Metabolic and Regulatory Roles of Leucine in Neural Cells

Dietary leucine transported into the brain parenchyma serves several functions. Most prominent is the role of leucine as a metabolic precursor of fuel molecules, α-ketoisocaproate and ketone bodies. As alternatives to glucose, these compounds are forwarded by the producing astrocytes to the adjacent neural cells. Leucine furthermore participates in the maintenance of the nitrogen balance in the glutamate/glutamine cycle pertinent to the neurotransmitter glutamate. Leucine also serves as a regulator of the activity of some enzymes important for brain energy metabolism. Another role of leucine as an informational molecule is in mTOR signaling that participates in the regulation of food ingestion. The importance of leucine for brain function is stressed by the fact that inborn errors in its metabolism cause metabolic diseases often associated with neuropathological symptoms. In this overview, the current knowledge on the metabolic and regulatory roles of this essential amino acid in neural cells are briefly summarized. Special issue dedicated to Dr. Frode Fonnum.     

Effects of Ketone Bodies on Astrocyte Amino Acid Metabolism

Abstract: The effects of acetoacetate and 3-hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with ¹⁵N as a metabolic probe and gas chromatography-mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3-hydroxybutyrate (5 m M ) to the incubation medium did not alter the initial rate of appearance of [¹⁵N]glutamate in the glia, but it did inhibit transamination of glutamate to [¹⁵N]aspartate. Addition of acetoacetate also inhibited formation of [2-¹⁵N]glutamine, but 3-hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 m M ) was also associated with diminished production of [¹⁵N]aspartate and [2-¹⁵N]glutamine with [¹⁵N]glutamate as precursor. Studies with [2-¹⁵N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [¹⁵N]GABA to [2-¹⁵N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3-hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.     

Immunocytochemical localization of beta-methylcrotonyl-CoA carboxylase in astroglial cells and neurons in culture

Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.     

Glial Metabolism of Isoleucine

Isoleucine, together with leucine and valine, constitutes the group of branched-chain amino acids (BCAAs). BCAAs are transported from the blood into the brain parenchyma, where they can serve several distinct functions. Since brain tissue is known to oxidatively metabolize BCAAs to CO₂, they are considered as fuel material in brain energy metabolism. Also, in the case of leucine, cultured astrocytes have been reported to be able to completely oxidize BCAA. While the metabolism of leucine by astroglia-rich primary culture (APC) has already been studied in detail, the metabolic fates of isoleucine and valine in these cells remained to be identified. Therefore, in the present study an NMR analysis was performed of ¹³C-labelled metabolites generated in the catabolism of [U-¹³C]Ile by astrocytes and released by them into the incubation medium. APC potently removed isoleucine from the medium and metabolized it. The major isoleucine metabolites released from APC are 2-oxo-3-methylvalerate, 2-methylbutyrate, 3-hydroxy-2-methylbutyrate and propionate. To a lesser extent, APC generate and release also [2,3-¹³C]glutamine, [4,5-¹³C]glutamine and ¹³C-labelled isotopomers of lactate and citrate. These results show that APC can release into the extracellular milieu catabolites and several TCA cycle dependent metabolites resulting from the degradation of isoleucine. Special issue article in honor of Dr. George DeVries.     

Acetoacetate and Glucose as Lipid Precursors and Energy Substrates in Primary Cultures of Astrocytes and Neurons from Mouse Cerebral Cortex

Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3-14C]acetoacetate or [2-14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, D-3-hydroxybutyrate, and 14CO2, and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only approximately 20% in the case of cultured astrocytes. This indicates that the rate at which 14C-labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.     

Glial Metabolism of Valine

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the ¹³C-labelled metabolites that are generated by APC during catabolism of [U-¹³C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-¹³C]2-oxoisovalerate, [U-¹³C]3-hydroxyisobutyrate, [U-¹³C]2-methylmalonate, [U-¹³C]isobutyrate, and [U-¹³C]propionate as well as several TCA cycle-dependent metabolites including lactate. This article is dedicated to Dr. George DeVries.     

