Transamination of 3-phenylpyruvate in pancreatic B-cell mitochondria

High aminotransferase activities catalyzing the reaction between L-glutamate and the aromatic ketomonocarboxylic acid, 3-phenylpyruvate, were observed in the mitochondria from pancreatic B-cells. At very low concentrations of 3-phenylpyruvate, L-glutamine was an effective amino group donor. The aminotransferase activities for the aliphatic ketomonocarboxylic acids, pyruvate and 2-ketoisovalerate, were lower in B-cell mitochondria. High rates of transamination of 2-ketoisocaproate with L-glutamine were observed and may be an important prerequisite for the insulin secretory potency of this 2-keto acid. Since B-cell mitochondria are well supplied with L-glutamine and L-glutamate, 3-phenylpyruvate-induced 2-ketoglutarate production may explain the insulin secretory potency of 3-phenylpyruvate which is not a fuel for pancreatic islet cells.     

3-Ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids

The various neutral amino acids and aliphatic 2-keto acids exhibit differential effects on insulin secretion. The common denominator for all these effects is the 2-ketoglutarate generation in the pancreatic B-cell mitochondria. The neutral amino acidsl-leucine andl-norvaline and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, 2-ketovalerate, and 2-keto-3-methylvalerate all stimulate insulin secretion and increase 2-ketoglutarate generation in pancreatic B-cell mitochondria through activation of glutamate dehydrogenase and transamination withl-glutamate andl-glutamine, respectively. The neutral amino acidsl-valine,l-norleucine, andl-alanine and the aliphatic 2-keto acids 2-ketoisovalerate and pyruvate do not stimulate insulin secretion and do not increase 2-ketoglutarate generation in pancreatic B-cell mitochondria. Inhibition of 2-keto acid induced insulin secretion byl-valine andl-isoleucine is accompanied by reduced 2-ketoglutarate generation in pancreatic B-cell mitochondria. Thus intramitochondrial 2-ketoglutarate generation in pancreatic B-cells may regulate the insulin secretory potency of amino acids and 2-keto acids.     

The stimulus-secretion coupling of amino acid-induced insulin release synergistic effects of L-glutamine and 2-keto acids upon insulin secretion

1. L-Glutamine markedly enhances insulin release evoked in rat pancreatic islets by 2-ketoisocaproate or 2-ketocaproate. L-Glutamine exerts a lesser enhancing action in the presence of 2-ketovalerate or 2-ketoisovalerate, which are themselves poor insulin secretagogues. L-Glutamine fails to affect insulin release in the presence of 2- ketobutyrate, pyruvate and β-hydroxybutyrate. 2. The relase of insulin evoked by the combination of L-glutamine and 2-ketoisocaproate represents a sustained phenomenon. It coincides with a stimulation of ⁴⁵Ca net uptake by the islets, and is inhibited in the absence of extracellular Ca²⁺ and presence of either menadione or epinephrine. 3. L-Valine inhibits insulin releaseevoked by either 2-ketoisocaproate or 2-ketocaproate, whether in the presence or absence of L-glutamine, but does not abolish the enhancing action of L-glutamine. L-Valine fails to affect insulin release evoked by the combination of L-leucine and L-glutamine. 4. L-Isoleucine also inhibits 2-keto acid-induced insulin release. However, in contrast to L-valine, L-isoleucine fails to affect or slightly augments insulin release in the simultaneous presence of L-glutamine and either 2-ketoisocaproate or 2-ketocaproate. 5. L-Leucine causes a dose-related enhancement of insulin release evoked by the combination of 2-ketoisocaproate and L-glutamine. Likewise, in the presence of L-glutamine, L-leucine and 2-ketocaproate act synergistically upon insulin release. 6. The hypothesis is advances that the enhancing action of L-glutamine upon 2-keto acid-stimulated insulin release depends on the availability of the 2-keto acid to act as a partner in the conversion of L-glutamate derived from exogenous L-glutamine to 2-ketoglutarate by transamination reaction, rather than being attributable to activation of glutamate dehydrogenase as observed in islets exposed to both L-glutamine and L-leucine.     

Does leucine- and norleucine-induced insulin release depend on amino acid aminotransferase activity?

In the absence of another exogenous nutrient, L-leucine but not L-norleucine stimulates insulin release from rat pancreatic islets, although the corresponding keto acids, 2-ketoisocaproate and 2-ketocaproate, are equally potent secretagogues. Such a situation cannot be ascribed to the preferential transamination of L-leucine as compared to L-norleucine in islet homogenates. Indeed, in the presence of a suitable activator of glutamate dehydrogenase, L-leucine and L-norleucine stimulate secretion to the same extent. It is concluded that the rate of transamination of these amino acids in intact islet cells depends on the availability of a 2-keto acid partner rather than on the assayed amino acid aminotransferase activity.     

