The effect of ketone bodies on protein turnover in isolated skeletal muscle from the fed and fasted chick

1. The addition of 4 mM acetoacetate or DL-beta-hydroxybutyrate to the incubation medium decreased the rate of protein synthesis without influencing the rate of protein degradation in extensor digitorum communis (EDC) muscles from fed chicks and decreased the rates of protein synthesis and degradation in muscles from fasted chicks. 2. Ketone bodies markedly decreased intracellular concentrations of glutamine in EDC muscles from fed chicks by increasing glutamine oxidation. 3. The addition of 0.5 mM glutamine to incubation media containing 1.0 mM glutamine reversed the ketone body-induced decrease in intracellular glutamine concentration to the control value and blocked the inhibiting effect of ketone bodies on protein synthesis in skeletal muscles from fed chicks. 4. The addition of 5 mM pyruvate blocked the ability of ketone bodies to increase glutamine oxidation and prevented the associated decrease in intracellular glutamine concentration and the rate of protein synthesis in EDC muscles from fed chicks. 5. These results suggest that ketone bodies can act directly on skeletal muscle to inhibit the rate of protein synthesis in muscles from fed chicks by decreasing intracellular glutamine concentration by increasing its oxidation.     

The effect of ketone bodies on nitrogen metabolism in skeletal muscle.

1. The ketone bodies, D-beta-hydroxybutyrate and acetoacetate, inhibit glycolysis thereby reducing pyruvate availability which leads to a marked inhibition of branched-chain amino acid metabolism and alanine synthesis in skeletal muscles from fasted mammalian and avian species. 2. The rate of glutamine release from skeletal muscles from fasted birds is increased at the expense of alanine in the presence of elevated concentrations of ketone bodies because of an increase in the availability of glutamate for glutamine synthesis. 3. Ketone bodies inhibit both protein synthesis and protein degradation in skeletal muscles from fasted mammalian and avian species in vitro. The mechanisms involved remain unknown. 4. Inhibition of amino acid metabolism and protein turnover in skeletal muscle by ketone bodies may be an important survival mechanism during adaptation to catabolic states such as prolonged fasting.     

Formation of alanine and glutamine in chick (Gallus domesticus) skeletal muscle

1. Incubation of extensor digitorum communis muscles from fed chicks in the presence of plasma concentrations of branched-chain amino acids (BCAA) increased the formation of glutamate, glutamine and alanine. This effect was inhibited by 1.5 mM L-cycloserine. 2. 2-Oxoisocaproate (0.1 and 0.5 mM) increased the formation of leucine but decreased that of glutamate, glutamine and alanine. 3. NH4Cl (0.3 mM) increased the formation of glutamine, and decreased the release and intracellular concentrations of glutamate and alanine. 4. Our results demonstrate that alanine and glutamine are synthesized de novo in chick skeletal muscle and demonstrate the similarity in alanine and glutamine synthesis in skeletal muscle between the domestic fowl and mammals.     

Effects of ketone bodies on amino acid metabolism in isolated rat diaphragm.

