Inhibition of glutamate dehydrogenase and insulin secretion by KHG26377 does not involve ADP-ribosylation by SIRT4 or deacetylation by SIRT3

We investigated the mechanisms involved in KHG26377 regulation of glutamate dehydrogenase (GDH) activity, focusing on the roles of SIRT4 and SIRT3. Intraperitoneal injection of mice with KHG26377 reduced GDH activity with concomitant repression of glucose-induced insulin secretion. Consistent with their known functions, SIRT4 ribosylated GDH and reduced its activity, and SIRT3 deacetylated GDH, increasing its activity. However, KHG26377 did not affect SIRT4-mediated ADP-ribosylation/ inhibition or SIRT3-mediated deacetylation/activation of GDH. KHG26377 had no effect on SIRT4 protein levels, and did not alter total GDH, acetylated GDH, or SIRT3 protein levels in pancreatic mitochondrial lysates. These results suggest that the mechanism by which KHG26377 inhibits GDH activity and insulin secretion does not involve ADP-ribosylation of GDH by SIRT4 or deacetylation of GDH by SIRT3. [BMB Reports 2012; 45(8): 458-463].     

From pancreatic islets to central nervous system, the importance of glutamate dehydrogenase for the control of energy homeostasis

Glutamate dehydrogenase (GDH) is a mitochondrial enzyme linking the Krebs cycle to the multifunctional amino acid glutamate. Thereby, GDH plays a pivotal role between carbohydrate and protein metabolisms, controlling production and consumption of the messenger molecule glutamate in neuroendocrine cells. GDH activity is under the control of several regulators, conferring to this enzyme energy-sensor property. Indeed, GDH directly depends on the provision of the co-factor NADH/NAD⁺, rendering the enzyme sensitive to the redox status of the cell. Moreover, GDH is allosterically regulated by GTP and ADP. GDH is also regulated by ADP-ribosylation, mediated by a member of the energy-sensor family sirtuins, namely SIRT4. In the brain, GDH ensures the cycling of the neurotransmitter glutamate between neurons and astrocytes. GDH also controls ammonia metabolism and detoxification, mainly in the liver and kidney. In pancreatic β-cells, the importance of GDH as a key enzyme in the regulation of insulin secretion is now well established. Inhibition of GDH activity decreases insulin release, while activating mutations are associated with a hyperinsulinism syndrome. Although GDH enzyme catalyzes the same reaction in every tissue, its function regarding metabolic homeostasis varies greatly according to specific organs. In this review, we will discuss specificities of GDH regulation in neuroendocrine cells, in particular pancreatic islets and central nervous system.     

Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation

Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of l-glutamate to alpha-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutamine-stimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C, suggesting observation in MIN6 cells to be relevant in native beta-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to alpha-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion.     

Effects of a GTP-insensitive mutation of glutamate dehydrogenase on insulin secretion in transgenic mice

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 beta-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 15N flux from [2-15N]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. 15NH4Cl tracing studies showed 15N 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.     

Glutamate dehydrogenase in brain mitochondria: do lipid modifications and transient metabolon formation influence enzyme activity?

Metabolism of glutamate, the primary excitatory neurotransmitter in brain, is complex and of paramount importance to overall brain function. Thus, understanding the regulation of enzymes involved in formation and disposal of glutamate and related metabolites is crucial to understanding glutamate metabolism. Glutamate dehydrogenase (GDH) is a pivotal enzyme that links amino acid metabolism and TCA cycle activity in brain and other tissues. The allosteric regulation of GDH has been extensively studied and characterized. Less is known about the influence of lipid modifications on GDH activity, and the participation of GDH in transient heteroenzyme complexes (metabolons) that can greatly influence metabolism by altering kinetic parameters and lead to channeling of metabolites. This review summarizes evidence for palmitoylation and acylation of GDH, information on protein binding, and information regarding the participation of GDH in transient heteroenzyme complexes. Recent studies suggest that a number of other proteins can bind to GDH altering activity and overall metabolism. It is likely that these modifications and interactions contribute additional levels of regulation of GDH activity and glutamate metabolism.     

Evolution of glutamate dehydrogenase regulation of insulin homeostasis is an example of molecular exaptation

Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the oxidative deamination of glutamate to 2-oxoglutarate. While this enzyme does not exhibit allosteric regulation in plants, bacteria, or fungi, its activity is tightly controlled by a number of compounds in mammals. We have previously shown that this regulation plays an important role in insulin homeostasis in humans and evolved concomitantly with a 48-residue "antenna" structure. As shown here, the antenna and some of the allosteric regulation first appears in the Ciliates. This primitive regulation is mediated by fatty acids and likely reflects the gradual movement of fatty acid oxidation from the peroxisomes to the mitochondria as the Ciliates evolved away from plants, fungi, and other protists. Mutagenesis studies where the antenna is deleted support this contention by demonstrating that the antenna is essential for fatty acid regulation. When the antenna from the Ciliates is spliced onto human GDH, it was found to fully communicate all aspects of mammalian regulation. Therefore, we propose that glutamate dehydrogenase regulation of insulin secretion is a example of exaptation at the molecular level where the antenna and associated fatty acid regulation was created to accommodate the changes in organelle function in the Ciliates and then later used to link amino acid catabolism and/or regulation of intracellular glutamate/glutamine levels in the pancreatic beta cells with insulin homeostasis in mammals.     

Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria

Mitochondrial ADP-ribosylation leads to modification of two proteins of approximately 26 and 53 kDA: The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine-specific ADP-ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [14C]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP-ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD+ onto the acceptor site. ADP- ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP-ribosylation.     

Cytosolic and mitochondrial malic enzyme isoforms differentially control insulin secretion

In islet beta-cells and INS-1 cells both the high activity of malic enzyme and the correlation of insulin secretion rates with pyruvate carboxylase (PC) flux suggest that a pyruvate-malate cycle is functionally relevant to insulin secretion. Expression of the malic enzyme isoforms in INS-1 cells and rat islets was measured, and small interfering RNA was used to selectively reduce isoform mRNA expression in INS-1 cells to evaluate its impact on insulin secretion. The cytosolic NADP(+)-specific isoform (ME1) was the most abundant, with the mitochondrial isoforms NAD(+)-preferred (ME2) expressed at approximately 50%, and the NADP(+)-specific (ME3) at approximately 10% compared with ME1. Selective reduction (89 +/- 2%) of cytosolic ME1 mRNA expression and enzyme activity significantly reduced glucose (15 mM:41 +/- 6%, p < 0.01) and amino acid (4 mM glutamine +/- 10 mM leucine: 39 +/- 6%, p < 0.01)-stimulated insulin secretion. Selective small interfering RNA reduction (51 +/- 6%) of mitochondrial ME2 mRNA expression did not impact glucose-induced insulin secretion, but decreased amino acid-stimulated insulin secretion by 25 +/- 4% (p < 0.01). Modeling of the metabolism of [U-(13)C]glucose by its isotopic distribution in glutamate indicates a second pool of pyruvate distinct from glycolytically derived pyruvate in INS-1 cells. ME1 knockdown decreased flux of both pools of pyruvate through PC. In contrast, ME2 knockdown affected only PC flux of the pyruvate derived from glutamate metabolism. These results suggest a physiological basis for two metabolically and functionally distinct pyruvate cycles. The cycling of pyruvate by ME1 generates cytosolic NADPH, whereas mitochondrial ME2 responds to elevated amino acids and serves to supply sufficient pyruvate for increased Krebs cycle flux when glucose is limiting.     

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2h in medium containing either 1.0mM [(15)NH(4)(+)] or 100μM [(15)N]glutamate. For those exposed to [(15)N]glutamate an additional 100μM was added after 1h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The (15)N incorporation from [(15)NH(4)(+)] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [(15)N]glutamate, the reduced GDH activity led to an increased (15)N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.     

Intertissue Differences for the Role of Glutamate Dehydrogenase in Metabolism

The enzyme glutamate dehydrogenase (GDH) plays an important role in integrating mitochondrial metabolism of amino acids and ammonia. Glutamate may function as a respiratory substrate in the oxidative deamination direction of GDH, which also yields α-ketoglutarate. In the reductive amination direction GDH produces glutamate, which can then be used for other cellular needs such as amino acid synthesis via transamination. The production or removal of ammonia by GDH is also an important consequence of flux through this enzyme. However, the abundance and role of GDH in cellular metabolism varies by tissue. Here we discuss the different roles the house-keeping form of GDH has in major organs of the body and how GDH may be important to regulating aspects of intermediary metabolism. The near-equilibrium poise of GDH in liver and controversy over cofactor specificity and regulation is discussed, as well as, the role of GDH in regulation of renal ammoniagenesis, and the possible importance of GDH activity in the release of nitrogen carriers by the small intestine.     

Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenase in muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabetic state?

Glucose homeostasis is determined by insulin secretion from the ß-cells in pancreatic islets and by glucose uptake in skeletal muscle and other insulin target tissues. While glutamate dehydrogenase (GDH) senses mitochondrial energy supply and regulates insulin secretion, its role in the muscle has not been elucidated. Here we investigated the possible interplay between GDH and the cytosolic energy sensing enzyme 5′-AMP kinase (AMPK), in both isolated islets and myotubes from mice and humans. The green tea polyphenol epigallocatechin-3-gallate (EGCG) was used to inhibit GDH. Insulin secretion was reduced by EGCG upon glucose stimulation and blocked in response to glutamine combined with the allosteric GDH activator BCH (2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid). Insulin secretion was similarly decreased in islets of mice with ß-cell-targeted deletion of GDH (ßGlud1^−/−). EGCG did not further reduce insulin secretion in the mutant islets, validating its specificity. In human islets, EGCG attenuated both basal and nutrient-stimulated insulin secretion. Glutamine/BCH-induced lowering of AMPK phosphorylation did not operate in ßGlud1^−/− islets and was similarly prevented by EGCG in control islets, while high glucose systematically inactivated AMPK. In mouse C2C12 myotubes, like in islets, the inhibition of AMPK following GDH activation with glutamine/BCH was reversed by EGCG. Stimulation of GDH in primary human myotubes caused lowering of insulin-induced 2-deoxy-glucose uptake, partially counteracted by EGCG. Thus, mitochondrial energy provision through anaplerotic input via GDH influences the activity of the cytosolic energy sensor AMPK. EGCG may be useful in obesity by resensitizing insulin-resistant muscle while blunting hypersecretion of insulin in hypermetabolic states.     

¹³C-metabolic enrichment of glutamate in glutamate dehydrogenase mutants of Saccharomyces cerevisiae

Glutamate dehydrogenases (GDH) interconvert α-ketoglutarate and glutamate. In yeast, NADP-dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from α-ketoglutarate, while an NAD-dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in acetate/raffinose (YNAceRaf) to examine the role(s) of these enzymes during aerobic metabolism. In YNAceRaf the doubling time of wild type, gdh2Δ, and gdh3Δ cells was comparable at ～4 h. NADP-dependent GDH activity (Gdh1p+Gdh3p) in wild type, gdh2Δ, and gdh3Δ was decreased ～80% and NAD-dependent activity (Gdh2p) in wild type and gdh3Δ was increased ～20-fold in YNAceRaf as compared to glucose. Cells carrying the gdh1Δ allele did not divide in YNAceRaf, yet both the NADP-dependent (Gdh3p) and NAD-dependent (Gdh2p) GDH activity was ～3-fold higher than in glucose. Metabolism of [1,2-(13)C]-acetate and analysis of carbon NMR spectra were used to examine glutamate metabolism. Incorporation of (13)C into glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at <15% of wild type. Analysis of (13)C-enrichment of glutamate carbons indicates a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wild type. Further, the relative complexity of (13)C-isotopomers at early time points was noticeably greater in gdh3Δ as compared to wild type and gdh2Δ cells. These in vivo data show that Gdh1p is the primary GDH enzyme and Gdh2p and Gdh3p play evident roles during aerobic glutamate metabolism.     

Regulatory effects of 5'-deoxypyridoxal on glutamate dehydrogenase activity and insulin secretion in pancreatic islets

It has been known that glutamate, generated by glutamate dehydrogenase (GDH), acts as an intracellular messenger in insulin exocytosis in pancreatic beta cells. Here we demonstrate the correlation of GDH activity and insulin release in rat pancreatic islets perfused with 5'-deoxypyridoxal. Perfusion of islets with 5'-deoxypyridoxal, an effective inhibitor of GDH, reduced the islet GDH activity at concentration-dependent manner. Treatment of 5'-deoxypyridoxal up to 2 mM did not affect the cell viability. There was reduction in V(max) values on average about 60%, whereas no changes in K(m) values for substrates and coenzymes were observed. The concentration of GDH on the Western blot analysis and the level of GDH mRNA remained unchanged. The concentration of glutamate decreased by 52%, whereas the concentration of 2-oxoglutarate increased up to 2.3-fold in the presence of 5'-deoxypyridoxal. 5'-Deoxypyridoxal had no effects on inhibition by GTP and activation by ADP or L-leucine of islet GDH. In parallel with the inhibition of GDH activity, perfusion of islets with 5'-deoxypyridoxal reduced insulin release up to 2.5-fold. Although precise mechanism for correlation between GDH activity and insulin release remains to be studied further, our results suggest a possibility that the inhibitory effect of 5'-deoxypyridoxal on islet GDH activity may correlate with its effect on insulin release.     

GAD65-mediated glutamate decarboxylation reduces glucose-stimulated insulin secretion in pancreatic beta cells

Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell by generating signals that couple glucose sensing to insulin secretion. We have demonstrated previously that mitochondrially derived glutamate participates directly in the stimulation of insulin exocytosis. The aim of the present study was to impose altered cellular glutamate levels by overexpression of glutamate decarboxylase (GAD) to repress elevation of cytosolic glutamate. INS-1E cells infected with a recombinant adenovirus vector encoding GAD65 showed efficient overexpression of the GAD protein with a parallel increase in enzyme activity. In control cells glutamate levels were slightly increased by 7.5 mm glucose (1.4-fold) compared with the effect at 15 mm (2.3-fold) versus basal 2.5 mm glucose. Upon GAD overexpression, glutamate concentrations were no longer elevated by 15 mm glucose as compared with controls (-40%). Insulin secretion was stimulated in control cells by glucose at 7.5 mm (2.5-fold) and more efficiently at 15 mm (5.2-fold). INS-1E cells overexpressing GAD exhibited impaired insulin secretion on stimulation with 15 mm glucose (-37%). The secretory response to 30 mm KCl, used to raise cytosolic Ca(2+) levels, was unaffected. Similar results were obtained in perifused rat pancreatic islets following adenovirus transduction. This GAD65-mediated glutamate decarboxylation correlating with impaired glucose-induced insulin secretion is compatible with a role for glutamate as a glucose-derived factor participating in insulin exocytosis.     

Reduced expression of SIRT1 is associated with diminished glucose-induced insulin secretion in islets from calorie-restricted rats

Alterations in food intake such as caloric restriction modulate the expression of SIRT1 and SIRT4 proteins that are involved in pancreatic β-cell function. Here, we search for a possible relationship between insulin secretion and the expression of SIRT1, SIRT4, PKC and PKA in islets from adult rats submitted to CR for 21 days. Rats were fed with an isocaloric diet (CTL) or received 60% (CR) of the food ingested by CTL. The dose-response curve of insulin secretion to glucose was shifted to the right in the CR compared with CTL islets (EC₅₀ of 15.1±0.17 and 10.5±0.11 mmol/L glucose). Insulin release by the depolarizing agents arginine and KCl was reduced in CR compared with CTL islets. Total islet insulin content and glucose oxidation were also reduced in CR islets. Leucine-stimulated secretion was similar in both groups, slightly reduced in CR islets stimulated by leucine plus glutamine but higher in CR islets stimulated by ketoisocaproate (KIC). Insulin secretion was also higher in CR islets stimulated by carbachol, compared with CTL islets. No differences in the rise of cytosolic Ca²⁺ concentrations stimulated by either glucose or KCl were observed between groups of islets. Finally, SIRT1, but not SIRT4, protein expression was lower in CR compared with CTL islets, whereas no differences in the expression of PKC and PKA proteins were observed. In conclusion, the lower insulin secretion in islets from CR rats was, at least in part, due to an imbalance between the expression of SIRT1 and SIRT4.     

SIRT1 attenuates high glucose-induced insulin resistance via reducing mitochondrial dysfunction in skeletal muscle cells

Insulin resistance is often characterized as the most critical factor contributing to the development of type 2 diabetes mellitus (T2DM). Sustained high glucose is an important extracellular environment that induces insulin resistance. Acquired insulin resistance is associated with reduced insulin-stimulated mitochondrial activity as a result of increased mitochondrial dysfunction. Silent information regulator 1 (SIRT1) is one member of the SIRT2 (Sir2)-like family of proteins involved in glucose homeostasis and insulin secretion in mammals. Although SIRT1 has a therapeutic effect on metabolic deterioration in insulin resistance, it is still not clear how SIRT1 is involved in the development of insulin resistance. Here, we demonstrate that pcDNA3.1 vector-mediated overexpression of SIRT1 attenuates insulin resistance in the high glucose-induced insulin-resistant skeleton muscle cells. These beneficial effects were associated with ameliorated mitochondrial dysfunction. Further studies have demonstrated that SIRT1 restores mitochondrial complex I activity leading to decreased oxidative stress and mitochondrial dysfunction. Furthermore, SIRT1 significantly elevated the level of another SIRT which is named SIRT3, and SIRT3 siRNA-suppressed SIRT1-induced mitochondria complex activity increments. Taken together, these results showed that SIRT1 improves insulin sensitivity via the amelioration of mitochondrial dysfunction, and this is achieved through the SIRT1–SIRT3–mitochondrial complex I pathway.     

Regulation of human eIF4E by 4E-BP1: binding analysis using surface plasmon resonance

The mitochondria play a pivotal role in regulating glucose-induced insulin secretion in the pancreatic beta cell. We have recently demonstrated that glutamate derived from mitochondria participates directly in the stimulation of insulin exocytosis. In the present study, mitochondria isolated from the beta cell line INS-1E generated glutamate when incubated with the tricarboxylic acid cycle intermediate succinate. The generation of glutamate correlated with stimulated mitochondrial activity monitored as oxygen consumption and was inhibited by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Glutamate is formed by the mitochondrial enzyme glutamate dehydrogenase from alpha-ketoglutarate. Transient overexpression of glutamate dehydrogenase in INS-1E cells resulted in potentiation of glucose-stimulated hormone secretion without affecting basal release. These results further point to glutamate as an intracellular messenger playing a key role in the control of insulin exocytosis.