The stimulus-secretion coupling of amino acid-induced insulin release. XI. Kinetics of deamination and transamination reactions

L-Leucine and its nonmetabolized analogue, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH) activate glutamate dehydrogenase in pancreatic islets, whether the reaction velocity is measured in the direction of glutamate synthesis or glutamate deamination. The rate of glutamate oxidative deamination is increased by ADP and inhibited by 2-ketoglutarate, NH4+ and GTP. The islet homogenate catalyzes the transamination between L-glutamate and either 2-ketoisocaproate or pyruvate, and between 2-ketoglutarate and L-leucine, L-aspartate, L-alanine, L-isoleucine, L-valine, L-norvaline or L-norleucine, but not b (+/-) BCH. The glutamate-aspartate transaminase is preferentially located in mitochondria relative to other transaminases. The parallel effects of L-leucine and BCH on glutamate dehydrogenase and their vastly different abilities to act as transamination partners may account for both analogies and discrepancies in the metabolic and functional responses of the islets to these two branched-chain amino acids.     

The stimulus-secretion coupling of amino acid-induced insulin release

Summary Aminooxyacetate, an inhibitor of cytosolic transamination reactions, inhibited insulin release evoked by either 2-ketoisocaproate or L-leucine in rat pancreatic islets incubated in the presence of L-glutamine or L-asparagine. As a rule, aminooxyacetate also inhibited the oxidation of these nutrient secretagogues and impaired the respiratory response of the islets to the combinations of nutrients. However, the oxidative and secretory response to the combination of L-leucine and L-glutamine was less severely affected by aminooxyacetate than that evoked by the three other combinations of exogenous nutrients. These findings reinforce the view that the stimulus-secretion coupling of insulin release in response to L-leucine and 2-ketoisocaproate in association with either L-glutamine or L-asparagine tightly depends on the oxidation of these nutrient secretagogues, on their effect upon O₂ uptake and, within limits, on the intracellular site of generation of reducing equivalents in the pancreatic islet cells.This paper is the 16th in a series.     

TRANSAMINATION OF AMINO ACIDS IN HOMOGENATES OF RAT BRAIN

Abstract— The aminotransferase activity of homogenates of brains from adult and neonatal rats has been investigated. Aminotransferase activity was demonstrated wtih 15 of 22 amino acids incubated with seven keto acids. The basic amino acids exhibited little or no activity.

• 1 The greatest activity was obtained when glutamate or aspartate was incubated with α-ketoglutarate or oxaloacetate. Significant activity was also observed when the neutral aliphatic and aromatic amino acids were incubated with these two keto acids.
• 2 Activity with pyruvate was obtained principally upon incubation with glutamate and alanine. Most of the other amino acids that underwent transamination with α-ketoglutarate also did so with pyruvate, although at a lower rate.
• 3 When phenylpyruvate was added to the medium, glutamate, phenylalanine and tyrosine transaminated most actively.
• 4 Incubations with 11 amino acids and glyoxylic acid demonstrated aminotransferase activity, with glutamate and ornithine being the most active substrates.
• 5 α-Ketoisocaproate and α-ketoisovalerate accepted amino groups primarily from the branched-chain amino acids. Except for glutamate, activity with other amino acids was low or not detectable.
• 6 A comparison of aminotransferase activity in the newborn brain with that in the adult brain showed that the greatest change in activity occurred for glutamate with pyruvate or for alanine with α-ketoglutarate, these activities increasing about 10-fold from birth to adulthood; during this time activities with most other amino acids increased two- to threefold. Amino transfers from the branched-chain amino acids showed no increase with maturation, and some reactions, such as that with methionine and a number of keto acids, decreased from birth to adulthood.
• 7 Our results correspond in general to previous studies of aminotransferase activity in brain and in liver. However, our study also indicates a possible second aminotransferase acting on the branched-chain amino acids, the presence of aminotransferase activity for methionine and asparagine, and relatively high aminotransferase activity for glutamine or ornithine when incubated with glyoxylic acid rather than other keto acids. Moreover, phenylpyruvate and glyoxylate are active in amino transfers and may serve as substrates for a number of aminotransferases.

     

Functional significance of aromatic amino acid: aromatic keto acid aminotransferases in rat brain and liver: competition for tryptophan between aminotransferases and tryptophan hydroxylase in vitro.

