beta-2-Aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. A new activator of glutaminase in intact rat liver mitochondria

beta-(+/-)-2-Aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (BCH) stimulated, in a concentration-dependent manner, the formation of glutamate by mitochondria isolated from rat liver and incubated with 20 mM glutamine. Maximum enhancement was seen with 10 mM BCH while 5 mM leucine was without effect. The initial lag in the rate of glutamate formation was not eliminated by BCH. Preincubation of the mitochondria without glutamine also did not abolish the lag period; to the contrary, it resulted in a progressive deactivation of the glutaminase. The decrease in enzyme activity during the preincubation without glutamine was partially reversed by the addition of either 10 mM BCH or 1.4 mM NH4Cl and was essentially abolished by their combined action. The apparently sigmoid rise in the activity of glutaminase with increasing concentration of glutamine became hyperbolic in the presence of 1.4 mM NH4Cl. BCH stimulated the NH4Cl-activated glutaminase in the entire range of glutamine concentrations studied (2-40 mM) without changing the S50 value. In mitochondria disrupted by repeated cycles of freezing and thawing, the enzymatic activity was maximal even in the absence of BCH. It is postulated that BCH is a potent activator of mitochondrial glutaminase and that manifestation of its action requires intact organelle structure. In addition, it is concluded that BCH-induced stimulation of glutamine catabolism in isolated hepatocytes (Zaleski, J., Wilson, D. F., and Erecinska, M. (1986) J. Biol. Chem. 261, 14082-14090) is the consequence of activation of the mitochondrial glutaminase.     

Effect of alloxan-diabetes on gluconeogenesis and ureogenesis in isolated rabbit liver cells.

1. Neither alloxan-diabetes nor starvation affected the rate of glucose production in hepatocytes incubated with lactate, pyruvate, propionate or fructose as substrates. In contrast, glucose synthesis with either alanine or glutamine was increased nearly 3- and 12-fold respectively, in comparison with that in fed rabbits. 2. The addition of amino-oxyacetate resulted in about a 50% decrease in glucose formation from lactate in hepatocytes isolated from fed, alloxan-diabetic and starved rats, suggesting that both mitochondrial and cytosolic forms of rabbit phosphoenolpyruvate carboxykinase function actively during gluconeogenesis. 3. Alloxan-diabetes resulted in about 2-3-fold stimulation of urea production from either amino acid studied or NH4Cl as NH3 donor, whereas starvation caused a significant increase in the rate of ureogenesis only in the presence of alanine as the source of NH3. 4. As concluded from changes in the [3-hydroxybutyrate]/[acetoacetate] ratio, in hepatocytes from diabetic animals the mitochondrial redox state was shifted toward oxidation in comparison with that observed in liver cells isolated from fed rabbits.     

Stimulation by glucagon of the incorporation of U-14C-labeled substrates into glucose by isolated hepatocytes from fed rats.

The effect of glucagon on the incorporation of U-14C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2--4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH4+ stimulated the incorporation of 20 mM [U-14C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporation into glucose from 20 mM [U-14C] lactate by NH4+ plus glucagon was synergistic. This suggested that glucagon also stimulated the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-14C] alanine into glucose by beta-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.     

Stimulation by glucagon of the incorporation of U-14 C-labeled substrates into glucose by isolated hepatocytes from fed rats

The effect of glucagon on the incorporation of U-¹⁴ C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2–4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH₄⁺ stimulated the incorporation of 20 mM [U-¹⁴ C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporatio into glucose from 20 mM [U-¹⁴ C] lactate by NH₄⁺ plus glucagon was synergistic. This suggested that glucagon also stimulates the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-¹⁴ C] alanine into glucose by β-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.     

Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids

Katz et al. [Katz, J., Golden, S. & Wals, P.A. (1976) Proc. Natl Acad. Sci. USA 73, 3433-3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made. 1. Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase. 2. Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents. 3. 3-Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate. 4. The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen. K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683-14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase. It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959].     

Glutamine metabolism of isolated rat hepatocytes. Evidence for catecholamine activation of alpha-ketoglutarate dehydrogenase

