Glutamate dehydrogenase (GDH) is regulated by both positive (leucine and ADP) and negative (GTP and ATP) allosteric factors. We hypothesized that the phosphate potential of ␤-cells regulates the sensitivity of leucine stimulation. These predictions were tested by measuring leucine-stimulated insulin secretion in perifused rat islets following glucose depletion and by tracing the nitrogen flux of [2-15 N]glutamine using stable isotope techniques. The sensitivity of leucine stimulation was enhanced by long time (120-min) energy depletion and inhibited by glucose pretreatment. After limited 50-min glucose depletion, leucine, not ␣-ketoisocaproate, failed to stimulate insulin release. ␤-Cells sensitivity to leucine is therefore proposed to be a function of GDH activation. Leucine increased the flux through GDH 3-fold compared with controls while causing insulin release. High glucose inhibited flux through both glutaminase and GDH, and leucine was unable to override this inhibition. These results clearly show that leucine induced the secretion of insulin by augmenting glutaminolysis through activating glutaminase and GDH. Glucose regulates ␤-cell sensitivity to leucine by elevating the ratio of ATP and GTP to ADP and P i and thereby decreasing the flux through GDH and glutaminase. These mechanisms provide an explanation for hypoglycemia caused by mutations of GDH in children.In addition to glucose, amino acids and other metabolic fuels are important stimulants of insulin secretion from pancreatic ␤-cells. Leucine, which has been studied intensively, may stimulate insulin release through two different mechanisms. The first involves transamination of leucine to ␣-ketoisocaproate (KIC) 1 and subsequent mitochondrial oxidation. The second promotes insulin release via allosteric activation of glutamate dehydrogenase (GDH) causing oxidation of glutamate to the Krebs cycle intermediate, ␣-ketoglutarate, plus ammonia. The importance of the latter mechanism has been highlighted recently by the discovery of a dominant form of congenital hyperinsulinism associated with mutations of GDH leading to a gain of enzyme activity, because sensitivity to inhibition by GTP and ATP is impaired (1-3). Affected children have increased ␤-cell responsiveness to leucine and are susceptible to acute hypoglycemia following a high protein meal (4). The involvement of GDH may explain the observation that, in contrast to other amino acids, leucine-stimulated insulin secretion (LSIS) is suppressed by high glucose. For example, Gao et al. (5) reported that glucose inhibits leucine stimulation of glutaminolysis and insulin secretion in isolated mouse islets, presumably by increasing intracellular ATP and GTP while decreasing ADP and thus inhibiting GDH activity.GDH has also been proposed by Maechler and Wollheim (6) to play an essential role in glucose-mediated insulin secretion by acting in the reverse direction to catalyze production of glutamate, which is hypothesized to work as a cofactor in the process leading to exocytosis of insulin granules. T...

Children with hypoglycemia due to recessive loss of function mutations of the ␤-cell ATP-sensitive potassium (K ATP ) channel can develop hypoglycemia in response to protein feeding. We hypothesized that amino acids might stimulate insulin secretion by unknown mechanisms, because the K ATP channel-dependent pathway of insulin secretion is defective. We therefore investigated the effects of amino acids on insulin secretion and intracellular calcium in islets from normal and sulfonylurea receptor 1 knockout (SUR1؊/؊) mice. Even though SUR1؊/؊ mice are euglycemic, their islets are considered a suitable model for studies of the human genetic defect. SUR1؊/؊ islets, but not normal islets, released insulin in response to an amino acid mixture ramp. This response to amino acids was decreased by 60% when glutamine was omitted. Insulin release by SUR1؊/؊ islets was also stimulated by a ramp of glutamine alone. Glutamine was more potent than leucine or dimethyl glutamate. Basal intracellular calcium was elevated in SUR1؊/؊ islets and was increased further by glutamine. In normal islets, methionine sulfoximine, a glutamine synthetase inhibitor, suppressed insulin release in response to a glucose ramp. This inhibition was reversed by glutamine or by 6-diazo-5-oxo-L-norleucine, a non-metabolizable glutamine analogue. High glucose doubled glutamine levels of islets. Methionine sulfoximine inhibition of glucose stimulated insulin secretion was associated with accumulation of glutamate and aspartate. We hypothesize that glutamine plays a critical role as a signaling molecule in amino acid-and glucosestimulated insulin secretion, and that ␤-cell depolarization and subsequent intracellular calcium elevation are required for this glutamine effect to occur.

