The effect of ketone bodies on alanine and glutamine metabolism in isolated skeletal muscle from the fasted chick.

The effects of ketone bodies on the metabolism of alanine and glutamine were studied in isolated extensor digitorum communis (EDC) muscles from 24 h-fasted chicks. (1) Acetoacetate and DL-beta-hydroxybutyrate (4 mM) markedly inhibit branched-chain amino acid (BCAA) transamination and alanine formation. (2) Ketone bodies (1 and 4 mM) increase the intracellular concentration and release of glutamate and glutamine, suggesting that inhibition of BCAA transamination does not limit intracellular availability of glutamate for alanine synthesis. (3) Ketone bodies (1 and 4 mM) do not affect glucose uptake by muscles, but decrease the rate of glycolysis as well as the intracellular concentration and release of pyruvate in muscles. (4) Addition of 12 mM-glucose increases the formation of alanine in muscles incubated in the absence of ketone bodies, but has no effect in muscles incubated in the presence of 4 mM ketone bodies. (5) Addition of 5 mM-pyruvate to the media prevents the inhibiting effect of ketone bodies on BCAA transamination and alanine synthesis. These results suggest that ketone bodies decrease alanine synthesis by limiting the intracellular availability of pyruvate, owing to inhibition of glycolysis, and inhibit BCAA transamination by decreasing the intracellular concentration of amino-group acceptors such as pyruvate in EDC muscles from fasted chicks.     

Mechanism responsible for the hypoglycemic actions of dichloroacetate and 2-chloropropionate

Dichloroacetate, an activator of the pyruvate dehydrogenase complex, is known to lower blood glucose, lactate, pyruvate, and alanine when given to diabetic and 24 h fasted rats. Under certain conditions, especially when pyruvate carboxylase is made rate limiting for want of bicarbonate, dichloroacetate effectively inhibits glucose synthesis from lactate by isolated hepatocytes. 2-Chloropropionate also activates the pyruvate dehydrogenase complex, lowers blood glucose, lactate, and pyruvate in 24 h fasted rats, but stimulates gluconeogenesis from lactate or alanine by isolated hepatocytes. Dichloroacetate is catabolized to glyoxylate and thence to oxalate by liver cells, whereas 2-chloropropionate cannot be catabolized to these products. Glyoxylate and oxalate are potent inhibitors of glucose synthesis from lactate, pyruvate, and alanine, but not from dihydroxyacetone. Inhibition is much more pronounced in a bicarbonate-deficient medium, in which pyruvate carboxylase is probably rate limiting for gluconeogenesis. It seems likely, therefore, that the inhibition of lactate gluconeogenesis by dichloroacetate is actually caused by oxalate, which inhibits pyruvate carboxylation. Nevertheless, the major effect of dichloroacetate, and probably the sole effect of 2-chloropropionate, on blood glucose concentration is to limit substrate availability in the blood for hepatic gluconeogenesis. Since oxalic acid stone formation and renal dysfunction may prove to be side effects of any therapeutic application of dichloroacetate, we suggest that further studies on the treatment of hyperglycemia and lactic acidosis with pyruvate dehydrogenase activators be carried out with 2-chloropropionate rather than dichloroacetate.     

Kinetics of the alanine aminotransferase reaction in the mitochondrial and cell sap fractions of rat brain.