Using low concentrations of substrates and cofactors, a comparison was made of the relative rates by which aminotransferases catalysed transaminations between aromatic amino acids and aromatic or aliphatic keto acids. Tryptophan aminotransferase in homogenates of rat midbrain and liver transaminated phenylpyruvate at a rate 70 to 150-fold greater than the rate with α-ketoglutarate at low concentrations of substrates. Phenylalanine aminotransferase in liver and midbrain also was more active with aromatic keto acids than with aliphatic keto acids. However, tyrosine aminotransferase in dialysed homogenates of midbrain transaminated α-ketoglutarate and phenylpyruvate at approximately equal rates. Fresh homogenates of midbrain contained an inhibitor which markedly decreased tyrosine aminotransferase activity with α-ketoglutarate but not with phenylpyruvate. Tyrosine aminotransferase in homogenates of rat liver transaminated α-ketoglutarate and phenylpyruvate at equal rates below 10 μM keto acid, but above 10 μM, transamination of α-ketoglutarate was favoured. With homogenates of liver, transamination of α-ketoglutarate, but not phenylpyruvate, by tyrosine was increased 650% by exogenous pyridoxal phosphate. Since tryptophan aminotransferase in the brain may compete with tryptophan hydroxylase for available tryptophan, a comparison was made of the relative activities of tryptophan hydroxylase and tryptophan aminotransferase. At concentrations above 7.5 μM phenylpyruvate, transamination was 8 to 17-fold greater than the rate of hydroxylation of 50 μM tryptophan.     

Mechanism of 3-phenylpyruvate-induced insulin release. Secretory, ionic and oxidative aspects.

1. 3-Phenylpyruvate caused a dose-related stimulation of insulin release from rat pancreatic islets deprived of exogenous nutrient or incubated in the presence of 5.6 or 8.3 mM-D-glucose. 2. 3-Phenylpyruvate inhibited insulin release evoked by high concentrations of D-glucose (16.7 or 27.8 mM) or 4-methyl-2-oxopentanoate (10.0 mM). This inhibitory effect appeared to be attributable to impairment of 2-oxo-acid transport into the mitochondria, with resulting inhibition of D-glucose, pyruvate or 4-methyl-2-oxopentanoate oxidation. 3. 3-Phenylpyruvate failed to affect the oxidation of, and secretory response to, L-leucine, and did not augment insulin release evoked by a non-metabolized analogue of the latter amino acid. 4. L-Glutamine augmented 3-phenylpyruvate-induced insulin release. The release of insulin evoked by the combination of 3-phenylpyruvate and L-glutamine represented a sustained phenomenon, abolished in the absence of extracellular Ca2+ or the presence of menadione and potentiated by theophylline. 5. Whether in the presence or in the absence of L-glutamine, the secretory response to 3-phenylpyruvate coincided with an increase in O2 uptake, a decrease in K+ conductance, a stimulation of both Ca2+ inflow and 45Ca2+ net uptake and an increase in cyclic AMP content. 6. It is concluded that the release of insulin induced by 3-phenylpyruvate displays features classically encountered when the B-cell is stimulated by nutrient secretagogues, and is indeed attributable to an increase in nutrient catabolism.     

Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.     

Stimulus-secretion coupling of arginine-induced insulin release. Functional response of islets to L-arginine and L-ornithine

L-Arginine and L-ornithine stimulate insulin release from pancreatic islets exposed to D-glucose. This coincides with an increased outflow of 86Rb and 45Ca from prelabelled islets and an increased net uptake of 45Ca by the islets. In the presence of D-glucose, L-lysine stimulates insulin secretion to the same extent as L-arginine or L-ornithine, but the hormonal release is not further enhanced by combinations of these cationic amino acids. L-Arginine or L-ornithine failed to enhance insulin release evoked by either L-leucine or 2-ketoisocaproate. The inhibitor of ornithine decarboxylase D,L-alpha-difluoromethyl ornithine failed to affect the metabolism and insulinotropic action of D-glucose in pancreatic islets, and only caused a partial inhibition of the secretory response to either L-arginine or L-ornithine. The latter amino acids inhibited modestly but significantly D-glucose utilization and oxidation by pancreatic islets. These and complementary findings suggest that the secretory response to L-arginine and L-ornithine is not attributable to any major change in the overall oxidative catabolism of nutrients, but involves mainly a biophysical component, such as the depolarization of the plasma membrane by these cationic amino acids.     