Obligate role for ketone body oxidation in neonatal metabolic homeostasis

To compensate for the energetic deficit elicited by reduced carbohydrate intake, mammals convert energy stored in ketone bodies to high energy phosphates. Ketone bodies provide fuel particularly to brain, heart, and skeletal muscle in states that include starvation, adherence to low carbohydrate diets, and the neonatal period. Here, we use novel Oxct1(-/-) mice, which lack the ketolytic enzyme succinyl-CoA:3-oxo-acid CoA-transferase (SCOT), to demonstrate that ketone body oxidation is required for postnatal survival in mice. Although Oxct1(-/-) mice exhibit normal prenatal development, all develop ketoacidosis, hypoglycemia, and reduced plasma lactate concentrations within the first 48 h of birth. In vivo oxidation of (13)C-labeled β-hydroxybutyrate in neonatal Oxct1(-/-) mice, measured using NMR, reveals intact oxidation to acetoacetate but no contribution of ketone bodies to the tricarboxylic acid cycle. Accumulation of acetoacetate yields a markedly reduced β-hydroxybutyrate:acetoacetate ratio of 1:3, compared with 3:1 in Oxct1(+) littermates. Frequent exogenous glucose administration to actively suckling Oxct1(-/-) mice delayed, but could not prevent, lethality. Brains of newborn SCOT-deficient mice demonstrate evidence of adaptive energy acquisition, with increased phosphorylation of AMP-activated protein kinase α, increased autophagy, and 2.4-fold increased in vivo oxidative metabolism of [(13)C]glucose. Furthermore, [(13)C]lactate oxidation is increased 1.7-fold in skeletal muscle of Oxct1(-/-) mice but not in brain. These results indicate the critical metabolic roles of ketone bodies in neonatal metabolism and suggest that distinct tissues exhibit specific metabolic responses to loss of ketone body oxidation.     

Cerebral Metabolic State During the Ethanol Withdrawal Reaction in the Rat

Abstract: A severe ethanol withdrawal reaction was induced in rats by means of repeated intragastric intubation during a 4-day period. At the peak of the withdrawal reaction cerebral cortical tissue was frozen in situ for analysis of glycogen, glucose, phosphocreatine, creatine, ATP, ADP, AMP, lactate, pyruvate, GAB A, β-hydroxybutyrate, acetoacetate, cAMP and cGMP. Blood glucose concentration was also measured. The level of brain glycogen was decreased during ethanol withdrawal. Brain glucose concentration was increased, probably secondary to the increase in blood glucose concentration. The calculated NADH/NAD⁺ ratio was slightly increased during the withdrawal and brain ATP concentration and adenine nucleotide pool size were decreased. The adenylate energy charge remained unchanged. The overall changes in the metabolites were in agreement with the previously shown metabolic activation during ethanol withdrawal. The brain concentrations of ketone bodies (β-hydroxybutyrate and acetoacetate) during withdrawal did not deviate from controls, indicating that no abnormal ketone metabolism had developed as a consequence of the long-lasting ethanol intoxication. No changes were observed in the concentrations of GABA, cAMP, or cGMP in the rat cerebral cortex during ethanol withdrawal.     

Metabolism of [U-<Superscript>13</Superscript>C]Leucine in Cultured Astroglial Cells

Leucine is rapidly metabolized in astroglial primary cultures. Therefore, it is considered as valuable fuel in brain energy metabolism. Only few of the leucine metabolites generated and released by astroglial cells have been identified. Therefore, a more detailed study was performed analyzing by NMR techniques the ¹³C-labeled metabolites, which were released by astroglial primary cultures during the degradation of [U-¹³C]leucine. Confirming a former radioactive study this analysis revealed ¹³C-labeled 2-oxoisocaproate and ketone bodies. Additionally, ¹³C-labeled alanine, citrate, glutamine, lactate and succinate were identified. Their detailed isotopomer analysis proves that ¹³C-labeled acetyl-CoA enters the tricarboxylic acid cycle, that intermediates with a characteristic ¹³C-labeling pattern can be withdrawn at several positions of the cycle, and that in the case of lactate and alanine there appears to be a participation of an active phosphoenolpyruvate carboxykinase and/or malic enzyme pathway. Thus, astroglial cells generate and release into the extracellular fluid not only the leucine catabolites 2-oxoisocaproate and ketone bodies, but also several tricarboxylic acid cycle dependent metabolites.Special issue dedicated to Dr. Lawrence F. Eng.M. Gabriele Bixel and Jörn Engelmann contributed equally to this work.     