1. A reversible transamination reaction between L-glutamate and pyruvate, or L-alanine and 2-oxoglutarate, takes place in the mitochondrial and cell sap fractions of rat brain. 2. The maximum rate of the transamination reaction in both subfractions was observed in the presence of a keto- substrate concentration of 2.5 mM only, but an amino- donor concentration of 20 mM. 3. The apparent Menten-Michaelis constants for pyruvate and 2-oxoglutarate were of a 10(-4) M and for L-glutamate and L-alanine of a 10(-3) M order and were approximately the same for both fractions. 4. The ratio of the initial rate of the L-alanine + 2-oxoglutarate to the L-glutamate + pyruvate transamination reaction in the cell sap and mitochondrial fractions amounted to up to 2. 5. The apparent equilibrium constant derived from the Haldane equation was 7.01 for cell sap alanine aminotransferase and 4 for the mitochondrial enzyme. 6. Increasing pyridoxal-5'-phosphate concentrations in the incubation medium were accompanied by only non-significant stimulation of alanine aminotransferase activity in the mitochondrial and cell sap fractions. 7. A comparison of the kinetic data obtained on mitochondrial and cell sap alanine aminotransferases in vitro with the actual substrate concentrations in the transamination reaction in nervous tissue in vivo indicates that the direction of the transamination reaction in situ seems to be determined simply by compartmentation and by dynamic changes in amino- and keto- substrates in the mitochondrial and cell sap spaces.     

Metabolic interactions of dichloroacetate and insulin in experimental diabetic ketoacidosis.

1. The infusion of sodium dichloroacetate into rats with severe diabetic ketoacidosis over 4h caused a 2mM decrease in blood glucose, and small falls in blood lactate and pyruvate concentrations. Similar findings had been reported in normal rats (Blackshear et al., 1974). In contrast there was a marked decrease in blood ketone-body concentration in the diabetic ketoacidotic rats after dichloroacetate treatment. 2. The infusion of insulin alone rapidly decreased blood glucose and ketone bodies, but caused an increase in blood lactate and pyruvate. 3. Dichloroacetate did not affect the response to insulin of blood glucose and ketone bodies, but abolished the increase of lactate and pyruvate seen after insulin infusion. 4. Neither insulin nor dichloroacetate stimulated glucose disappearance after functional hepatectomy, but both agents decreased the accumulation in blood of lactate, pyruvate and alanine. 5. Dichloroacetate inhibited 3-hydroxybutyrate uptake by the extra-splachnic tissues; insulin reversed this effect. Ketone-body production must have decreased, as hepatic ketone-body content was unchanged by dicholoracetate yet blood concentrations decreased. 6. It was concluded that: (a) dichloroacetate had qualitatively similar effects on glucose metabolism in severely ketotic rats to those observed in non-diabetic starved animals; (b) insulin and dichloroacetate both separately and together, decreased the net release of lactate, pyruvate and alanine from the extra-splachnic tissues, possibly through a similar mechanism; (c) insulin reversed the inhibition of 3-hydroxybutyrate uptake caused by dichloroacetate; (d) dichloroacetate inhibited ketone-body production in severe ketoacidosis.     

Factors regulating amino acid release from extrasplanchnic tissues in the rat. Interactions of alanine and glutamine.

1. Factors regulating the release of alanine and glutamine in vivo were investigated in starved rats by removing the liver from the circulation and monitoring blood metabolite changes for 30 min. 2. Alanine and glutamine were the predominant amino acids released into the circulation in this preparation. 3. Dichloroacetate, an activator of pyruvate dehydrogenase, inhibited net alanine release: it also interfered with the metabolism of the branched-chain amino acids valine, leucine and isoleucine. 4. L-Cycloserine, an inhibitor of alanine aminotransferase, decreased alanine accumulation by 80% after functional hepatectomy, whereas methionine sulphoximine, an inhibitor of glutamine synthetase, decreased glutamine accumulation by the same amount. 5. It was concluded that: (a) the alanine aminotransferase and the glutamine synthetase pathways respectively were responsible for 80% of the alanine and glutamine released into the circulation by the extrasplanchnic tissues, and extrahepatic proteolysis could account for a maximum of 20%; (b) alanine formation by the peripheral tissues was dependent on availability of pyruvate and not of glutamate; (c) glutamate availability could influence glutamine formation subject, possibly, to renal control.     