TRANSAMINATIONS BETWEEN AMINO ACIDS AND KETO ACIDS ELEVATED IN PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE

Abstract— The transamination between amino acids and aliphatic and aromatic keto acids has been investigated in homogenates of human and rat brain. Tryptophan, phenylalanine and 3,4-dihydroxyphenylalanine (DOPA) at concentrations of 3.6 min and below trans-aminated aromatic keto acids more rapidly than α-ketoglutarate; lower K_m values were found for tryptophan and phenylalanine in the presence of the aromatic keto acid. Rat brain and liver arninotransferases exhibited similar affinities for tryptophan in the presence of different keto acids. Branched chain keto acids were also acceptors of the amino groups of tryptophan and DOPA. In brain homogenates α-ketoglutarate and p -hydroxyphenyl-pyruvate were transaminated by tyrosine and 5-hydroxytryptophan at about equal rates, whereas a-ketoglutarate was transaminated more rapidly with aliphatic amino acids. At concentrations of 1.6 m DOPA and 0.8 mM p -hydroxyphenylpyruvate, transamination was 6-fold greater than the rate of formation of dopamine. The dihydroxyphenylpyruvate formed during arninotransfer from DOPA by brain tissue was not readily decarboxylated, whereas 65–70 per cent of the indolepyruvate formed from tryptophan was decarboxylated. We suggest that an increased rate or degree of transamination between tryptophan and aromatic and branched chain keto acids may explain the increased excretion of non-hydroxylated indolic acids in phenylketonuria and'maple syrup urine'disease, respectively. Increased aminotransfers from tryptophan and DOPA may reduce the amounts of precursors available for the synthesis of serotonin and catecholamines, both of which are at low levels in the sera of untreated phenylketonurics.     

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction of L-asparagine and L-leucine in pancreatic islets

Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. L-Asparagine augmented the oxidation of L-leucine, an effect possibly attributable to activation of 2-ketoisocaproate dehydrogenase. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.     

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction L-asparagine and L-leucine in pancreatic islets

1. Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. 2. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. 3. L-Asparagine augmented the oxidation of L-leucine, and effect possibly attributable to activaion of 2-ketoisocaproate dehydrogenase. 4. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. 5. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.     

Functional differences in the catabolism of branched-chain L-amino acids in cultured normal and maple syrup urine disease fibroblasts

Possible functional differences in the catabolism of the four branched-chain L-amino acids in maple syrup urine disease were assessed using cultured human skin fibroblast stains. Transamination and oxidative decarboxylation were comparatively studied in 90-min incubations with 1 mmole/liter of 1-14C-labeled substrates. In normal cell strains (n = 5), apparent transamination rates (sum of branched-chain 2-oxo[14C]acid and 14CO2 release; means expressed in nmole/90 min/mg of cell protein) were in the order L-leucine (32) greater than L-valine (17) greater than or equal to L-isoleucine (14) greater than L-allo-isoleucine (8); 14CO2 production was in the order L-valine (9) greater than L-isoleucine (6) greater than or equal to L-leucine (5) greater than L-allo-isoleucine (2). In variant (n = 5) as well as classical (n = 2) MSUD cell lines, branched-chain 2-oxo-[14C]acid release rates were generally comparable to the control values. As compared to the 14CO2 release in controls (= 100%), branched-chain 2-oxo acid dehydrogenase activity in MSUD fibroblasts was individually reduced and varied considerably between strains (residual activity 2-38%). Within individual strains, only small differences in the residual decarboxylation activity were observed in incubations with L-valine, L-leucine, and L-isoleucine. It was remarkably high, however, when L-allo-isoleucine was applied as a substrate. With the exception of L-allo-isoleucine, apparent total transamination rates of branched-chain L-amino acids were therefore distinctly lower in MSUD cells than in normal cells.     

Growth hormone and liver mitochondria. Effects on cytochromes and some enzymes.

