Inhibition Of Pyruvate Dehydrogenase Research Articles

Effect of As2O3 on gluconeogenesis.

1) The effect of As2O3 and As2O5 on gluconeogenesis from various substrates in the liver and kidney of rats was investigated. 2) A concentration-dependent inhibition by As2O3 was found. The effect was not dependent on the amount of investigated material (hepatocytes or kidney tubules). For either hepatocytes or kidney tubules the extent of inhibition depended strongly on the substrate used. The highest degree of inhibition was observed in incubations with pyruvate. The inhibition of glucose formation was accompanied to a lesser extent by a diminution in O2 consumption and ATP content. The effect was also dependent on the substrate used. Maximum effect was found in incubations with pyruvate. 3) Oleate, 0.5 mmol/l, increased gluconeogenesis from pyruvate. The effect was not abolished by As2O3. 4) A decrease in the content of acetyl-CoA, 3-hydroxybutyrate, and reduced glutathione was found in suspensions of isolated rat kidney tubules or hepatocytes incubated with As2O3. 5) About 10 times higher concentrations of As2O5 were necessary to induce a similar extent of inhibition of gluconeogenesis, decrease in O2 consumption, and in ATP content as compared with As2O3. The extent of the As2O5 effect depended on the concentration of the toxicant and on the substrate used. Gluconeogenesis from pyruvate exhibited the highest sensitivity to As2O5. 6) All findings can be largely explained by inhibition of pyruvate dehydrogenase as the central target for arsenicals. The subsequent depletion of acetyl CoA results in impaired formation of reducing equivalents in the citric acid cycle, decrease in high energy phosphates and, acetyl CoA being a strong positive modulator of pyruvate carboxylase, in gluconeogenesis inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

C4-Dicarboxylate Metabolism in Free-living and Bacteroid Forms of Rhizobium leguminosarum MNF3841

The transport and catabolism of C4-dicarboxylic acids have been further studied in Rhizobium leguminosarum MNF3841. Uptake of [14C]succinate was induced in free-living cells of strain MNF3841 within 12 min of exposure to succinate and reached a maximum rate within 60 min. Free-living cells of strain MNF3841 oxidizing fumarate accumulated pyruvate when treated with arsenite, an inhibitor of pyruvate dehydrogenase. Generation of pyruvate from C4-dicarboxylates was accomplished by malic enzyme. Although malic enzyme was present in free-living cells grown on sucrose, higher activities were observed when fumarate or l-arabinose was the growth substrate. In crude extracts, malic enzyme activity required either NAD+ or NADP+ as cofactor, together with Mn2+, and was stimulated by K+. Bacteroids from pea nodules transported [14C]succinate immediately after isolation, and contained twofold higher activities of both malic enzyme and pyruvate dehydrogenase than those found in fumarate-grown free-living cells. These data indicate that C4-dicarboxylates are available to nodule bacteroids and that they are catabolized through the tricarboxylic acid cycle using malic enzyme and pyruvate dehydrogenase to generate acetyl-CoA.

Interaction of gossypol with sperm macromolecules and enzymes

The interaction of gossypol with sperm macromolecules is an important process for the gossypol action. It is of our interests to study the nature of this interaction in order to understand the mechanism of action of gossypol. Our experimental results support the thesis that gossypol inhibits sperm motility by blocking ATP production and utilization. The drug acts on the mitochondria, suppressing oxygen consumption, inhibiting pyruvate dehydrogenase and ATPase activities and probably on the motility apparatus by blocking dyne in ATPase activity and preventing protein phosphorylation. The nature of the chemical bond in the interaction of gossypol and macromolecules is through the Schiff base formation and by the hydrophobic and nucleophilic attraction.

Metabolism of 1-aminoethylphosphinate generates acetylphosphinate, a potent inhibitor of pyruvate dehydrogenase.

The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate.

Pyruvate transport by thermogenic-tissue mitochondria.

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.