1. Diaphragms from 48h-starved rats were incubated in Krebs-Ringer bicarbonate medium at 37degreesC for 30min and then transferred into new medium and incubated for 1, 2 and 3 h. 2. The amount of free amino acids found at the end of each time of incubation was larger than the amount at the beginning of incubation, indicating that in this system proteolysis is prevailing. 3. The diaphragms was releasing mainly alanine and glutamine into the incubation medium. 4. Within the periods of incubation the release and metabolism of free amino acids was proceeding at a constant rate. 5. Addition of sodium DL-3-hydroxybutyrate decreased the tissue content of several amino acids, among which were tyrosine and phenylalanine, suggesting that proteolysis was decreased by ketone bodies. 6. In the presence of glucose (10mM) and branched-chain amino acids (0.5mM), sodium DL-3-hydroxybutyrate at concentrations of 4 or 6 mM resulted in 30% decrease in tissue alanine content and a 20% decline in alanine release. Release of taurine and glutamine was decreased by 19 and 16% respectively with 6 mM-sodium DL-3-hydroxybutyrate. Addition of sodium acetoacetate (1-3mM) also resulted in a 20-35% decrease in tissue content of alanine, glutamine and taurine and in a 15-24% decrease of alanine and glutamine release. Smaller decreases (less than 15%) in the release of glycine, threonine, proline, serine and aspartate were also observed in the presence of sodium DL-3-hydroxybutyrate or sodium acetoacetate. 7. Substitution of pyruvate (1.0mM) for glucose in the presence of acetoacetate restored alanine and glutamine production to control values. In the presence of acetoacetate, pyruvate also increased the tissue content of aspartate by 77% and decreased the tissue content of glutamate by 30%. 8. It is suggested that in diaphragms from starved rats, ketone bodies (a) in the absence of other substrates inhibit protein catabolism and (b) in the presence of glucose and branched-chain amino acids decrease alanine and glutamine production, by inhibiting glycolysis.     

The effect of glutamine on protein turnover in chick skeletal muscle in vitro.

The effect of glutamine on the rates of protein synthesis and degradation was studied in isolated chick extensor digitorum communis muscles incubated in the presence of plasma concentrations of amino acids. Addition of 0.5-15 mM-glutamine increases (P less than 0.01) intracellular glutamine concentrations by 31-670%. There is a positive relationship (r = 0.975, P less than 0.01) between intracellular glutamine concentration and the rate of muscle protein synthesis measured by the incorporation of [3H]phenylalanine. The stimulating effect of 15 mM-glutamine on protein synthesis was decreased from 58 to 19% in muscles incubated in the absence of tyrosine. The rates of protein degradation, estimated from [3H]phenylalanine release from muscle proteins prelabelled in vivo, decreased (P less than 0.05) by 15-30% in the presence of 4-15 mM-glutamine when compared with muscles incubated in the presence of physiological concentrations of glutamine (0.5-1 mM). Glutamine concentrations ranging from 2 to 15 mM appear to have an overall anabolic effect on chick skeletal muscles incubated in vitro.     

Effect of pyruvate, octanoate and glucose on leucine degradation in skeletal muscle from fed and fasted chicks

1. Pyruvate at 5 mM decreased the rate of leucine oxidative decarboxylation and increased the rate of 2-oxoisocaproate production in extensor digitorum communis (EDC) muscles from both fed and 24-hr fasted chicks. Pyruvate at 5 mM increased the net rate of leucine transamination in EDC muscle from fed chicks and had no effect in EDC muscles from 24-hr fasted chicks. 2. Octanoate at 0.2 and 1 mM markedly increased the rates of net leucine transamination, leucine oxidative decarboxylation and oxidation of decarboxylated leucine carbons 2-6 in EDC muscles from fed chicks, but had no effect on these parameters of leucine degradation in muscles from 24-hr fasted chicks. 3. Glucose at 5 and 12 mM decreased the rates of leucine oxidative decarboxylation and oxidation of decarboxylated leucine carbons 2-6, and increased the net rate of 2-oxoisocaproate production as compared to control (no glucose) group in muscles from fed chicks. Glucose had no effect on these parameters of leucine degradation in muscles from 24-hr fasted chicks.     

Metabolism of ketone bodies, oleate and glucose in lymphocytes of the rat.