A reliable and reproducible assay was developed for measuring mitochondrial α-keto acid decarboxylase activity using ferricyanide as the electron acceptor. This method permitted the functional isolation and investigation of the decarboxylase step of the branched-chain α-keto acid dehydrogenases in rat liver mitochondria. Pyruvate and α-ketoglutarate decarboxylases are known to be separate and distinct enzymes from the branched-chain α-keto acid decarboxylases and were studied as controls. The relative specific activities of rat liver mitochondrial decarboxylases as measured by the ferricyanide assay showed that pyruvate and α-ketoglutarate were decarboxylated twice as rapidly as α-ketoisovalerate and four to ten times as fast as α-keto-β-methylvalerate and α-ketoisocaproate. The three branched-chain α-keto acids individually inhibit pyruvate and α-ketoglutarate decarboxylases. Inactivation of mitochondrial branched-chain α-keto acid decarboxylase activity by freezing and thawing and by prolonged storage resulted in a proportional decrease in decarboxylase activity toward each of the three branched-chain α-keto acids. However, hypophysectomy was found to increase decarboxylase activity with α-keto-β-methylvalerate to four times normal and with α-ketoisovalerate to three times normal, but the activity with α-ketoisocaproate was not changed. Hypophysectomy did not alter mitochondrial decarboxylase activity with pyruvate, α-ketoglutarate, or α-ketovalerate. The finding that hypophysectomy differentially alters the mitochondrial decarboxylase activity with the three branched-chain α-keto acids suggests either that there is more than one substrate-specific enzyme with branched-chain α-keto acid decarboxylase activity or that there is a modification of one enzyme such that the catalytic activity is selectively altered toward the three substrates.     

Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell.

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.     

Mechanism of 3-phenylpyruvate-induced insulin release. Metabolic aspects.

1. The metabolism and metabolic effects of 3-phenylpyruvate were examined in rat pancreatic islets. 2. Islet homogenates catalysed transamination reactions between 3-phenylpyruvate and L-glutamate, L-leucine, L-norleucine or L-valine. 3-Phenylpyruvate failed to activate glutamate dehydrogenase. 3. 3-Phenylpyruvate rapidly entered into islet cells, was extensively converted into phenylalanine but slowly oxidized. 4. The conversion of phenylpyruvate into phenylalanine coincided with a fall in the content of several amino acids (especially glutamate and aspartate) in the islets and incubation medium, the accumulation of 2-oxoglutarate and a modest fall in the NH4+ production rate. 5. 3-Phenylpyruvate failed to affect 14CO2 output from islets prelabelled with [U-14C]palmitate, but augmented 14CO2 output from islets prelabelled or incubated with L-[U-14C]glutamine. 6. In the presence of L-glutamine, 3-phenylpyruvate augmented the ATP/ADP ratio and NAD(P)H islet content, and caused a rapid and sustained decrease in the outflow of radioactivity from islets prelabelled with [2-3H]adenosine. 7. These data support the view that the insulin-releasing capacity of 3-phenylpyruvate coincides with an increase in the catabolism of endogenous amino acids acting as 'partners' in transamination reactions leading to the conversion of 3-phenylpyruvate into phenylalanine.     

Regulation of valine catabolism in Pseudomonas putida

The activities of six enzymes which take part in the oxidation of valine by Pseudomonas putida were measured under various conditions of growth. The formation of four of the six enzymes was induced by growth on d- or l-valine: d-amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-hydroxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. Branched-chain amino acid transaminase and isobutyryl-CoA dehydrogenase were synthesized constitutively. d-Amino acid dehydrogenase and branched-chain keto acid dehydrogenase were induced during growth on valine, leucine, and isoleucine, and these enzymes were assumed to be common to the metabolism of all three branched-chain amino acids. The segment of the pathway required for oxidation of isobutyrate was induced by growth on isobutyrate or 3-hydroxyisobutyrate without formation of the preceding enzymes. d-Amino acid dehydrogenase was induced by growth on l-alanine without formation of other enzymes required for the catabolism of valine. d-Valine was a more effective inducer of d-amino acid dehydrogenase than was l-valine. Therefore, the valine catabolic pathway was induced in three separate segments: (i) d-amino acid dehydrogenase, (ii) branched-chain keto acid dehydrogenase, and (iii) 3-hydroxyisobutyrate dehydrogenase plus methylmalonate semialdehyde dehydrogenase. In a study of the kinetics of formation of the inducible enzymes, it was found that 3-hydroxyisobutyrate and methylmalonate semialdehyde dehydrogenases were coordinately induced. Induction of enzymes of the valine catabolic pathway was studied in a mutant that had lost the ability to grow on all three branched-chain amino acids. Strain PpM2106 had lowered levels of branched-chain amino acid transaminase and completely lacked branched-chain keto acid dehydrogenase when grown in medium which contained valine. Addition of 2-ketoisovalerate, 2-ketoisocaproate, or 2-keto-3-methylvalerate to the growth medium of strain PpM2106 resulted in induction of normal levels of branched-chain keto acid dehydrogenase; therefore, the branched-chain keto acids were the actual inducers of branched-chain keto acid dehydrogenase.     

Role of Branched-Chain Aminotransferase Isoenzymes and Gabapentin in Neurotransmitter Metabolism

Abstract: Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with α-ketoglutarate to form the branched-chain α-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-¹⁴C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to α-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an α-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from α-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.