Stimulation of flux through hepatic pyruvate dehydrogenase by 3-mercaptopicolinate

Isolated hepatocytes from 24-h-starved rats were used to assess the possible effect of Ahe hypoglycaemic agent 3-mercaptopicolinate on flux through the hepatic pyruvate dehydrogenase complex. Increasing the extraceIIular pyruvate concentration from 1 mM to 2 mM or 5 mM resulted in an increase in flux through pyruvate dehydrogenase and the tricarboxylic acid cycle as measured by¹⁴CO₂ evolution from [1-¹⁴C]pyruvate and [3-¹⁴C]pyruvate. Gluconeogenesis was inhibited by 3-mercaptopicolinate from both 1 mM and 2 mM pyruvate, but significant increases in malate and citrate concentrations only occurred in cells incubated with 1 mM pyruvate. Flux through pyruvate dehydrogenase was stimulated by 3-mercaptopicolinate with 1 mM pyruvate but was unaltered with 2 mM pyruvate. Dichloroacetate stimulated flux through pyruvate dehydrogenase with no effect on gluconeogenesis in the presence of I mM pyruvate. There was no effect of 3-mercaptopicolinate, administered in vivo, to 24-h-starved rats on the activity of pyruvate dehydrogenase in freeze-clamped heart or liver tissue, although the drug did decrease blood glucose concentration and increase the blood concentrations of lactate and alanine. Dichloroacetate, administered in vivo to 24-h-starved rats, increased the activity of pyruvate dehydrogenase in freeze-clamped heart and liver, and caused decreases in the blood concentrations of glucose, lactate , and alanine. The results suggest that 3-mercaptopicolinate increases flux through hepatocyte pyruvate dehydrogenase by an indirect mechanism.     

13C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism

The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance 13C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The L-alanine level increases further upon incubation with the non-permissive substrate D-glucose. L-Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with D-glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on D-fructose and L-glutamate, alanine also accumulates within 3 h upon transfer to D-glucose.     

Observations on enzymes of ammonia assimilation in two different strains of Cyanidium caldarium.

Two strains of Cyanidium caldarium, one able to utilize nitrate as a substrate, and the other not, were tested for the presence of enzymes of ammonia assimilation. The nitrate-assimilating strain exhibits glutamate dehydrogenase activity. By contrast, the other strain lacks glutamate dehydrogenase; it possesses high alanine dehydrogenase and L-alanine aminotransferase activities which suggest that this strain may incorporate ammonia through reductive amination of pyruvate and may form glutamate from 2-ketoglutarate by a transamination reaction with alanine. Neither strain reveals glutamate synthase activity. Both strains contain similar levels of glutamine synthetase.     

Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids

1. Monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, 2-chloropropionate, 2,2'-dichloropropionate and 3-chloropropionate were inhibitors of pig heart pyruvate dehydrogenase kinase. Dichloroacetate was also shown to inhibit rat heart pyruvate dehydrogenase kinase. The inhibition was mainly non-competitive with respect to ATP. The concentration required for 50% inhibition was approx. 100mum for the three chloroacetates, difluoroacetate and 2-chloropropionate and 2,2'-dichloropropionate. Dichloroacetamide was not inhibitory. 2. Dichloroacetate had no significant effect on the activity of pyruvate dehydrogenase phosphate phosphatase when this was maximally activated by Ca(2+) and Mg(2+). 3. Dichloroacetate did not increase the catalytic activity of purified pig heart pyruvate dehydrogenase. 4. Dichloroacetate, difluoroacetate, 2-chloropropionate and 2,2'-dichloropropionate increased the proportion of the active (dephosphorylated) form of pyruvate dehydrogenase in rat heart mitochondria with 2-oxoglutarate and malate as respiratory substrates. Similar effects of dichloroacetate were shown with kidney and fat-cell mitochondria. Glyoxylate, monochloroacetate and dichloroacetamide were inactive. 5. Dichloroacetate increased the proportion of active pyruvate dehydrogenase in the perfused rat heart, isolated rat diaphragm and rat epididymal fat-pads. Difluoroacetate and dichloroacetamide were also active in the perfused heart, but glyoxylate, monochloroacetate and trichloroacetate were inactive. 6. Injection of dichloroacetate into rats starved overnight led within 60 min to activation of pyruvate dehydrogenase in extracts from heart, psoas muscle, adipose tissue, kidney and liver. The blood concentration of lactate fell within 15 min to reach a minimum after 60 min. The blood concentration of glucose fell after 90 min and reached a minimum after 120 min. There was no significant change in plasma glycerol concentration. 7. In epididymal fatpads dichloroacetate inhibited incorporation of (14)C from [U-(14)C]glucose, [U-(14)C]fructose and from [U-(14)C]lactate into CO(2) and glyceride fatty acid. 8. It is concluded that the inhibition of pyruvate dehydrogenase kinase by dichloroacetate may account for the activation of pyruvate dehydrogenase and pyruvate oxidation which it induces in isolated rat heart and diaphragm muscles, subject to certain assumptions as to the distribution of dichloroacetate across the plasma membrane and the mitochondrial membrane. 9. It is suggested that activation of pyruvate dehydrogenase by dichloroacetate could contribute to its hypoglycaemic effect by interruption of the Cori and alanine cycles. 10. It is suggested that the inhibitory effect of dichloroacetate on fatty acid synthesis in adipose tissue may involve an additional effect or effects of the compound.     