Isolated incubated lymphocytes utilized acetoacetate, 3-hydroxybutyrate or oleate at about 0.5 mumol/min per g dry wt. These rates were not markedly affected by concanavalin A or by starvation of the donor animal. When ketone bodies replaced glucose in the culture medium, they could not support lymphocyte proliferation when cells were cultured for 48 h. Addition of oleate (0.5 mM) to isolated lymphocytes increased the rate of O2 consumption markedly, suggesting that it could contribute about 30% to O2 consumption. The rate of oleate uptake and the stimulated rate of O2 consumption were maximal at 0.5 M-oleate; this is in contrast with the effect in some other tissues, in which the rate of fatty acid oxidation is linear with concentration up to about 2 mM. Since the normal plasma concentration of fatty acid in the fed state is about 0.5 mM, this suggests that lymphocytes can utilize fatty acids at a maximal rate in the fed state. Ketone bodies or oleate decreased the rate of glucose utilization by incubated lymphocytes; ketone bodies decreased the rate of pyruvate oxidation and increased the intracellular concentration of hexose monophosphate and citrate, suggesting that 6-phosphofructokinase is inhibited by citrate, and hexokinase by glucose 6-phosphate. These effects may be important not so much in conserving glucose in the whole animal but in maintaining the concentrations of glycolytic intermediates necessary for biosynthetic processes during proliferation.     

Metabolic effects of valproate on dog renal cortical tubules

The effect of valproate (0.01-10 mM), an antiepileptic drug inducing hyperammonemia in humans, was studied in vitro on a suspension of renal cortical tubules (greater than 85% proximal tubules) obtained from six normal dogs. When these tubules were incubated with 1 mM glutamine, the addition of valproate accelerated glutamine uptake, ammoniagenesis, and the production of alanine, lactate, and pyruvate. With 5 mM glutamine, a rise in glutamate accumulation, a much greater synthesis of alanine, an important aspartate production, and a striking accumulation of lactate and pyruvate were observed. With 1 or 5 mM lactate, lactate utilization and gluconeogenesis were markedly reduced with increasing concentrations of valproate. Oxygen consumption was reduced by only 15-20% by 10 mM valproate. The accelerated glutamine utilization resulting from valproate could not be prevented by aminooxyacetate, an inhibitor of transamination. Valproate also reduced various enzymatic activities, a finding that could not explain its metabolic effects. Four sites of action may explain these various metabolic changes: (i) a stimulation of mitochondrial glutamine transport, (ii) an increase in the flux of glutamate to malate, and (iii) a reduction in the net oxidation of pyruvate and (iv) in the flux through pyruvate carboxylase.     

Glutamine synthesis in the developing porcine placenta

Glutamine plays a vital role in fetal carbon and nitrogen metabolism and exhibits the highest fetal:maternal plasma ratio among all amino acids in pigs. Such disparate glutamine levels between mother and fetus suggest that glutamine may be actively synthesized and released into the fetal circulation by the porcine placenta. We hypothesized that branched-chain amino acid (BCAA) metabolism in the placenta plays an important role in placental glutamine synthesis. This hypothesis was tested by studying conceptuses from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, or 110 of gestation (n = 6 per day). Placental tissue was analyzed for amino acid concentrations, BCAA transport, BCAA degradation, and glutamine synthesis as well as the activities of related enzymes (including BCAA transaminase, branched-chain alpha-ketoacid dehydrogenase, glutamine synthetase, glutamate-pyruvate transaminase, and glutaminase). On all days of gestation, rates of BCAA transamination were much greater than rates of branched-chain alpha-ketoacid decarboxylation. The glutamate generated from BCAA transamination was primarily directed to glutamine synthesis and, to a much lesser extent, alanine production. Placental BCAA transport, BCAA transamination, glutamine synthesis, and activities of related enzymes increased markedly between Days 20 and 40 of gestation, as did glutamine in fetal allantoic fluid. Accordingly, placental BCAA levels decreased after Day 20 of gestation in association with a marked increase in BCAA catabolism and concentrations of glutamine. There was no detectable catabolism of glutamine in pig placenta throughout pregnancy, which would ensure maximum output of glutamine by this tissue. These novel results demonstrate glutamine synthesis from BCAAs in pig placentae, aid in explaining the abundance of glutamine in the fetus, and provide valuable insight into the dynamic role of the placenta in fetal metabolism and nutrition.     

Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential.