“Stable” effects of insulin and isoproterenol on adipocyte pyruvate dehydrogenase

Insulin, at a concentration of 1 mU/ml, stimulated glycogen synthase and pyruvate dehydrogenase about threefold in isolated rat adipocytes. Upon the removal of insulin, glycogen synthase activity remained in the activated state for 10 min and thereafter rapidly returned to basal level. On the other hand, insulin-stimulated pyruvate dehydrogenase activity remained elevated for at least 30 min. Isoproterenol (10 −8 m) stimulated phosphorylase and inhibited pyruvate dehydrogenase through the activation of β-adrenergic receptors. Addition of the β-antagonist, propranolol (10 −5 m), after isoproterenol reversed the action of isoproterenol on phosphorylase but not its action on pyruvate dehydrogenase. Dibutyryl cyclic AMP, when added to intact adipocytes, produced an effect on pyruvate dehydrogenase similar to that induced by isoproterenol. Our results indicate that both insulin and the β-agonist have a unique action on pyruvate dehydrogenase which is different from their effects on other enzymes such as glycogen synthase and phosphorylase.

Aerobic glycolysis: A study of human articular cartilage

Cartilage generally is one of those tissues that exhibit aerobic glycolysis. In a previous study on rat epiphyseal cartilage it had been suggested that this phenomenon is related to potentially excessive production of pyruvate and acetyl coenzyme A, the latter derived from fatty acid oxidation and inhibiting pyruvate dehydrogenase activity. The present study has shown that, in human articular cartilage, the contribution from fatty acid oxidation is too small to account for this phenomenon although the total potential production of pyruvate could still be in excess of the requirements for acetyl coenzyme A for the Krebs' cycle. Of greater relevance may be the apparent correlations that have been found between the activities of lactate and glyceraldehyde 3-phosphate dehydrogenases (r = 0 X 82: 0.01 greater than p greater than 0.001) and between those of lactate and glucose 6-phosphate dehydrogenases (r = 0.92; p less than 0.001).

Arsenite toxicity and arsenite tolerance in the cyanobacterium Synechococcus leopoliensis

Sodium arsenite at concentrations above 50 μM inhibited the growth of Synechococcus leopoliensis UTEX 625 in a defined culture medium. Inhibition was transitory, with growth resuming after a lag period the duration of which depended on the arsenite concentration. Cells grown for several hours in the presence of 10 μM arsenite became tolerant to concentrations of arsenite that inhibited the growth of untreated cells. Neither sensitive nor tolerant cells chemically modified the external arsenite detectably within the experimental period. At a concentration of 200 μM, arsenite temporarily halted growth of sensitive cells but did not affect that of tolerant cells. This concentration of arsenite inhibited net photosynthesis in both sensitive and tolerant cells. At the same time it selectively decreased the incorporation of carbon in the light into α-amino acids, especially glutamate, in sensitive but not in tolerant cells. Simultaneously, incorporation of carbon into pyruvic acid markedly increased. The activity of the partially purified pyruvate dehydrogenase complex of S. leopoliensis was abolished by 45 μM arsenite. It is concluded that inhibition of pyruvate dehydrogenase by arsenite is sufficient to explain its inhibition of growth in this organism.

The Mr-50 000 polypeptide of mammalian pyruvate dehydrogenase complex participates in the acetylation reactions.

The mammalian pyruvate dehydrogenase complex, Mr 8.5 X 10(6), contains an additional tightly bound 50 000-Mr polypeptide, component X, which copurifies with the intact assembly. Small amounts of the individual E2 and X polypeptides were obtained by elution of the protein bands from SDS/polyacrylamide gels. One-dimensional peptide mapping studies with 125I-labelled lipoyl acetyltransferase (E2) and component X subunits indicate that these two proteins are structurally distinct entities. Similar analysis of purified subunits, initially radiolabelled in the intact complex in the presence of [2-14C]pyruvate and N-ethyl-[2,3-14C]maleimide confirm that distinct 14C-labelled peptides are generated from these two species. These protein-chemical data supplement recent immunological findings, which demonstrate that component X is not a proteolytic fragment of the larger lipoyl acetyltransferase (Mr 70 000) subunit. Incubation of the native PDC in the presence of [2-14C]pyruvate leads to rapid uptake of radiolabel, presumably as acetyl groups, into both E2 and protein X. Specific incorporation of acetyl groups declines to a similar extent on both polypeptides after inhibiting pyruvate dehydrogenase (E1) activity by phosphorylation or omitting thiamine diphosphate (TPP) from the assay mixture. Addition of CoASH promotes the parallel deacetylation of both lipoyl acetyltransferase and protein X in a reaction which displays sensitivity to N-ethylmaleimide.