Effect of hyperphenylalaninaemia on lipid synthesis from ketone bodies by rat brain.

The effect of hyperphenylalaninaemia on the metabolism of ketone bodies in vivo and in vitro by developing rat brain was investigated. The incorporation in vivo of [14C]acetoacetate into cerebral lipids was decreased by both chronic (for 3 days) and acute (for 6h) hyperphenylalaninaemia induced by injecting phenylalanine into 1-week-old rats. In studies in vitro it was observed that the incorporation of the radioactivity from [14C]acetoacetate and 3-hydroxy[14C]butyrate into cerebral lipids was inhibited by phenyl-pyruvate, but not by phenylalanine. Phenylpyruvate also inhibited the incorporation of 3H from 3H2O into lipids by brain slices metabolizing either 3-hydroxybutyrate or acetoacetate in the presence of glucose. These findings suggest that the decrease in the incorporation in vivo of [14C]acetoacetate into cerebral lipids in hyperphenylalaninaemic rats is most likely caused by phenylpyruvate and not by phenylalanine. Phenylpyruvate as well as phenylalanine had no inhibitory effects on ketone-body-catabolizing enzymes, namely 3-hydroxybutyrate dehydrogenase, 3-oxo acid CoA-transferase and acetoacetyl-CoA thiolase, in rat brain. Phenylpyruvate but not phenylalanine inhibited the activity of the 2-oxoglutarate dehydrogenase complex from rat and human brain. These findings suggest that the metabolism of ketone bodies is impaired in brains of untreated phenylketonuric patients, and in turn may contribute to the diminution of mental development and function associated with phenylketonuria.     

Ketosis (acetoacetate) can generate oxygen radicals and cause increased lipid peroxidation and growth inhibition in human endothelial cells

Elevated level of cellular lipid peroxidation can increase the incidence of vascular disease. The mechanism by which ketosis causes accelerated cellular damage and vascular disease in diabetes is not known. This study was undertaken to test the hypothesis that elevated levels of ketone bodies increase lipid peroxidation in endothelial cells. Human umbilical venous endothelial cells (HUVEC) were cultured for 24 h at 37^oC with ketone bodies (acetoacetate, β-hydroxybutyrate). Acetoacetate, but not β-hydroxybutyrate, caused an increase in lipid peroxidation and growth inhibition in cultured HUVEC. To determine whether ketone bodies generate oxygen radicals, studies using cell-free buffered solution were performed. They showed a significant superoxide dismutase (SOD) inhibitable reduction of cytochrome C by acetoacetate, but not by β-hydroxybutyrate, suggesting the generation of superoxide anion radicals by acetoacetate. Additional studies show that Fe²⁺ potentiates oxygen radical generation by acetoacetate. Thus, elevated levels of ketone body acetoacetate can generate oxygen radicals and cause lipid peroxidation in endothelial cells, providing a possible mechanism for the increased incidence of vascular disease in diabetes.     