1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[1-14C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through alpha-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222, 813-820] and Preuss [(1972) Am. J. Physiol. 222, 1395-1397]. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenous-difference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.     

Regulation of leucine catabolism by metabolic fuels in mammary epithelial cells

Lactation is associated with elevated catabolism of branched-chain amino acids (BCAA) in mammary glands to produce glutamate, glutamine, alanine, aspartate, and asparagine. This study determined effects of metabolic fuels on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 0.5 mM l-leucine and either l-[1-¹⁴C]leucine or l-[U-¹⁴C]leucine. The medium also contained 0–5 mM d-glucose, 0–2 mM l-glutamine, 0–4 mM dl-β-hydroxybutyrate, or 0–2 mM oleic acid. Rates of leucine decarboxylation were 60 % lower, but rates of α-ketoisocaproate production were 34 % higher, in the presence of 2 mM glucose than in its absence. All variables of leucine catabolism did not differ between 2 and 5 mM glucose or between 0 and 4 mM dl-β-hydroxybutyrate. Compared with 0–0.25 mM glutamine, 0.5 and 2 mM l-glutamine reduced leucine transport, transamination, and decarboxylation. In contrast, increasing the concentration of oleic acid from 0 to 2 mM dose-dependently stimulated leucine transamination, decarboxylation, and oxidation of carbons 2–6. Oleic acid also enhanced the abundance of cytosolic BCAA transaminase, while reducing the phosphorylated level (inactive state) of the E1α subunit of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. Thus, hypoglycemia or ketosis in early lactation does not likely affect BCAA metabolism in mammary epithelial cells. Increasing circulating levels of BCAA and oleic acid may have great potential to increase the syntheses of glutamate, glutamine, aspartate, alanine, and asparagine by lactating mammary glands, thereby leading to enhanced production of milk for suckling neonates.     

The metabolic response of the kidney to acute sodium lactate alkalosis

In vivo studies were performed in the dog to verify if sodium lactate had an important effect on the metabolism of glutamine by the kidney. The animals were infused with 0.6 M sodium lactate to induce acute metabolic alkalosis with plasma bicarbonate of 29.7 mM. During these experiments, it was demonstrated that the renal uptake of glutamine increased by 46%, while the renal production of ammonia was unchanged. The renal production of alanine rose from 6.0 to 16.8 mumol/min. Plasma concentration of lactate increased from 1.3 to 19.2 mM, while that of pyruvate increased from 0.075 to 0.454 mM. In the renal tissue, alpha-ketoglutarate, malate, oxaloacetate, lactate, pyruvate, citrate, and alanine increased significantly. Similar changes were found in the liver and skeletal muscle. The observed changes are best described by transamination of pyruvate and glutamate under the influence of alanine aminotransferase (GPT). It can be calculated that this reaction was responsible for 76% of the production of ammonia from glutamine, the latter being necessary to provide glutamate for the synthesis of alanine. Dogs infused with 0.3 M sodium bicarbonate instead of sodium lactate with the same degree of acute metabolic alkalosis, showed a depression of 40% in the renal uptake of glutamine with a 38% decrease in renal ammoniagenesis and a 20% fall in the production of alanine. The present studies demonstrate that the production of ammonia from glutamine is not necessarily related to changes in acid-base balance, but may be associated with biochemical alterations related to the synthesis of alanine by the kidney.     

Alanine and glutamine synthesis and release from skeletal muscle. I. Glycolysis and amino acid release.

