed oscillatory electrical activity and cytosolic Ca 2 + ([ C a 2 + ] i) oscillations in response to glucose concentrations of 5.6 mmol/l compared with islets from the control group at 11.1 mmol/l. All the above changes were fully reversible both in vitro (after 48-h culture of islets from the Px14 group) and in vivo (after regeneration of ␤-cell mass in islets studied 60 days after Px). No significant differences in the input resistance and ATP inhibition of ATP-sensitive K + (K AT P) channels were found between ␤-cells from the Px14 and control groups. The dose-response curve for glucoseinduced MTT (C,N-d i p h e n y l-N-4,5-dimethyl thiazol 2 yl tetrazolium bromide) reduction showed a significant displacement to the left in islets from the Px14 group (P < 0.001). These results indicate that increased glucose sensitivity in terms of insulin secretion and Ca 2 + s i gnaling was not due to intrinsic modifications of K AT P channel properties, and suggest that the changes are most likely to be found in the glucose metabolism.