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia.

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.

Role of branched-chain 2-oxo acid dehydrogenase and pyruvate dehydrogenase in 2-oxobutyrate metabolism.

Purified branched-chain 2-oxo acid dehydrogenase (BCODH) and pyruvate dehydrogenase (PDH) had apparent Km values (microM) for 2-oxobutyrate of 26 and 114, with a relative Vmax. (% of Vmax. for 3-methyl-2-oxobutyrate and pyruvate) of 38 and 45% respectively. The phosphorylation state of both complexes in extracts of mitochondria from rat liver, kidney, heart and skeletal muscle was shown to influence oxidative decarboxylation of 2-oxobutyrate. Inhibitory antibodies to BCODH and an inhibitor of PDH (3-fluoropyruvate) were used with mitochondrial extracts to determine the relative contribution of both complexes to oxidative decarboxylation of 2-oxobutyrate. Calculated rates of 2-oxobutyrate decarboxylation in mitochondrial extracts, based on the kinetic constants given above and the activities of both complexes, were the same as the measured rates. Hydroxyapatite chromatography of extracts of mitochondria from rat liver revealed only two peaks of oxidative decarboxylation of 2-oxobutyrate, with one peak associated with PDH and the other with BCODH. Competition studies with various 2-oxo acids revealed a different inhibition pattern with mitochondrial extracts from liver compared with those from heart or skeletal muscle. We conclude that both intramitochondrial complexes are responsible for oxidative decarboxylation of 2-oxobutyrate. However, the BCODH is probably the more important complex, particularly in liver, on the basis of kinetic analyses, activity or phosphorylation state of both complexes, competition studies, and the apparent physiological concentration of pyruvate, 2-oxobutyrate and the branched-chain 2-oxo acids.

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase.

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.

Dexamethasone permits the release of an inhibitor of pyruvate dehydrogenase from Reuber H-35 hepatoma cells in response to insulin.

Reuber H-35 rat hepatoma cells respond to physiological levels of insulin as a growth factor. Glucocorticoids antagonize this response. A chemical mediator of insulin action which activates mitochondrial pyruvate dehydrogenase has also been isolated from these cells. The present report demonstrates that if the H35 cells are incubated with glucocorticoid before treatment with insulin, they produce not only the stimulator, but also inhibitory mediator. Cells exposed to the glucocorticoid but not to insulin do not produce the inhibitory mediator. Therefore, insulin interaction with the cell is necessary to elicit this negative modifier of pyruvate dehydrogenase. A time course of the response suggests that the effect of the glucocorticoid is time dependent. The inhibitory mediator can be separated from the stimulatory mediator by molecular sieve chromatography. These results suggest a biochemical basis for glucocorticoid-mediated insulin resistance.

Unidirectional actions of insulin and Ca2+-dependent hormones on adipocyte pyruvate dehydrogenase.