Brain uptake and metabolism of ketone bodies in animal models

As a consequence of the high fat content of maternal milk, the brain metabolism of the suckling rat represents a model of naturally occurring ketosis. During the period of lactation, the rate of uptake and metabolism of the two ketone bodies, beta-hydroxybutyrate and acetoacetate is high. The ketone bodies enter the brain via monocarboxylate transporters whose expression and activity is much higher in the brain of the suckling than the mature rat. beta-Hydroxybutyrate and acetoacetate taken up by the brain are efficiently used as substrates for energy metabolism, and for amino acid and lipid biosynthesis, two pathways that are important for this period of active brain growth. Ketone bodies can represent about 30-70% of the total energy metabolism balance of the immature rat brain. The active metabolism of ketone bodies in the immature brain is related to the high activity of the enzymes of ketone body metabolism. Thus, the use of ketone bodies by the immature rodent brain serves to spare glucose for metabolic pathways that cannot be fulfilled by ketones such as the pentose phosphate pathway mainly. The latter pathway leads to the biosynthesis of ribose mandatory for DNA synthesis and NADPH which is not formed during ketone body metabolism and is a key cofactor in lipid biosynthesis. Finally, ketone bodies by serving mainly biosynthetic purposes spare glucose for the emergence of various functions such as audition, vision as well as more integrated and adapted behaviors whose appearance during brain maturation seems to critically relate upon active glucose supply and specific regional increased use.     

Factors affecting concentrations of acetoacetate and d-3-hydroxybutyrate in haemolymph and tissues of the adult desert locust

The ketone bodies acetoacetate and d-3-hydroxybutyrate are found in the haemolymph, the fat body, and the flight muscles of the adult desert locust. Acetoacetate is the major ketone body in the haemolymph and the flight muscles, but in the fat body d-3-hydroxybutyrate usually predominates. The concentration of acetoacetate in the haemolymph varies with age, and increases during starvation and flight and also after the injection of corpus cardiacum homogenate; it is little affected by stress and there are no differences between the sexes. Ketone bodies appear to be formed in the fat body and are oxidized by the fat body, the flight muscles, and the testes. All the tissues oxidize acetoacetate much more readily than d-3-hydroxybutyrate, and the flight muscles of fed locusts oxidize acetoacetate much more readily than the fat body or the testes. In starved locusts the ability of the fat body and the flight muscles to oxidize ketone bodies is greatly reduced, but utilization by the testes remains normal. Thus the flight muscles appear to be the major consumers of ketone bodies in fed locusts, and the testes the major consumers in starved locusts. It is suggested that ketone bodies are formed in the fat body during the mobilization of the triglyceride lipid reserves, and are either oxidized by the fat body or transported by the haemolymph to the flight muscles and other tissues to be used as a respiratory fuel.     

Ketone-body utilization by homogenates of adult rat brain

The regulation of ketone-body metabolism and the quantitative importance of ketone bodies as lipid precursors in adult rat brain has been studied in vitro. Utilization of ketone bodies and of pyruvate by homogenates of adult rat brain was measured and the distribution of¹⁴C from [3-¹⁴C]ketone bodies among the metabolic products was analysed. The rate of ketone-body utilization was maximal in the presence of added Krebs-cycle intermediates and uncouplers of oxidative phosphorylation. The consumption of acetoacetate was faster than that ofd-3-hydroxybutyrate, whereas, pyruvate produced twice as much acetyl-CoA as acetoacetate under optimal conditions. Millimolar concentrations of ATP in the presence of uncoupler lowered the consumption of ketone bodies but not of pyruvate. Indirect evidence is presented suggesting that ATP interferes specifically with the mitochondrial uptake of ketone bodies. Interconversion of ketone bodies and the accumulation of acid-soluble intermediates (mainly citrate and glutamate) accounted for the major part of ketone-body utilization, whereas only a small part was oxidized to CO₂. Ketone bodies were not incorporated into lipids or protein. We conclude that adult rat-brain homogenates use ketone bodies exclusively for oxidative purposes.     

Ketone body utilization in duodenum. Differential effect of fasting on lipogenesis from acetoacetate and 3-hydroxybutyrate

The effect of fasting and refeeding on oxidation, lipogenesis and amino acid synthesis from ketone bodies has been studied in neonatal chick duodenal mucosa. Oxidation and amino acid synthesis were higher from acetoacetate and were stimulated by fasting from both 3-hydroxybutyrate and acetoacetate. On the contrary, lipogenesis was always higher from 3-hydroxybutyrate and fasting reduced lipogenesis rate from acetoacetate (by 66%) but not from 3-hydroxybutyrate. Results suggests the existence of a cytosolic fast-dependent acetoacetyl-CoA synthetase in chick duodenal mucosa which is involved in phospholipid synthesis.     

