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...

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.

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Leucine or the nonmetabolized leucine analog +/- 2-amino-2-norbornane-carboxylic acid (BCH) (both at 10 mmol/l) induced biphasic insulin secretion in the presence of 2 mmol/l glutamine (Q2) in cultured mouse islets pretreated for 40 min without glucose but with Q2 present. The beta-cell response consisted of an initial peak of 20- to 25-fold above basal and a less marked secondary phase. However, BCH produced only a delayed response, while leucine was totally ineffective when islets were pretreated with 25 mmol/l glucose plus Q2. With Q2, 10 mmol/l BCH or leucine caused a nearly threefold increase, a twofold increase, or had no effect on cytosolic Ca2+ levels in islets pretreated for 40 min with 0, 5, or 15 mmol/l glucose, respectively. Thus, pretreatment of islets with high glucose inhibited BCH- and leucine-induced cytosolic Ca2+ changes and insulin release. Glucose decreased glutamine oxidation in cultured rat islets when BCH was present at 10 mmol/l, but not in its absence, with a lowest effective level of approximately 0.1 mmol/l, a maximum of 18-30 mmol/l, and an inhibitory concentration, 50%, of approximately 3 mmol/l. The data are consistent with the hypothesis that glucose inhibits glutaminolysis in pancreatic beta-cells in a concentration-dependent manner and hence blocks leucine-stimulated insulin secretion. We postulate that in the basal interprandial state, glutaminolysis of beta-cells is partly turned on because glutamate dehydrogenase (GDH) is activated by a decreased P-potential due to partial fuel depletion and sensitization to endogenous activators such as leucine. Additionally, it may contribute significantly to basal insulin release, which is known to be responsible for about half of the insulin released daily. The data explain "leucine-hypersensitivity" of beta-cells during hypoglycemia and contribute to the elucidation of the GDH-linked syndrome of hyperinsulinism associated with elevated serum ammonia levels. Thus, understanding the precise regulation and role of beta-cell glutaminolysis is probably central to our concept of normal blood glucose control.

It was reported previously that isolated human islets from individuals with type 2 diabetes mellitus (T2DM) show reduced glucose-stimulated insulin release. To assess the possibility that impaired bioenergetics may contribute to this defect, glucose-stimulated respiration (Vo(2)), glucose usage and oxidation, intracellular Ca(2+), and insulin secretion (IS) were measured in pancreatic islets isolated from three healthy and three type 2 diabetic organ donors. Isolated mouse and rat islets were studied for comparison. Islets were exposed to a "staircase" glucose stimulus, whereas IR and Vo(2) were measured. Vo(2) of human islets from normals and diabetics increased sigmoidally from equal baselines of 0.25 nmol/100 islets/min as a function of glucose concentration. Maximal Vo(2) of normal islets at 24 mM glucose was 0.40 ± 0.02 nmol·min(-1)·100 islets(-1), and the glucose S(0.5) was 4.39 ± 0.10 mM. The glucose stimulation of respiration of islets from diabetics was lower, V(max) of 0.32 ± 0.01 nmol·min(-1)·100 islets(-1), and the S(0.5) shifted to 5.43 ± 0.13 mM. Glucose-stimulated IS and the rise of intracellular Ca(2+) were also reduced in diabetic islets. A clinically effective glucokinase activator normalized the defective Vo(2), IR, and free calcium responses during glucose stimulation in islets from type 2 diabetics. The body of data shows that there is a clear relationship between the pancreatic islet energy (ATP) production rate and IS. This relationship was similar for normal human, mouse, and rat islets and the data for all species fitted a single sigmoidal curve. The shared threshold rate for IS was ∼13 pmol·min(-1)·islet(-1). Exendin-4, a GLP-1 analog, shifted the ATP production-IS curve to the left and greatly potentiated IS with an ATP production rate threshold of ∼10 pmol·min(-1)·islet(-1). Our data suggest that impaired β-cell bioenergetics resulting in greatly reduced ATP production is critical in the molecular pathogenesis of type 2 diabetes mellitus.