Effects of norepinephrine on gluconeogenesis and ureogenesis from glutamine by hepatocytes from fasted rats were assessed. Comparisons were made to asparagine metabolism and to the effects of NH4Cl and dibutyryl cyclic AMP. With asparagine as substrate, aspartate content was very high but norepinephrine, dibutyryl cyclic AMP, or NH4Cl had little effect on gluconeogenesis or ureogenesis. Metabolism of asparagine could be greatly enhanced by the combination of oleate, ornithine, and NH4Cl. However, even under these conditions, asparatate content remained high, and norepinephrine and dibutyryl cyclic AMP had little influence on glucose or urea synthesis. With glutamine as substrate, aspartate content was much lower, but was greatly elevated by norepinephrine, dibutyryl cyclic AMP, or NH4Cl. Each of these effectors strongly stimulated glucose and urea formation from glutamine. NH4Cl stimulation was accompanied by an increased glutamate and decreased alpha-ketoglutarate content. This suggests the mechanism for NH4Cl stimulation is a near-equilibrium adjustment to ammonia by glutamate dehydrogenase and aspartate aminotransferase rather than a principal involvement of glutaminase. Although both norepinephrine and dibutyryl cyclic AMP lowered alpha-ketoglutarate to the same extent, norepinephrine more rapidly increased aspartate content and led to a smaller accumulation of glutamate than did dibutyryl cyclic AMP. Moreover, only norepinephrine led to a rapid increase in succinyl-CoA concentration. The catecholamine effect could not be explained by specific changes in cytosolic or mitochondrial redox states. The results suggest that alpha-ketoglutarate dehydrogenase is a site of catecholamine action in rat liver. Since purified alpha-ketoglutarate dehydrogenase is known to be Ca2+ stimulated and Ca2+ flux is involved in catecholamine action, these findings also suggest that mitochondrial Ca2+ is elevated by catecholamines.     

Arginine Catabolism by Leishmania donovani Promastigotes

ABSTRACT Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of α -aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than α -aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine. It is suggested that the effects of glucose and 2-deoxyglucose on arginine catabolism depend largely upon the nature of their metabolites, whereas the effects of the various amino acids examined depend largely on the extent to which they interfere with or enhance arginine transport into the cells.     

Fetal hepatic and umbilical uptakes of glucogenic substrates during a glucagon-somatostatin infusion

To test the hypothesis that fetal hepatic glutamate output diverts the products of hepatic amino acid metabolism from hepatic gluconeogenesis, ovine fetal hepatic and umbilical uptakes of glucose and glucogenic substrates were measured before and during fetal glucagon-somatostatin (GS) infusion and during the combined infusion of GS, alanine, glutamine, and arginine. Before the infusions, hepatic uptake of lactate, alanine, glutamine, arginine, and other substrates was accompanied by hepatic output of pyruvate, aspartate, serine, glutamate, and ornithine. The GS infusion induced hepatic output of 1.00 +/- 0.07 mol glucose carbon/mol O(2) uptake, an equivalent reduction in hepatic output of pyruvate and glutamate carbon, a decrease in umbilical glucose uptake and placental uptake of fetal glutamate, an increase in hepatic alanine and arginine clearances, and a decrease in umbilical alanine, glutamine, and arginine uptakes. The latter result suggests that glucagon inhibits umbilical amino acid uptake. We conclude that fetal hepatic pyruvate and glutamate output is part of an adaptation to placental function that requires the fetal liver to maintain both a high rate of catabolism of glucogenic substrates and a low rate of gluconeogenesis.     

A quantitative analysis of the metabolic pathways of hepatic glucose synthesis in vivo with 13C-labeled substrates

A quantitative analysis of the major metabolic pathways of hepatic glucose synthesis in fasted rats was conducted. [2-13C]Acetate was administered intraintestinally into awake fasted rats. 13C NMR and GC-MS analysis were used to quantitate the isotopic enrichments of glutamate, glutamine, lactate, alanine and the newly synthesized liver glucose. By measuring the ratio of carbon atoms in glutamate molecules derived from acetyl-CoA to carbon atoms in the glucose molecule derived from oxaloacetate and gluconeogenic substrates, such as lactate and alanine, the relative activities of the Krebs cycle and gluconeogenesis were quantified. Our results indicate that the percentage of glucose carbons originating by 'metabolic exchange' with the oxaloacetate pool, via the Krebs cycle, is less than 7%.     

Arginine catabolism by Leishmania donovani promastigotes.

Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of alpha-aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than alpha-aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Ammonia overloading in hepatocytes isolated from liver of fetal and adult rats

Ammonia overloading was investigated during glucose and fructose metabolism in isolated hepatocytes under a variety of metabolic conditions. In all assay conditions, the glycolytic flux and oxygen uptake was not modified by 10 mM ammonia. In hepatocytes isolated from rats fed as libitum, the presence of ammonia caused a decrease in the production of lactate (pyruvate); this effect was not observed in anaerobic incubations, in hepatocytes isolated from starved animals, or in fetal hepatocytes. In spite of an overproduction of urea, ammonia detoxification also takes place by the synthesis of alanine, glutamate and aspartate. Addition of 1 mM aminooxyacetate, an inhibitor of aminotransferases, to the incubation medium prevents the formation of these amino acids, and also prevents the decrease of lactate in hepatocytes isolated from fed animals.     

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.     

