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.

Short-term fasting, seizure control and brain amino acid metabolism.

The ketogenic diet is an effective treatment for seizures, but the mechanism of action is unknown. It is uncertain whether the anti-epileptic effect presupposes ketosis, or whether the restriction of calories and/or carbohydrate might be sufficient. We found that a relatively brief (24 h) period of low glucose and low calorie intake significantly attenuated the severity of seizures in young Sprague-Dawley rats (50-70 gms) in whom convulsions were induced by administration of pentylenetetrazole (PTZ). The blood glucose concentration was lower in animals that received less dietary glucose, but the brain glucose level did not differ from control blood [3-OH-butyrate] tended to be higher in blood, but not in brain, of animals on a low-glucose intake. The concentration in brain of glutamine increased and that of alanine declined significantly with low-glucose intake. The blood alanine level fell more than that of brain alanine, resulting in a marked increase ( approximately 50%) in the brain:blood ratio for alanine. In contrast, the brain:blood ratio for leucine declined by about 35% in the low-glucose group. When animals received [1-(13)C]glucose, a metabolic precursor of alanine, the appearance of (13)C in alanine and glutamine increased significantly relative to control. The brain:blood ratio for [(13)C]alanine exceeded 1, indicating that the alanine must have been formed in brain and not transported from blood. The elevated brain(alanine):blood(alanine) could mean that a component of the anti-epileptic effect of low carbohydrate intake is release of alanine from brain-to-blood, in the process abetting the disposal of glutamate, excess levels of which in the synaptic cleft would contribute to the development of seizures.

Brain amino acid requirements and toxicity: the example of leucine.

Glutamic acid is an important excitatory neurotransmitter of the brain. Two key goals of brain amino acid handling are to maintain a very low intrasynaptic concentration of glutamic acid and also to provide the system with precursors from which to synthesize glutamate. The intrasynaptic glutamate level must be kept low to maximize the signal-to-noise ratio upon the release of glutamate from nerve terminals and to minimize the risk of excitotoxicity consequent to excessive glutamatergic stimulation of susceptible neurons. The brain must also provide neurons with a constant supply of glutamate, which both neurons and glia robustly oxidize. The branched-chain amino acids (BCAAs), particularly leucine, play an important role in this regard. Leucine enters the brain from the blood more rapidly than any other amino acid. Astrocytes, which are in close approximation to brain capillaries, probably are the initial site of metabolism of leucine. A mitochondrial branched-chain aminotransferase is very active in these cells. Indeed, from 30 to 50% of all alpha-amino groups of brain glutamate and glutamine are derived from leucine alone. Astrocytes release the cognate ketoacid [alpha-ketoisocaproate (KIC)] to neurons, which have a cytosolic branched-chain aminotransferase that reaminates the KIC to leucine, in the process consuming glutamate and providing a mechanism for the "buffering" of glutamate if concentrations become excessive. In maple syrup urine disease, or a congenital deficiency of branched-chain ketoacid dehydrogenase, the brain concentration of KIC and other branched-chain ketoacids can increase 10- to 20-fold. This leads to a depletion of glutamate and a consequent reduction in the concentration of brain glutamine, aspartate, alanine, and other amino acids. The result is a compromise of energy metabolism because of a failure of the malate-aspartate shuttle and a diminished rate of protein synthesis.

Response of brain amino acid metabolism to ketosis.

Our objective was to study brain amino acid metabolism in response to ketosis. The underlying hypothesis is that ketosis is associated with a fundamental change of brain amino acid handling and that this alteration is a factor in the anti-epileptic effect of the ketogenic diet. Specifically, we hypothesize that brain converts ketone bodies to acetyl-CoA and that this results in increased flux through the citrate synthetase reaction. As a result, oxaloacetate is consumed and is less available to the aspartate aminotransferase reaction; therefore, less glutamate is converted to aspartate and relatively more glutamate becomes available to the glutamine synthetase and glutamate decarboxylase reactions. We found in a mouse model of ketosis that the concentration of forebrain aspartate was diminished but the concentration of acetyl-CoA was increased. Studies of the incorporation of 13C into glutamate and glutamine with either [1-(13)C]glucose or [2-(13)C]acetate as precursor showed that ketotic brain metabolized relatively less glucose and relatively more acetate. When the ketotic mice were administered both acetate and a nitrogen donor, such as alanine or leucine, they manifested an increased forebrain concentration of glutamine and GABA. These findings supported the hypothesis that in ketosis there is greater production of acetyl-CoA and a consequent alteration in the equilibrium of the aspartate aminotransferase reaction that results in diminished aspartate production and potentially enhanced synthesis of glutamine and GABA.

Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine.

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol x min(-1) x (mg of protein)(-1). Experiments with an increasing concentration (0- 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol x min(-1) x (mg of protein)(-1) for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol x min(-1) x g(-1)) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway.

Ketogenic diet, brain glutamate metabolism and seizure control.

We do not know the mode of action of the ketogenic diet in controlling epilepsy. One possibility is that the diet alters brain handling of glutamate, the major excitatory neurotransmitter and a probable factor in evoking and perpetuating a convulsion. We have found that brain metabolism of ketone bodies can furnish as much as 30% of glutamate and glutamine carbon. Ketone body metabolism also provides acetyl-CoA to the citrate synthetase reaction, in the process consuming oxaloacetate and thereby diminishing the transamination of glutamate to aspartate, a pathway in which oxaloacetate is a reactant. Relatively more glutamate then is available to the glutamate decarboxylase reaction, which increases brain [GABA]. Ketosis also increases brain [GABA] by increasing brain metabolism of acetate, which glia convert to glutamine. GABA-ergic neurons readily take up the latter amino acid and use it as a precursor to GABA. Ketosis also may be associated with altered amino acid transport at the blood-brain barrier. Specifically, ketosis may favor the release from brain of glutamine, which transporters at the blood-brain barrier exchange for blood leucine. Since brain glutamine is formed in astrocytes from glutamate, the overall effect will be to favor the release of glutamate from the nervous system.

Role of the glutamate dehydrogenase reaction in furnishing aspartate nitrogen for urea synthesis: studies in perfused rat liver with 15N.

The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.

Metabolism of brain amino acids following pentylenetetrazole treatment.

We studied the effects of pentylenetetrazole (PTZ) on brain amino acid metabolism in mice. Administration of this convulsant did not change forebrain concentrations of amino acids, but when treated animals also received an injection of [15N]leucine, which served as a tracer of brain nitrogen metabolism, total (14N+15N) forebrain [leucine] exceeded control and [glutamate] and [aspartate] were less than control, as were forebrain concentrations of [15N]glutamate and [2-15N]glutamine. These data suggest greater uptake of [15N]leucine but diminished transamination of leucine to glutamate in experimental mice. In contrast to the [15N]leucine studies, which were associated with increased brain [leucine], the administration of [15N]alanine did not alter levels of alanine, glutamate or glutamine. However, label appeared in [2-15N]glutamine much more readily with [15N]alanine than with [15N]leucine as precursor and the ratio of enrichment in [2-15N]glutamine/[15N]alanine was much higher than that in [2-15N]glutamine/[15N]leucine, a finding that is compatible with preferential metabolism of alanine in astrocytes, which are the primary site of brain glutamine synthetase. We conclude that PTZ treatment favors the uptake of selected amino acids such as leucine but also diminishes transamination of leucine to yield glutamate via branched-chain amino acid transaminase. PTZ treatment may favor the "reverse" transamination of 2-keto-isocaproate (KIC), the ketoacid of leucine, to form leucine and to consume glutamate. A net result of these processes may be to enable the brain more readily to dispose of the glutamate that is released from neurons during convulsive activity.

Regulation of urea synthesis by agmatine in the perfused liver: studies with 15N.

Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of l-[guanidino-15N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15N-labeled glutamine and unlabeled ammonia or 15NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15N2]agmatine is formed in the liver from perfusate l-[guanidino-15N2]arginine ( approximately 90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15N-labeled NAG and [15N]urea from 15N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15N-labeled NAG and [15N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.