The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free Ca(2+) ([Ca(2+)](c)) in beta-cells play a key role in the control of insulin secretion, and because oscillations of these two factors have been observed in single isolated islets and beta-cells, pulsatile insulin secretion could theoretically result from [Ca(2+)](c) or metabolism oscillations. We could not detect metabolic oscillations independent from [Ca(2+)](c) changes in beta-cells, and imposed metabolic oscillations were poorly effective in inducing oscillations of secretion when [Ca(2+)](c) was kept stable, which suggests that metabolic oscillations are not the direct regulator of the oscillations of secretion. By contrast, tight temporal and quantitative correlations between the changes in [Ca(2+)](c) and insulin release strongly suggest that [Ca(2+)](c) oscillations are the direct drivers of insulin secretion oscillations. Metabolism may play a dual role, inducing [Ca(2+)](c) oscillations (via changes in ATP-sensitive K(+) channel activity and membrane potential) and amplifying the secretory response by increasing the efficiency of Ca(2+) on exocytosis. The mechanisms underlying the oscillations of insulin secretion by the isolated pancreas and those observed in vivo remain elusive. It is not known how the functioning of distinct islets is synchronized, and the possible role of intrapancreatic ganglia in this synchronization requires confirmation. That pulsatile insulin secretion is beneficial in vivo, by preventing insulin resistance, is suggested by the greater hypoglycemic effect of exogenous insulin when it is infused in a pulsatile rather than continuous manner. The observation that type 2 diabetic patients have impaired pulsatile insulin secretion has prompted the suggestion that such dysregulation contributes to the disease and justifies the efforts toward understanding of the mechanism underlying the pulsatility of insulin secretion both in vitro and in vivo.

Rapid and sustained stimulation of ␤-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each ␤-cell rather than heterogeneity in the time-course of the response between ␤-cells or islets. There is no evidence indicating that biphasic secretion can be attributed to an intrinsically biphasic metabolic signal. In contrast, the biphasic rise in cytoplasmic Ca

Hypertrophy is one mechanism of pancreatic ␤-cell growth and is seen as an important compensatory response to insulin resistance. We hypothesized that the induction of protective genes contributes to the survival of enlarged (hypertrophied) ␤-cells. Here, we evaluated changes in stress gene expression that accompany ␤-cell hypertrophy in islets from hyperglycemic rats 4 weeks after partial pancreatectomy (Px). A variety of protective genes were upregulated, with markedly increased expression of the antioxidant genes heme oxygenase-1 and glutathione peroxidase and the antiapoptotic gene A20. Cu/Zn-superoxide dismutase (SOD) and Mn-SOD were modestly induced, and Bcl-2 was modestly reduced; however, several other stress genes (catalase, heat shock protein 70, and p53) were unaltered. The increases in mRNA levels corresponded to the degree of hyperglycemia and were reversed in Px rats by 2-week treatment with phlorizin (treatment that normalized hyperglycemia), strongly suggesting the specificity of hyperglycemia in eliciting the response. Hyperglycemia in Px rats also led to activation of nuclear factor-B in islets. The profound change in ␤-cell phenotype of hyperglycemic Px rats resulted in a reduced sensitivity to the ␤-cell toxin streptozotocin. Sensitivity to the toxin was restored, along with the ␤-cell phenotype, in islets from phlorizin-treated Px rats. Furthermore, ␤-cells of Px rats were not vulnerable to apoptosis when further challenged in vivo with dexamethasone, which increases insulin resistance. In conclusion, ␤-cell adaptation to chronic hyperglycemia and, hence, increased insulin demand is accompanied by the induction of protective stress genes that may contribute to the survival of hypertrophied ␤-cells. Diabetes 51:413-423, 2002 E xpansion of islet ␤-cell mass represents an important compensatory mechanism to maintain normoglycemia in the face of insulin resistance and obesity (1-5). An increase in ␤-cell mass accompanies insulin resistance induced in rats by glucocorticoid treatment (6) and in mice by mutation of the insulin receptor or insulin receptor substrate-1 (1). In humans, there is evidence that ␤-cell mass is increased in obese subjects compared with lean control subjects (7). In rodents, there is substantial evidence that increases in ␤-cell size (hypertrophy), replication, and neogenesis occur in situations of an increased insulin demand. For example, partial pancreatectomy (Px) in rats induces active neogenesis and ␤-cell replication within the first 7 days (8,9), and by 4 weeks, moderate chronic hyperglycemia produces ␤-cell hypertrophy (3,10). Hypertrophy has also been found in rats after chronic glucose infusion (11), in prediabetic ZDF-fa/fa rats with impaired glucose tolerance (2), and in pregnancy (12).During the progression to type 2 diabetes, the capacity for compensatory ␤-cell expansion may be overwhelmed, leading to decompensation (2,4,5,13-15). Reduction in ␤-cell mass secondary to an increased rate of apoptosis has been implicated (2,13,15). Recent experiments show that ...