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.     

Metabolism of [U-13C]Aspartate by Astroglial Cultures: Nuclear Magnetic Resonance Analysis of the Culture Media

In brain the amino acid L-aspartate serves roles as: (1) putative transmitter, (2) protein precursor, (3) donor of atoms for the biosynthesis of pyrimidine and purine bases, and (4) fuel for energy metabolism. Astrocytes dominate aspartate clearance in brain, and in culture they take up aspartate and quickly metabolize it. In brain, only astrocytes were shown to express the enzymes for de novo pyrimidine biosynthesis. To gain more details about the spectrum of metabolites generated from aspartate and subsequently released by cultured astrocytes a ¹³C-nuclear magnetic resonance analysis was performed of [U-¹³C]aspartate supplemented incubation media exposed to astroglial cultures. The results show that astrocytes readily metabolize aspartate and release into their culture media ¹³C-isotopomers of lactate, glutamine, citrate and alanine. Despite the presence in astroglial cells of two tandem enzymes of pyrimidine biosynthesis and their mRNAs, pyrimidine nucleotide-related heterocyclic compounds such as dihydroorotate and orotate could not be detected in the culture media.     

Ketone body synthesis from leucine by adipose tissue from different sites in the rat

Leucine is catabolized to ketone bodies in adipose tissue, but the contribution of this output to overall ketone metabolism is not known. The intent of the present study was to determine the capacity of different adipose tissues to synthesize ketone bodies from leucine. The amino acid was readily converted into acetoacetate in epididymal, perirenal, and omental fat tissues. In rats fed ad libitum, the rate of acetoacetate synthesis in omental fat (about 2 mumol g tissue-1h-1) was at least 8 times higher than in epididymal or perirenal fat. In omental fat, the rates of acetoacetate formation from alpha-ketoisocaproic acid were 47-55% lower than from leucine at all concentrations examined. There was no significant synthesis of beta-hydroxybutyrate from leucine or alpha-ketoisocaproic acid. After oxidative decarboxylation, a greater proportion (about three-fourths) of leucine in omental fat was metabolized to acetoacetate than to CO2 production through the Krebs cycle. Although addition of glucose, pyruvate, or carnitine did not affect the production of acetoacetate, fasting for 24 h stimulated acetoacetate synthesis from leucine and alpha-ketoisocaproic acid in omental fat. The high rate of leucine conversion to acetoacetate in omental fat was related to high activities of leucine aminotransferase and branched-chain alpha-keto acid dehydrogenase. Moreover, protein content and cytochrome c oxidase activity of omental mitochondria were, respectively, 13 and 12 times higher than in epididymal mitochondria. In contrast, fat content of epididymal adipose tissue was 21 times that of omental adipose tissue. Epididymal depot consisted of 2.0% protein and 75.8% fat, whereas omental depot contains 17.2% protein and 3.6% fat, resembling that of liver and muscle. The results suggest that the high ketogenic capacity of omental fat stems in part from an augmented mitochondrial mass and high activity of branched-chain alpha-keto acid dehydrogenase.     

Glucose, glutamine and ketone body utilisation by resting and concanavalin A activated rat splenic lymphocytes

The utilisation of glucose, glutamine, acetoacetate and D-3-hydroxybutyrate were investigated over 72 h of incubation of rat splenic lymphocytes, with and without concanavalin A. Lymphocytes consumed both ketone bodies; acetoacetate was consumed preferentially. The ketone bodies reduced glucose consumption by 30-50%, but had little effect on lactate production. Glutamine uptake was concentration dependent up to 4 mM, and consumption was increased in the presence of concanavalin. Glutamine stimulated glucose consumption and lactate production in both resting and activated cells. Complete oxidation contributed 65% of glucose-derived ATP, but less than 40% of glutamine-derived ATP. Glutamine metabolism makes only a minor contribution to lymphocyte ATP generation.     

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.