The synthesis and release of alanine and glutamine were investigated with an intact rat epitrochlaris muscle preparation. This preparation will maintain on incubation for up to 6 hours, tissue levels of phosphocreatine, ATP, ADP, lactate, and pyruvate closely approximating those values observed in gastrocnemius muscles freeze-clamped in vivo. The epitrochlaris preparation releases amino acids in the same relative proportions and amounts as a perfused rat hindquarter preparation and human skeletal muscle. Since amino acids were released during incubation without observable changes in tissue amino acids levels, rates of alanine and glutamine release closely approximate net amino acid synthesis. Large increases in either glucose uptake or glycolysis in muscle were not accompanied by changes in either alanine or glutamine synthesis. Insulin increased muscle glucose uptake 4-fold, but was without effect on alanine and glutamine release. Inhibition of glycolysis by iodacetate did not decrease the rate of alanine synthesis. The rates of alanine and glutamine synthesis and release from muscle decreased significantly during prolonged incubation despite a constant rate of glucose uptake and pyruvate production. Alanine synthesis and release were decreased by aminooxyacetic acid, an inhibitor of alanine aminotransferase. This inhibition was accompanied by a compensatory increase in the release of other amino acids, such as aspartate, an amino acid which was not otherwise released in appreciable quantities by muscle. The release of alanine, pyruvate, glutamate, and glutamine were observed to be interrelated events, reflecting a probable near-equilibrium state of alanine aminotransferase in skeletal muscle. It is concluded that glucose metabolism and amino acid release are functionally independent processes in skeletal muscle. Alanine release reflects the de novo synthesis of the amino acid and does not arise from the selective proteolysis of an alanine-rich storage protein. It appears that the rate of alanine and glutamine synthesis in skeletal muscle is dependent upon the transformation and metabolism of amino acid precursors.     

Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with (15)N

We focused on the role of plasma membrane glutamate uptake in modulating the intracellular glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in determining the fate of the intracellular glutamate in the proximal tubule-like LLC-PK(1)-F(+) cell line. We used high-affinity glutamate transport inhibitors D-aspartate (D-Asp) and DL-threo-beta-hydroxyaspartate (THA) to block extracellular uptake and then used [(15)N]glutamate or [2-(15)N]glutamine to follow the metabolic fate and distribution of glutamine and glutamate. In monolayers incubated with [2-(15)N]glutamine (99 atom %excess), glutamine and glutamate equilibrated throughout the intra- and extracellular compartments. In the presence of 5 mM D-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33% (P < 0.05) as the extracellular enrichment increased by 39% (P < 0.005). With glutamate uptake blocked, intracellular glutamate concentration decreased by 37% (P < 0.0001), in contrast to intracellular glutamine concentration, which remained unchanged. Both glutamine disappearance from the media and the estimated intracellular GA flux increased with the fall in the intracellular glutamate concentration. The labeled glutamate and NH formed from [2-(15)N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux. However, labeled alanine formation was reduced by 37%, indicating inhibition of transamination. Although both D-Asp and THA alone accelerated the GA and GDH flux, only THA inhibited transamination. These results are consistent with glutamate transport both regulating and being regulated by glutamine and glutamate metabolism in epithelial cells.     

Ketone bodies inhibit leucine degradation in chick skeletal muscle

1. DL-beta-hydroxybutyrate (4 mM) increased the net rate of leucine transamination and the net rate of 2-oxoisocaproate (KIC) production in extensor digitorum communis muscles from fed chicks. 2. DL-beta-hydroxybutyrate at 1 and 4 mM inhibited leucine oxidative decarboxylation in muscles from fed chicks. 3. Acetoacetate at 1 and 4 mM inhibited leucine oxidative decarboxylation and total leucine oxidation, but increased net KIC production in muscles from fed chicks. 4. Both DL-beta-hydroxybutyrate and acetoacetate at 1 and 4 mM inhibited the net rate of leucine transamination and the rates of leucine oxidative decarboxylation and total leucine oxidation in muscles from 24-hr fasted chicks.     

Effects of branched-chain amino acids on muscles under hyperammonemic conditions

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1-¹⁴C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.     