Although type 2 diabetes mellitus is associated with insulin resistance, many individuals compensate by increasing insulin secretion. Putative mechanisms underlying this compensation were assessed in the present study by use of 4-day glucose (GLC; 35% Glc, 2 ml/h) and lipid (LIH; 10% Intralipid + 20 U/ml heparin; 2 ml/h) infusions to rats. Within 2 days of beginning the infusion of either lipid or glucose, plasma glucose profiles were normalized (relative to saline-infused control rats; SAL; 0.45% 2 ml/h). During glucose infusion, plasma glucose was maintained in the normal range by an approximately twofold increase in plasma insulin and an approximately 80% increase in beta-cell mass. During LIH infusion, glucose profiles were also maintained in the normal range. Plasma insulin responses during feeding were doubled, and beta-cell mass increased 54%. For both groups, the increase in beta-cell mass was associated with increased beta-cell proliferation (98% increase during GLC and 125% increase during LIH). At the end of the 4-day infusions, no significant changes were observed in islet-specific gene transcription (i.e., the expression of islet hormone genes, glucose metabolism genes, and insulin transcription factors were unaffected). Two days after termination of the infusions, the glucose-stimulated plasma insulin response was increased approximately 67% in glucose-infused animals. No sustained effect on insulin secretory capacity was observed in the LIH animals. The increase in plasma insulin response after glucose infusion was achieved in the absence of any change in insulin clearance. We conclude that, in rats, an increase in insulin demand after an increase in glucose appearance or free fatty acid leads to an increase in beta-cell mass, mediated in part by an increase in beta-cell proliferation, and that these compensatory changes lead to increased insulin secretion, normal plasma glucose levels, and the maintenance of normal islet gene expression.

IntroductionWhether adenine nucleotides in pancreatic B cells serve as second messengers during glucose stimulation of insulin secretion remains disputed. Our hypothesis was that the actual changes in ATP and ADP are obscured by the large pool of adenine nucleotides (ATP/ADP ratio close to 1) in insulin granules. Therefore, mouse islets were degranulated acutely with a cocktail of glucose, KCl, forskolin, and phorbol ester or during overnight culture in RPMI-1640 medium containing 10 mM glucose. When these islets were then incubated in 0 glucose + azide (to minimize cytoplasmic and mitochondrial adenine nucleotides), their content in ATP+ADP+AMP was decreased in proportion to the decrease in insulin stores. After incubation in 10 mM glucose (no azide), the ATP/ADP ratio increased from 2.4 to > 8 in cultured islets, and only from 2 to < 4 in fresh islets. These differences were not explained by changes in glucose oxidation. The glucose dependency (0-30 mM) of the changes in insulin secretion and in the ATP/ADP ratio were then compared in the same islets. In nondegranulated, fresh islets, the ATP/ADP ratio increased between 0 and 10 mM glucose and then stabilized although insulin release kept increasing. In degranulated islets, the ATP/ADP ratio also increased between 0 and 10 mM glucose, but a further increase still occurred between 10 and 20 mM glucose, in parallel with the stimulation of insulin release. In conclusion, decreasing the granular pool of ATP and ADP unmasks large changes in the ATP/ADP ratio and a glucose dependency which persists within the range of stimulatory concentrations. The ATP/ADP ratio might thus serve as a coupling factor between glucose metabolism and insulin release. (J. Clin. Invest. 1995. 96:1738-1745 Measurements of adenine nucleotides in incubated islets. The aliquot of medium used for insulin assay was taken while the incubation tubes remained at 37°C. The islets were then incubated for another 5 min before the incubation was stopped by addition of 0.6 ml of icecold trichloroacetic acid to a final concentration of 5%. The tubes were vortex-mixed, left on ice for 15 min and centrifuged for 5 min in a microfuge (Eppendorf Inc., Fremont, CA). A fraction (0.5 ml) of the supernatant was then mixed with 1.5 ml diethylether, and the ether phase containing trichloracetic acid was discarded. This step was repeated three times to ensure complete elimination of trichloracetic acid. The extracts were eventually diluted with 0. Batches of five freshly isolated islets were incubated for 120 min in 1 ml medium containing 10 mM glucose or a stimulatory cocktail composed of 25 mM glucose, 30 mM KCI, 250 ,uM diazoxide, 1 pM forskolin, and 50 nM PMA. At the end of this incubation, the medium was removed and replaced, for 60 min, by 1 ml medium containing no glucose and 10 mM azide. At the end of this second incubation, certain batches were used for measurement of insulin content and others for measurement of adenine nucleotides. Values are means±SEM. Experiment A was repeated three times with eight...