Characteristics of hepatic serine-pyruvate aminotransferase in different mammalian species.

1. Serine-pyruvate aminotransferase was purified from mouse, rat, dog and cat liver. Each enzyme preparation was homogeneous as judged by polyacrylamide-disc-gel electrophoresis in the presence of sodium dodecyl sulphate. However, isoelectric focusing resulted in the detection of two or more active forms from enzyme preparations from dog, cat and mouse. A single active form was obtained with the rat enzyme. All four enzyme preparations had similar pH optima and molecular weights. 2. Both mouse and rat preparations catalysed transamination between a number of L-amino acids (serine, leucine, asparagine, methionine, glutamine, ornithine, histidine, phenylalanine or tyrosine) and pyruvate. Effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine as amino donor. The reverse transamination activity, with hydroxypyruvate and alanine as subtrates, was lower than with serine and pyruvate for both species. Serine-pyruvate aminotransferase activities were inhibited by isonicotinic acid hydrazide. 3. In contrast, both dog and cat enzyme preparations were highly specific for serine as amino donor with pyruvate, and utilized pyruvate and glyoxylate as effective amino acceptors. A little activity was detected with phenylpyruvate. The reverse activity was higher than with serine and pyruvate for both species. Serine-pyruvate amino-transferase activities were not inhibited by isonicotinic acid hydrazide.     

Amino acid oxidation and alanine production in rat hemidiaphragm in vitro. Effects of dichloroacetate.

Dichloroacetate (an activator of pyruvate dehydrogenase) stimulates 14CO2 production from [U-14C]glucose, but not from [U-14C]glutamate, [U-14C]aspartate, [U-14C]- and [1-14C]-valine and [U-14C]- and [1-14C]-leucine. It is concluded (1) that pyruvate dehydrogenase is not rate-limiting in the oxidation to CO2 of amino acids that are metabolized to tricarboxylic acid-cycle intermediates, and (2) that carbohydrate (and not amino acids) is the main carbon precursor in alanine formation in muscle.     

Pyruvate metabolism by aminopterin-inhibited Aerobacter aerogenes

1. The synthesis and utilization of both alanine (by reductive amination, oxidative deamination and transamination) and valine (by transamination only) in Aerobacter aerogenes are unaffected by aminopterin. These amino acids, which accumulate in aminopterin-treated cultures of this organism, are therefore considered to be formed as secondary products from the excess of pyruvate that also accumulates. 2. Oxidative metabolism of pyruvate and the synthesis of acetylmethylcarbinol by A. aerogenes cells are unaltered by growth in the presence of aminopterin. 3. Cells from static and anaerobic cultures that have been treated with the folic acid antagonist in the early exponential phase have a decreased ability to cleave pyruvate to acetate and formate, and to effect the exchange of formate with the carboxyl group of pyruvate. 4. 3-Methyl-2-oxobutanoate, the keto acid precursor of valine, cannot replace pyruvate as substrate in either the phosphoroclastic or the exchange reaction.     