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh ؊/؊ ). Congenital hyperinsulinism is the most common cause of persistent hypoglycemia in infants and children (1). Six genetic loci have been associated with the disorder. The most common of these disorders are due to inactivating mutations of the sulfonylurea receptor 1 (SUR1) 2 and Kir6.2 subunits of the ␤-cell ATP-dependent potassium (K ATP ) channel or to activating mutations of glutamate dehydrogenase (GDH) and glucokinase. Recently, several children have been described with a recessively inherited form of hyperinsulinism that is associated with deficiency of a mitochondrial fatty acid ␤-oxidation enzyme, short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) encoded by the HADH gene on 4q (2-4). SCHAD catalyzes the third step in the ␤-oxidation cycle for medium and short-chain 3-hydroxy fatty acyl-CoAs. Children afflicted with SCHAD deficiency have recurrent episodes of hypoglycemia that can be prevented by treatment with diazoxide (2, 3) and also have characteristic accumulations of fatty acid metabolites, including plasma 3-hydroxybutyrylcarnitine and urinary 3-hydroxyglutaric acid (2, 3). This form of abnormal insulin regulation is unique, because other genetic disorders of mitochondrial fatty acid oxidation do not cause hyperinsulinism (5). In addition, the genetic defect in SCHAD deficiency is expected to impair, rather than increase, the production of ATP, which normally serves as the triggering signal for insulin release. An important clue to the mechanism of insulin dysregulation in SCHAD deficiency has been recently provided by the report * This work was supported, in whole or in part, by National Institutes of Health Grants DK53012 (to C. A. S.), DK22122 (to F. M. M.), DK 53761 (to I. N.), HL075421 (to A. W. S.). This work was also supported by a fellowship award from Society for Inherited Metabolic Disorders (to A. P.

Pancreatic islet ␤-cell dysfunction is a signature feature of Type 2 diabetes pathogenesis. Consequently, knowledge of signals that regulate ␤-cell function is of immense clinical relevance. Transforming growth factor (TGF)-␤ signaling plays a critical role in pancreatic development although the role of this pathway in the adult pancreas is obscure. Here, we define an important role of the TGF-␤ pathway in regulation of insulin gene transcription and ␤-cell function. We identify insulin as a TGF-␤ target gene and show that the TGF-␤ signaling effector Smad3 occupies the insulin gene promoter and represses insulin gene transcription. In contrast, Smad3 small interfering RNAs relieve insulin transcriptional repression and enhance insulin levels. Transduction of adenoviral Smad3 into primary human and non-human primate islets suppresses insulin content, whereas, dominant-negative Smad3 enhances insulin levels. Consistent with this, Smad3-deficient mice exhibit moderate hyperinsulinemia and mild hypoglycemia. Moreover, Smad3 deficiency results in improved glucose tolerance and enhanced glucose-stimulated insulin secretion in vivo. In ex vivo perifusion assays, Smad3-deficient islets exhibit improved glucosestimulated insulin release. Interestingly, Smad3-deficient islets harbor an activated insulin-receptor signaling pathway and TGF-␤ signaling regulates expression of genes involved in ␤-cell function. Together, these studies emphasize TGF-␤/Smad3 signaling as an important regulator of insulin gene transcription and ␤-cell function and suggest that components of the TGF-␤ signaling pathway may be dysregulated in diabetes.

Insulin secretion by pancreatic ␤-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED 50 values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the ␤-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH.