Regulation of glutamate metabolism and insulin secretion by glutamate dehydrogenase in hypoglycemic children^,

Abstract

In addition to its extracellular roles as a neurotransmitter/sensory molecule, glutamate serves important intracellular signaling functions via its metabolism through glutamate dehydrogenase (GDH). GDH is a mitochondrial matrix enzyme that catalyzes the oxidative deamination of glutamate to α-ketoglutarate in a limited number of tissues in humans, including the liver, the kidney, the brain, and the pancreatic islets. GDH activity is subject to complex regulation by negative (GTP, palmitoyl-coenzyme A) and positive (ADP, leucine) allosteric effectors. This complex regulation allows GDH activity to be modulated by changes in energy state and amino acid availability. The importance of GDH regulation has been highlighted by the discovery of a novel hypoglycemic disorder in children, the hyperinsulinism-hyperammonemia syndrome, which is caused by dominantly expressed, activating mutations of the enzyme that impair its inhibition by GTP. Affected children present in infancy with hypoglycemic seizures after brief periods of fasting or the ingestion of a high-protein meal. Patients have characteristic persistent 3- to 5-fold elevations of blood ammonia concentrations but do not display the usual neurologic symptoms of hyperammonemia. The mutant GDH enzyme shows impaired responses to GTP inhibition. Isolated islets from mice that express the mutant GDH in pancreatic β cells show an increased rate of glutaminolysis, increased insulin release in response to glutamine, and increased sensitivity to leucine-stimulated insulin secretion. The novel hyperinsulinism-hyperammonemia syndrome indicates that GDH-catalyzed glutamate metabolism plays important roles in 3 tissues: in β cells, the regulation of amino acid–stimulated insulin secretion; in hepatocytes, the modulation of amino acid catabolism and ammoniagenesis; and in brain neurons, the maintenance of glutamate neurotransmitter concentrations.

INTRODUCTION

This conference, held on the 100th anniversary of the discovery of glutamate as the basis for umami, or the deliciousness taste, focused on the important roles of glutamate as an extracellular sensory and neurotransmitter molecule. Given these important extracellular roles of glutamate, it is perhaps not surprising that there should be regulation of the intracellular metabolism of this key amino acid. Oxidation of glutamate by glutamate dehydrogenase (GDH) not only is highly regulated but also subserves a signaling function with important implications for normal homeostasis and for the pathophysiology of disease in humans. Our concepts of the role of GDH dramatically changed a decade ago with the discovery that a disease of hypoglycemia in infants and young children was associated with activating mutations of GDH (1–3). The following sections highlight some of these new concepts of GDH and its regulatory role, which have evolved from the discovery of this novel disease, and the implications for understanding the role of glutamate and other amino acids in fuel-stimulated insulin secretion by pancreatic β cells.

GDH: THE ENZYME

GDH catalyzes the “reversible” dehydrogenation of glutamate to α-ketoglutarate and ammonia with either NAD⁺ or NADP⁺ as a cofactor (Figure 1). X-ray crystallography has shown that the enzyme exists as a homo-hexamer in both lower phyla and animals and is composed of 2 trimers stacked together, base to base (4). In prokaryotes, such as bacteria, the enzyme is not allosterically regulated and operates in the “reverse” or synthetic direction to generate glutamate from ammonia plus a carbohydrate skeleton (5). Therefore, in these species, through the network of transamination reactions that convert glutamate to other amino acids, GDH serves the anabolic function of synthesizing amino acids from simple precursors for purposes such as protein synthesis.

FIGURE 1.

Glutamate metabolism by glutamate dehydrogenase (GDH). GDH oxidative deamination of glutamate is controlled by multiple allosteric inhibitors, particularly GTP, and by activators, particularly ADP and leucine. These allow control of glutamate oxidation by the intracellular energy potential and by the supply of amino acids (through leucine) and, possibly, also fatty acids. α-KG, α-ketoglutarate.

During evolution, this synthetic role of GDH has been reversed to support the catabolic function of energy production from amino acid oxidation. This switch occurs in eukaryotic species that depend on exogenous sources of amino acids for protein synthesis. To support this change in function, the enzyme acquired a corresponding complex array of structural changes to accommodate a wide array of both positive and negative allosteric signals that modulate responses to changes in the environment (5). Each monomer of GDH contains a catalytic cleft for binding glutamate and the NAD(P)⁺ cofactor, plus a long antenna-like projection that extends from the back side of the catalytic cleft hinge region and intertwines with the antennas of 2 adjacent subunits (6). The antenna provides a means for intercommunication between subunits that allows the cooperative regulation of catalytic activity of each trimer, with presumably further communication across the binding surface between the bases of the 2 trimers. The antenna loop first appears on GDH in ciliates and becomes longer in higher species of animals, including rodents, cows, and humans. Presumably, the function of this special loop of amino acids is to mediate allosteric control of GDH because the GDH reaction is one of the few paths for uncoupling amino nitrogen from amino acids. In the absence of allosteric control, unregulated GDH enzyme activity in the oxidative direction would deplete supplies of glutamate and, via transamination, all the other amino acids that are needed for protein synthesis and growth. In ciliates and lower animals, the antenna is present, and enzymatic activity is responsive to inhibition by fatty acids but does not respond to nucleotides. In higher animals, GDH acquires responsiveness to the cellular energy potential, as mediated through GTP and ADP, and also to leucine, which serves as a marker of amino acid abundance (5).

The 3 key allosteric regulators of mammalian GDH (Figure 1) are GTP, a very potent inhibitor of enzyme activity; ADP, a less potent activator of activity; and leucine, a lower potency activator. In this way, the flux of glutamate into the tricarboxylic acid cycle for energy generation is modulated by the mitochondrial energy potential (the ratio of GTP to ADP) and also by the supply of amino acids. When energy potential is high, amino acid oxidation is not required, and GDH is shut down. When energy potential is low, GDH is activated to sustain energy generation through the oxidation of amino acids. After a protein meal, increases in the concentrations of leucine, an abundant essential amino acid in dietary proteins, can activate GDH to dispose of surplus amino acids not needed for protein synthesis. Other potentially important regulators of GDH activity include long-chain fatty acyl-coenzyme A, which are relatively potent inhibitors, and SIRT4, one of the class of sirtuins that mediates the longevity effects of caloric restriction in a variety of subcellular compartments (7). In addition, as we have recently shown, the common polyphenol in green tea, epigallocatechin-3-gallate, is a potent allosteric inhibitor of GDH (8).

The GDH reaction is well positioned to control the interface between amino acid pools and carbohydrate metabolism in the tricarboxylic acid cycle. Intracellular glutamate concentrations are very high (5–10 mmol/L) compared with extracellular concentrations (<30–50 μmol/L). Although amino acids can be converted to glutamate via transamination reactions, glutamine serves as the major precursor for glutamate via the phosphate-dependent glutaminase, which is also regulated by the energy potential. Thus, flux into glutamate comes from glutamine, especially after a meal when intestinal production of glutamine from ammonia generated by intestinal flora is high. Note that oxidation of glutamate is suppressed when glucose metabolism is increased because of the elevated phosphate potential. Recent work suggests that inhibitory control of GDH is primarily provided by GTP generated in the tricarboxylic acid cycle by the GTP isoform of succinyl–coenzyme A synthetase (9).

ACTIVATING MUTATIONS OF GDH: THE HYPERINSULINISM-HYPERAMMONEMIA SYNDROME

This genetic disorder was first described in 1998 and has subsequently been recognized as one of the more common forms of congenital hyperinsulinism (2, 3). A typical pedigree of 3 generations with the disorder, indicating the dominant pattern of expression, is shown in Figure 2. The proband presented with hypoglycemic symptoms late in the first year of life. Her affected father had had a history of seizures since early childhood that were treated with antiepileptic drugs; his hypoglycemia escaped recognition until his daughter was diagnosed. The affected grandfather never had recognized symptoms of hypoglycemia, although close questioning revealed episodes consistent with hypoglycemic spells since childhood. The 3 affected persons all had persistent 3- to 5-fold elevations of plasma ammonia but did not show the usual symptoms of hyperammonemia, such as lethargy or coma. The patient and her father achieved good control of hypoglycemia on treatment with diazoxide, a thiazide-related drug that suppresses insulin release from pancreatic β cells by activating the plasma membrane ATP-dependent potassium channel (see Figure 3). More than 100 other patients with this hyperinsulinism-hyperammonemia (HI-HA) syndrome have been identified; ≈80% of the cases are familial, but 20% have de novo mutations (2). It has been reported that HI-HA patients have a propensity for an unusual seizure disorder, termed generalized epilepsy, which suggests that the expression of the abnormal GDH in the brain has specific consequences that are not yet defined (10).

FIGURE 2.

Pedigree with hyperinsulinism-hyperammonemia syndrome. Note the elevations of plasma ammonia in the 3 affected persons. Mutation analysis revealed heterozygosity for an Arg269Cys missense mutation in all 3 affected persons. Gel heteroduplex analysis of genomic DNA–polymerase chain reaction amplicons shows that mutation carriers are heterozygous for a mutation in exon 7. Circles, females; boxes, males; solid symbols, affected individuals.

FIGURE 3.

Mechanism of hyperinsulinism and hyperammonemia in hyperinsulinism-hyperammonemia syndrome. In the β cell, oxidation of fuels, such as glucose, increases the ATP:ADP ratio to inhibit the ATP-dependent potassium channel, which triggers the influx of calcium to release insulin from stored granules. Amino acids feed into this triggering pathway via glutamate dehydrogenase (GDH) oxidation of glutamate under the control of GTP and ADP, as well as leucine. In the liver, ammonia is generated from glutamate via GDH; glutamate also generates N-acetylglutamate to regulate ammonia detoxification into urea. GK, glucokinase; SUR, sulfonylurea receptor; K_ATP channel, ATP-dependent potassium channel; Kir, potassium pore; CPS, carbamyl-phosphate synthetase.

The mechanisms of excessive insulin secretion and of elevated concentrations of plasma ammonia in HI-HA are diagrammed in Figure 3. In the pancreatic β cell, metabolic fuels, such as glucose, stimulate insulin secretion by increasing the ATP:ADP ratio, which leads to closure of a plasma membrane ATP-dependent potassium channel, membrane depolarization, opening of a voltage-gated calcium channel, and an increase in cytosolic calcium, which leads to release of insulin from stored granules. Amino acids stimulate insulin secretion via this pathway by increasing oxidation of glutamate through GDH because leucine, an abundant essential amino acid, is an allosteric activator of GDH enzyme activity. Normally, GDH activity is suppressed in the basal state by the potent inhibitory effects of GTP and ATP. But activating mutations, which impair the inhibitory effects of GTP and ATP, cause a constitutive oversecretion of insulin and a heightened sensitivity to leucine-stimulated insulin release. In the liver (Figure 3), activation of GDH leads to hyperammonemia in 2 ways: first, by directly increasing ammonia production from oxidation of glutamate; and second, by lowering the set point for ureagenesis because the reduction of hepatocyte concentrations of glutamate decrease production of N-acetylglutamate, a required allosteric activator of the rate-controlling step in ureagenesis, carbamyl-phosphate synthetase.

GDH expression is restricted to the pancreatic islets, the liver, the kidney, and the brain. There have not been obvious consequences related to abnormal GDH activity in the kidneys of patients with HI-HA. However, there may be adverse effects of the activation of GDH in the brains of these patients because they appear to have a greater risk for developmental delay and seizures than persons with other forms of hyperinsulinemic hypoglycemia. In addition, they have an unusual propensity for developing absence seizures (generalized epilepsy), which is not a feature of other forms of hyperinsulinism (11).

Clinically, the major manifestations of GDH-activating mutations are fasting hypoglycemia and hypoglycemia induced by a high-protein meal (12). The development of hypoglycemia after an oral protein load is often much more rapid and dramatic than that after fasting. Evidence of GDH activation can also be shown by the acute insulin response to stimulation by an intravenous bolus infusion of leucine (13).

GDH MUTATIONS IN HI-HA SYNDROME

A total of 14 amino acid residues affected by GDH-activating mutations have been identified in patients with the HI-HA syndrome. Some occur in the GTP allosteric binding site and interfere directly with effector binding. Others occur along the limbs of the antenna loop to interfere with the process of GTP inhibition. The effects of these mutations on GDH sensitivity to GTP has been studied in both the isolated lymphocytes from patients and the expressed enzyme. For example, in the H454Y mutant GDH patient cells, the ED₅₀ (equivalent dose producing 50% inhibition) for GTP inhibition is increased 6-fold (from 40 to 250 nmol/L); in the pure mutant enzyme, the ED₅₀ is increased 1000-fold (1).

REGULATION OF INSULIN SECRETION BY WILD-TYPE AND MUTANT GDH

To determine the effects of the HI-HA GDH mutations on insulin regulation, we generated a transgenic mouse line expressing the H454Y GDH mutation in pancreatic islets under the control of the rat insulin promoter (14). Mice from this line have hypoglycemia in vivo, which is similar to that of affected humans. The GDH activation produces dramatically increased responses either to a complete amino acid mixture or to glutamine alone. The activating mutation also increases the sensitivity to leucine-stimulated insulin secretion, which is similar to what is observed in patients.

To investigate the effects of the HI-HA–activating mutations on flux through GDH, we carried out tracer studies in isolated islets from the H454Y transgenic mouse by using 2-N¹⁵-glutamine to measure flux through GDH (14). The transgenic islets showed increased flux in the basal state, which was increased further by the addition of leucine. Flux through GDH was suppressed in these transgenic islets by the addition of glucose to increase production of the allosteric inhibitors, GTP and ATP. A similar suppressive effect of glucose on the insulin response to leucine also occurs in patients with HI-HA (13). These observations support the concept that GDH operates exclusively in the oxidative deamination direction in vivo and is highly responsive to the energy state of the cell. As a clinical correlate, the data suggest that, in addition to treatment with diazoxide to control hyperinsulinism, HI-HA patients may benefit from carbohydrate loading to reduce their hypersensitivity to protein-induced hypoglycemia.

POSSIBLE ROLE OF GDH IN AMPLIFYING GLUCOSE-STIMULATED INSULIN SECRETION

In studies of another form of congenital hyperinsulinism due to recessive mutations of the plasma membrane ATP-dependent potassium channel, we found that patients with this disease share with HI-HA patients a marked sensitivity to protein-induced hypoglycemia, but are not responsive to leucine stimulation (15). To understand this discrepancy, we examined responses to amino acids of isolated islets from mice with a knockout of the SUR1 subunit of the KATP channel (16). These islets showed marked stimulation of insulin release by a physiologic mixture of amino acids, but no response to leucine. The effect of amino acids could be fully reproduced by glutamine alone, and this effect could not be suppressed by inhibition of glutaminase. In addition, glutamine was found to potentiate the insulin response to glucose in wild-type islets. We interpret these observations to mean that glutamine may serve as a specific amplifier of insulin secretion downstream of the elevation in cytosolic calcium. As shown in Figure 4, the rise in high-energy phosphate potential during glucose-stimulated insulin secretion not only shuts down GDH oxidation of glutamate, but also activates the production of glutamine via the ATP-dependent glutamine synthetase. We speculate that this elevation of glutamine acts at some distal site to amplify the release of insulin when cytosolic calcium concentrations are high.

FIGURE 4.

Role of glutamine in amplifying glucose-stimulated insulin secretion. High glucose raises the β cell ATP:ADP ratio, which not only triggers membrane depolarization and elevation of cytosolic calcium but also inhibits glutamate oxidation and redirects glutamate toward synthesis of glutamine. Glutamine then amplifies insulin release at some as-yet-unknown site. GLUT2, glucose transporter 2; G6P, glucose-6-phosphate; GS, glutamine synthetase; GDH, glutamate dehydrogenase; K_ATP channel, ATP-dependent potassium channel; Ψ, plasma membrane depolarization.

CONCLUSIONS

Intracellular glutamate plays a central role in processes regulating the switches in the choice of metabolic fuels in individual cells and control of whole-body nitrogen balance. Glutamate dehydrogenase and its multiple stimulatory and inhibitory allosteric regulators provide the mechanism for this finely tuned control of glutamate oxidation. In contrast to previous suggestions that the GDH reaction could operate in the reductive amination direction, the findings in patients with the HI-HA syndrome indicate that the reaction operates essentially exclusively in the direction of glutamate oxidation. The effects of GDH activation mutations have been studied primarily in pancreatic β cells, but it is likely that these mutations have consequences in the brain, which remain to be studied. It is intriguing to speculate that the intramitochondrial glutamate regulatory system through GDH and other enzymes preceded the evolution of plasma membrane systems for responding to extracellular glutamate, which eventually allowed the highly refined umami taste systems. (Other articles in this supplement to the Journal include references 17–45.)