Inhibition of L-alanine aminotransferase by lithium salts.

Pharmacologic concentrations of either LiCl, LiN03 and LiF or the lithium salts of pyruvic or glutamic acids inhibit the formation of alanine arising from the transamination of glutamate in the presence of pyruvate. Lithium pyruvate is the most effective inhibitor, while the addition of K+ to the incubation reaction can effectively reduce this inhibition. Some possible modes of action of the lithium ion is presented.     

Stimulation of mitochondrial pyruvate transport in rat renal cortex by phenylephrine

Phenylephrine effect on liver and kidney cortex mitochondrial pyruvate concentration was investigated. While in liver the alpha 1-adrenergic agent produced a decrease in pyruvate content, a significant increase was observed in kidney, even in the presence of 0.5 mM alpha-cyano-4-hydroxy-cinnamate. These changes were not observed when pyruvate was formed by intramitochondrial transamination of alanine, suggesting a role for the pyruvate transport across mitochondrial membranes in the regulation of mitochondrial pyruvate metabolism in kidney cortex. This was corroborated measuring the phenylephrine effect on pyruvate carboxylation.     

C-NMR analysis of Aspergillus mutants disturbed in pyruvate metabolism

The metabolic consequences of two defects in pyruvate metabolism of the hyphal fungus Aspergillus nidulans have been investigated by natural abundance ¹³C-NMR spectroscopy. A pyruvate dehydrogenase complex (pdh) mutant, grown on acetate, accumulates alanine upon starvation which is derived from mannitol reserves. The -alanine level increases further upon incubation with the non-permissive substrate -glucose. -Glutamate is absent from these spectra as it is required both for the transamination of pyruvate and as a reaction on an impaired energy metabolism in such a pdh-deficient strain. A pyruvate carboxylase (pyc) mutant, grown upon acetate, only starts to accumulate alanine after a long incubation period with -glucose, due to the long-lasting presence of phosphoenolpyruvate carboxykinase and malic enzyme, which are both induced by growth on acetate. When this strain is grown on -fructose and -glutamate, alanine also accumulates within 3 h upon transfer to -glucose.     

Inactivation of pig heart alanine aminotransferase by beta-chloroalanine.

The substrate analog β-chloro-l-alanine rapidly inactivates the pig heart alanine aminotransferase. Inactivation is dependent upon formation of an intermediate. The substrate alanine protects the enzyme by competing with the inhibitor for the formation of this intermediate. Halide and carboxylate anions accelerated the rate of inactivation once the enzyme-inhibitor complex was formed. This acceleration appears to mimic the action with the substrate, for the rate of exchange transamination between unlabeled alanine and labeled pyruvate is similarly accelerated. When labeled inhibitor was used, the inactive enzyme became labeled. The spectral changes which occur resemble, in many respects, those which occur with aspartate aminotransferase when its active-site lysine undergoes alkylation by β-chloroalanine. We conclude that chloroalanine fulfills the criteria for a “suicide substrate.”     

Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia

Alanine dehydrogenase [L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of L-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0-11.5 for oxidative deamination of alanine. The apparent Km values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.     

Alternative oxidase in durum wheat mitochondria. Activation by pyruvate, hydroxypyruvate and glyoxylate and physiological role.

In order to gain a first insight into the alternative oxidase (AO) function in durum wheat mitochondria (DWM), we investigated some activation pathways of this enzyme in DWM purified from both etiolated shoots and green leaves. AO was activated when DWM were added with either pyruvate, known as an AO activator in other plant mitochondria, or alanine plus 2-oxoglutarate, which can generate intramitochondrial pyruvate and glutamate via transamination. In contrast, no AO activity was observed during oxidation of malate plus glutamate or succinate (which can generate malate). In this regard DWM differ from other plant mitochondria. Moreover, DWM were found: (i) to have a very low malic enzyme (ME) activity, (ii) to release oxaloacetate rather than pyruvate during malate oxidation and (iii) to poorly oxidise malate in the absence of glutamate, which removes oxaloacetate via transamination. Therefore, we show that, unlike other plant mitochondria, no pyruvate is generated inside DWM from malate via ME, allowing no AO activity. Other AO activators, alternative to pyruvate, were checked by evaluating the capability of several compounds to induce oxygen uptake and/or electrical membrane potential (Delta Psi) in cyanide-treated DWM. Hydroxypyruvate and glyoxylate, photorespiratory cycle intermediates, were found to be powerful AO activators, capable of inducing a maximal rate of cyanide-insensitive oxygen uptake 1.7 times and 2.3 times higher than pyruvate, respectively. These results suggest that in durum wheat a link may exist between AO activity and photorespiratory metabolism rather than malate metabolism. Moreover, we observed that AO activation resulted in both a partially coupled respiration and a reduction by half of the rate of superoxide anion generation; therefore, AO is expected to work as an antioxidative defence system when the photorespiratory cycle is highly active, as under environmental stress.     

Likely individuality of the enzymes catalyzing the phosphorylation of choline and ethanolamine.

Dichloroacetate has effects upon hepatic metabolism which are profoundly different from its effects on heart, skeletal muscle, and adipose tissue metabolism. With hepatocytes prepared from meal-fed rats, dichloroacetate was found to activate pyruvate dehydrogenase, to increase the utilization of lactate and pyruvate without effecting an increase in the net utilization of glucose, to increase the rate of fatty acid synthesis, and to decrease slightly [1-¹⁴C]oleate oxidation to ¹⁴CO₂ without decreasing ketone body formation. With hepatocytes isolated from 48-h-starved rats, dichloroacetate was found to activate pyruvate dehydrogenase, to have no influence on net glucose utilization, to inhibit gluconeogenesis slightly with lactate as substrate, and to stimulate gluconeogenesis significantly with alanine as substrate. The stimulation of fatty acid synthesis by dichloroacetate suggests that the activity of pyruvate dehydrogenase can be rate determining for fatty acid synthesis in isolated liver cells. The minor effects of dichloroacetate on gluconeogenesis suggest that the regulation of pyruvate dehydrogenase is only of marginal importance in the control of gluconeogenesis.     

A reexamination of the role of the cytosolic alanine aminotransferase in hepatic gluconeogenesis

The relative importance of the mitochondrial and cytosolic alanine aminotransferase isozymes for providing pyruvate from alanine for further metabolism in the mitochondrial compartment was examined in the isolated perfused rat liver. The experimental rationale employed depends upon the supposition that gluconeogenesis from alanine and the decarboxylation of infused [1-14C]alanine should be diminished by pyruvate transport inhibitors (e.g., alpha-cyanocinnamate) in proportion to the contribution of the cytosolic alanine aminotransferase for generating pyruvate. alpha-Cyanocinnamate inhibited the endogenous rate of glucose production in perfused livers derived from 24-h-fasted rats. The rate of [1-14C]alanine decarboxylation at low (1 mM) and high (10 mM) perfusate alanine concentrations was inhibited by 9.5 and 42%, respectively, in the presence of alpha-cyanocinnamate. In livers from fasted animals perfused with either 1 or 10 mM alanine, alpha-cyanocinnamate caused a substantial increase in the rates of both lactate and pyruvate production. Elevating the hepatic ketogenic rate during infusion of acetate in livers, perfused with alanine, stimulated both the rates of alanine decarboxylation and glucose production; the extent of stimulation of these two metabolic parameters was determined to be a function of the alanine concentration in the perfusate. The stimulation of the rate of alanine decarboxylation during acetate-induced ketogenesis was reversed by co-infusion of alpha-cyanocinnamate with simultaneous increases in the rates of lactate and pyruvate production. The results indicate that during rapid ketogenesis, cytosolic transamination of alanine contributes at least 19% (at 1 mM alanine) and 55% (at 10 mM alanine) of the pyruvate for gluconeogenesis.