Gluconeogenesis in rabbit liver. III. The influences of glucagon, epinephrine, alpha- and beta-adrenergic agents on gluconeogenesis in isolated hepatocytes

1. Gluconeogenesis from various substrates has been demonstrated in hepatocytes from 48 h fasted rabbits. Maximum rates of gluconeogenesis (expressed as mumol glucose formed/30 min per 10(8) cells) are: D-fructose, 9.86; dihydroxyacetone, 5.28; L-lactate, 5.26; L-lactate/pyruvate, 3.83; pyruvate, 3.32; glycerol, 2.92; L-alanine, 2.24. 2. Gluconeogenesis from L-lactate is enhanced 1.3--1.5-fold over control values by glucagon, L-epinephrine, L-norepinephrine, dibutyryl cyclic AMP, L-phenylephrine and L-isoproterenol. Glucogenesis from both dihydroxyacetone and D-fructose is stimulated 1.7--2.0-fold of control values by glucagon, epinephrine and dibutyryl cyclic AMP. 3. Gluconeogenesis from lactate is enhanced by both alpha- and beta-adrenergic stimulations based on findings with alpha- and beta-agonists and antagonists. 4. Enhancement of gluconeogenesis by epinephrine and norepinephrine is apparently due to both alpha- and beta-adrenergic effects, as either propranolol or phentolamine partially inhibits such enhancement. The consistently more pronounced inhibition produced by propranolol implies that stimulation of glucose formation by catecholamines is more strongly beta-adrenergic related. Epinephrine-induced glycogenolysis in rabbit hepatocytes is severely inhibited by propranolol but insensitive to phentolamine, suggesting that glycogen breakdown is solely beta-adrenergic related. These observations contrast with those of others that stimulation of both gluconeogenesis and glycogenolysis by catecholamines while sensitive to both alpha- and beta-adrenergic stimulation in rats, at least young rats, is primarily alpha-adrenergic mediated, especially in adult rats.     

Effect of the antiepileptic drug sodium valproate on glutamine and glutamate metabolism in isolated human kidney tubules

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.     

Alpha-adrenergic stimulation of glutamine metabolism in isolated rat hepatocytes.

The mechanisms by means of which phenylephrine stimulates glutamine metabolism were studied in isolated rat hepatocytes. In the first 2 min after phenylephrine addition there was a rapid fall in the concentrations of intracellular 2-oxoglutarate and glutamate, presumably owing to activation of 2-oxoglutarate dehydrogenase. This was followed 2-3 min later by activation of glutaminase and by increases in glutamate and 2-oxoglutarate. Activation of glutaminase by phenylephrine was due to direct stimulation of the enzyme rather than to reversal of inhibition by the decrease in 2-oxoglutarate and glutamate. The stimulation of glutaminase by phenylephrine is partly due to an increase in the affinity of the enzyme for ammonia, its essential activator. It is concluded that stimulation of steady-state flux through the pathway from glutamine to glucose and urea can only be achieved by stimulation of glutaminase, the first enzyme in the pathway.     

3-Aminopicolinate inhibits phosphoenolpyruvate carboxykinase in hepatocytes and increases release of gluconeogenic precursors from peripheral tissues

3- Aminopicolinate , a hyperglycemic agent that activates purified phosphoenolpyruvate carboxykinase in the presence of Fe2+, inhibits glucose synthesis from lactate, pyruvate, asparagine, monomethyl succinate, or glutamine but does not affect that from fructose, dihydroxyacetone, sorbitol, or glycerol in hepatocytes isolated from rats fasted for 24 h. Lactate production from monomethyl succinate by hepatocytes is also inhibited by 3- aminopicolinate . This compound elevates the concentrations of pyruvate, malate, and aspartate but decreases that of phosphoenolpyruvate in hepatocytes incubated with lactate plus pyruvate. In rats, the ability of 3- aminopicolinate to elevate blood glucose concentration is unimpaired by renalectomy . The drug does not significantly affect glycemia in functionally hepatectomized rats but accelerates blood lactate and pyruvate accumulation to higher maximum concentrations even when kidney function is also ablated. It is concluded that 3- aminopicolinate inhibits phosphoenolpyruvate carboxykinase in hepatocytes, that the reported stimulation of renal glutaminase and glutamine gluconeogenesis by this compound does not contribute significantly to its hyperglycemic property, and that the drug increases gluconeogenic substrate supply from peripheral tissues.     

Effect of natural amino acids on ethanol oxidation in isolated rat hepatocytes]

The effects of the various naturally occurring amino acids on ethanol oxidation in hepatocytes from starved rats was systematically studied. In order to minimize the non ADH pathways, the ethanol concentration used was 4 mmol/litre, the amino acids being added at the same concentration. In hepatocytes from fasted rats, alanine, arginine, asparagine, aspartate, citrulline, cysteine, glutamate, glutamine, glycine, histidine, hydroxyproline, ornithine and serine increase significantly ethanol consumption. The stimulatory effect of glutamine being much less pronounced than the asparagine one and proline being devoid of action, the influence of ammonium chloride addition on ethanol consumption in the presence of these amino acids was studied. Ammonium chloride determines an enhancement of ethanol oxidation in these conditions, the results showing no apparent correlation between intracellular glutamate concentration and ethanol oxidation rate, contrarily to previous data. In hepatocytes from fed rats, only alanine, asparagine, cysteine, glycine, hydroxyproline, ornithine and serine increase ethanol oxidation, although to a lesser extent than in cells from starved rats.     

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.     

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.