Glutamate dehydrogenase (GDH) plays an important role in insulin secretion as evidenced in children by gain of function mutations of this enzyme that cause a hyperinsulinism-hyperammonemia syndrome (GDH-HI) and sensitize ␤-cells to leucine stimulation. GDH transgenic mice were generated to express the human GDH-HI H454Y mutation and human wild-type GDH in islets driven by the rat insulin promoter. H454Y transgene expression was confirmed by increased GDH enzyme activity in islets and decreased sensitivity to GTP inhibition. The H454Y GDH transgenic mice had hypoglycemia with normal growth rates. H454Y GDH transgenic islets were more sensitive to leucine-and glutamine-stimulated insulin secretion but had decreased response to glucose stimulation. The fluxes via GDH and glutaminase were measured by tracing 15 N flux from [2-15 N]glutamine. The H454Y transgene in islets had higher insulin secretion in response to glutamine alone and had 2-fold greater GDH flux. High glucose inhibited both glutaminase and GDH flux, and leucine could not override this inhibition.15 NH 4 Cl tracing studies showed 15 N was not incorporated into glutamate in either H454Y transgenic or normal islets. In conclusion, we generated a GDH-HI disease mouse model that has a hypoglycemia phenotype and confirmed that the mutation of H454Y is disease causing. Stimulation of insulin release by the H454Y GDH mutation or by leucine activation is associated with increased oxidative deamination of glutamate via GDH. This study suggests that GDH functions predominantly in the direction of glutamate oxidation rather than glutamate synthesis in mouse islets and that this flux is tightly controlled by glucose.Glucose, fatty acids, and amino acids are fuels that stimulate pancreatic ␤-cell insulin secretion. Congenital hyperinsulinism (HI), 2 a group of disorders arising from mutations of genes encoding ␤-cell function, illustrates this basic phenomenon. For instance, gain of function mutations of glucokinase cause HI by lowering the threshold for glucosestimulated insulin secretion (GSIS) and highlight the role of glucokinase as the glucosensor of the ␤-cell (1, 2). Recently a form of HI due to loss of function mutations in the enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase has been identified (3-6). Although the biochemical mechanisms of short-chain 3-hydroxyacyl-CoA dehydrogenase-HI are unknown, this disorder provides evidence of a role for fatty acid metabolism in insulin secretion. The ATP-dependent potassium channel (K ATP ), encoded by the sulfonylurea receptor 1 (SUR1) and Kir 6.2, transduces the energy state of the ␤-cell. Loss of function mutations in the ATP-dependent potassium channel cause the most common form of HI (K ATP -HI) and confirm that the channel plays a key role in triggering insulin release (7-9). In 1998, we identified mutations of glutamate dehydrogenase (GDH) in children with a dominant form of hyperinsulinism (GDH-HI) and implicated this enzyme as a mediator of leucine-stimulated insulin secretion (LSIS) (10 -12). GDH-HI mut...

Culturing rat islets in high glucose (HG) increased 1-(14)C-alpha-ketoisocaproate (KIC) oxidation compared with culturing them in low glucose. Leucine caused insulin secretion (IS) in low glucose but not in HG rat islets, whereas KIC did so in both. Pretreatment with HG for 40 min abolished leucine stimulation of IS by mouse islets and prevented the cytosolic Ca(2+) rise without inhibiting IS and Ca(2+) increments caused by KIC. When islets were pretreated without glucose and glutamine, aminooxyacetic acid (AOA) markedly decreased KIC effects. When islets were pretreated without glucose and with glutamine, AOA potentiated leucine effects but attenuated KIC effects. AOA stimulated glutamine oxidation in the presence but not the absence of +/-2-amino-2-norbornane-carboxylic acid, a nonmetabolized leucine analog. Pretreatment with HG and glutamine partially reversed AOA inhibition of KIC effects. Glucose increased intracellular ATP and GTP, whereas it decreased ADP and GDP in beta HC9 cells. Glutamate dehydrogenase activity of beta HC9 cell extracts was increased by leucine and attenuated by GTP, but it was potentiated by ADP. In conclusion, leucine and KIC stimulated beta-cells via distinct mechanisms. Glutamate dehydrogenase is the sensor of leucine, whereas transamination plays an important role in KIC stimulation of pancreatic beta-cells.

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1 ؊/؊ ) and control mouse islets with D-[U-13 C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and ␥-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1 ؊/؊ islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue D,L-␤-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1 ؊/؊ and glyburide-treated SUR1 ؉/؉ islets. Glucose oxidation and pyruvate cycling was increased in SUR1 ؊/؊ islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1 ؊/؊ islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1 ؊/؊ islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.The pancreatic beta cells function as the predominant sensors and regulators of glucose, amino acid, and fatty acid levels of the mammalian organism, including man, by adjusting the minute to minute rate of insulin secretion such that these fuels are maintained at physiologically optimal blood concentrations under all nutritional conditions including feeding and fasting. This process of fuel-sensing and stimulation of insulin secretion requires that the various stimuli are transported into the beta cells and are metabolized to generate coupling factors that trigger and sustain the secretion of the hormone from large stores of insulin granules (1-4). The diverse specific pathways that allow access to metabolism for glucose, amino acids, and fatty acids converge to a complex network of intermediary metabolism represented by the citric acid cycle, a considerable variety of metabolite and cofactor shuttles including the GABA 2 shunt, and the processes of electron transport and oxidative phosphorylation, to mention just a few outstanding features of the biochemical maze of the beta cell.In the present study we have used uniformly labeled D-[13 C]glucose and GC-MS methods to explore the role of intermediary metabolism of pancreatic islets isolated from normal and sulfonylurea receptor 1 (SUR1) knock-out...