The estimation of rates of utilization of glucose and ketone bodies in the brain of the suckling rat using compartmental analysis of isotopic data

The brains of 18-day-old rats utilize glucose and ketone bodies. The rates of acetyl-CoA formation from these substrates and of glycolysis were determined in vivo from the labelling of intermediary metabolites after intraperitoneal injection of d-[2-(14)C]glucose, l(+)-[3-(14)C]- and l(+)-[U-(14)C]-lactate and d(-)-3-hydroxy[(14)C]butyrate. Compartmental analysis was used in calculating rates to allow for the rapid exchange of blood and brain lactate, the presence in brain of at least two pools each of glucose and lactate, and the incomplete equilibration of oxaloacetate with aspartate and of 2-oxoglutarate with glutamate. Results were as follows. 1. Glucose and ketone bodies labelled identical pools of tricarboxylate-cycle metabolites, and were in every way alternative substrates. 2. The combined rate of oxidation of acetyl-CoA derived from pyruvate (and hence glucose) and ketone bodies was 1.05mumol/min per g. 3. Ketone bodies contributed 0.11-0.53mumol/min per g in proportion to their concentration in blood, with a mean rate of 0.30mumol/min per g at 1.24mm. 4. Pyruvate and ketone bodies were converted into lipid at 0.018 and 0.008mumol/min per g respectively. 5. Glycolysis, at 0.48mumol/min per g, was more rapid in most rats than pyruvate utilization by oxidation and lipid synthesis, resulting in a net output of lactate from brain to blood. 6. Rates of formation of brain glutamate, glutamine and aspartate were also measured. Further information on the derivation of the models has been deposited as Supplementary Publication SUP 50034 (18 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant

Summary Intra- and extracellular metabolite concentrations were determined for hybridoma cells grown in tissue culture flasks in batch culture. Significant differences were found for intracellular lactate and amino acid concentrations depending on the culture medium. AB2-143.2 cells cultivated in Dulbecco's modified Eagle's (DME) medium supplemented with 50 mm lactate exhibited intracellular lactate and glutamine levels of 40 mm and 4 mm, respectively, whereas cells grown in standard DME medium had low intracellular glutamine and 20 mm lactate concentrations. For cells cultivated in medium supplemented with 6 mm pyruvate, intracellular lactate levels were estimated at approx. 40 mm, but these cells also showed an increased intracellular alanine concentration of 22 mm. The higher alanine pools probably result from increased transamination of pyruvate under these conditions.     

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transamination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.     

Acute hyperammonemia activates branched-chain amino acid catabolism and decreases their extracellular concentrations: different sensitivity of red and white muscle

Hyperammonemia is considered to be the main cause of decreased levels of the branched-chain amino acids (BCAA), valine, leucine, and isoleucine, in liver cirrhosis. In this study we investigated whether the decrease in BCAA is caused by the direct effect of ammonia on BCAA metabolism and the effect of ammonia on BCAA and protein metabolism in different types of skeletal muscle. M. soleus (SOL, slow-twitch, red muscle) and m. extensor digitorum longus (EDL, fast-twitch, white muscle) of white rat were isolated and incubated in a medium with or without 500 μM ammonia. We measured the exchange of amino acids between the muscle and the medium, amino acid concentrations in the muscle, release of branched-chain keto acids (BCKA), leucine oxidation, total and myofibrillar proteolysis, and protein synthesis. Hyperammonemia inhibited the BCAA release (81% in SOL and 60% in EDL vs. controls), increased the release of BCKA (133% in SOL and 161% in EDL vs. controls) and glutamine (138% in SOL and 145% in EDL vs. controls), and increased the leucine oxidation in EDL (174% of controls). Ammonia also induced a significant increase in glutamine concentration in skeletal muscle. The effect of ammonia on intracellular BCAA concentration, protein synthesis and on total and myofibrillar proteolysis was insignificant. The data indicates that hyperammonemia directly affects the BCAA metabolism in skeletal muscle which results in decreased levels of BCAA in the extracellular fluid. The effect is associated with activated synthesis of glutamine, increased BCAA oxidation, decreased release of BCAA, and enhanced release of BCKA. These metabolic changes are not directly associated with marked changes in protein turnover. The effect of ammonia is more pronounced in muscles with high content of white fibres.