Norepinephrine and epinephrine, in the presence of the beta-adrenergic antagonist propranolol (10(-5) M), stimulated adipocyte pyruvate dehydrogenase at low concentrations but inhibited the enzyme at higher concentrations. The alpha-adrenergic agonist, phenylephrine, rapidly stimulated pyruvate dehydrogenase activity in a dose-dependent manner with maximal stimulation observed at 10(-6) M. The stimulation of pyruvate dehydrogenase by phenylephrine was mediated via alpha 1-receptors. Inhibition of pyruvate dehydrogenase by catecholamines was mediated via beta-adrenergic receptors, since the beta-agonist, isoproterenol, and dibutyryl cAMP produced similar effects. Like insulin, alpha-adrenergic agonists increased the active form of pyruvate dehydrogenase without changing the total enzyme activity and cellular ATP concentration. The effects induced by maximally effective concentrations of insulin and alpha-adrenergic agonists were nonadditive. The ability of phenylephrine and methoxamine to stimulate pyruvate dehydrogenase and phosphorylase and to inhibit glycogen synthase was not affected by the removal of extracellular Ca2+. Similarly, the stimulation of pyruvate dehydrogenase and glycogen synthase by insulin was also observed under the same conditions. However, when intracellular adipocyte Ca2+ was depleted by incubating cells in a Ca2+-free buffer containing 1 mM ethylene glycol bis(beta-amino-ethyl ether)-N,N,N' -tetraacetic acid, the actions of alpha-adrenergic agonists, but not insulin, on pyruvate dehydrogenase were completely abolished. Vasopressin and angiotensin II also stimulated pyruvate dehydrogenase in a dose-dependent manner with enhancement of glucose oxidation and lipogenesis. Our results demonstrate that the Ca2+ -dependent hormones stimulate pyruvate dehydrogenase and lipogenesis in isolated rat adipocytes, and the action is dependent upon intracellular, but not extracellular, Ca2+.

Inhibition of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase by glyoxylate

The overall reaction catalyzed by the pyruvate dehydrogenase complex from rat epididymal fat tissue is inhibited by glyoxylate at concentrations greater than 10 μ m. The inhibition is competitive with respect to pyruvate; K i was found to be 80 μ m. Qualitatively similar results were observed using pyruvate dehydrogenase from rat liver, kidney, and heart. Glyoxylate also inhibits the pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat, with the inhibition being readily detectable using 50 μ m glyoxylate. These effects of glyoxylate are largely reversed by millimolar concentrations of thiols (especially cysteine) because such compounds form relatively stable adducts with glyoxylate. Presumably these inhibitions by low levels of glyoxylate had not been previously observed, because others have used high concentrations of thiols in pyruvate dehydrogenase assays. Since the inhibitory effects are seen with suspected physiological concentrations, it seems likely that glyoxylate partially controls the activity of pyruvate dehydrogenase in vivo.

Pyruvate inhibition of pyruvate dehydrogenase kinase is a physiological variable

Pyruvate inhibited pyruvate dehydrogenase kinase activity in mitochondria from adipose tissue, heart, brain and kidney of fed rats. Starvation for 24 h led to increased kinase activity in mitochondria from adipose tissue and heart but not from brain or kidney and to reduction of pyruvate inhibition of the enzyme from adipose tissue, heart and brain. Insulin injection into starved animals rapidly restored pyruvate inhibition without alteration of kinase activity in adipose tissue and heart mitochondria. Induction of streptozotocin diabetes resulted in loss of pyruvate inhibition of the kinase in heart mitochondria at 48 h but not at 24 h whereas a significant increase of kinase activity was seen at 24 h. It is concluded that the mechanisms which control fluctuations of pyruvate sensitivity of the kinase are different from the mechanisms which control fluctuations of the uninhibited kinase activity.

Hepatopancreas mitochondria from Mytilus edulis:

A method for the isolation of intact coupled mitochondria from the hepatopancreas of Mytilus edulis has been developed. These mitochondria prefer lipid substrates and accept a range of fatty acid chain lengths. Acetyl carnitine is also oxidized at high rates. Evidence for the presence of the tricarboxylate and the dicarboxylate transporters is presented. The absence of the pyruvate transporter or inhibition of pyruvate dehydrogenase is indicated by the absence of pyruvate oxidation. Glutamate is oxidized at high rates with a pH optimum of 7.5 based on the RCR. Cysteine oxidation may indicate the presence of the enzymes involved in the synthesis of taurine. Ornithine transport is also indicated, implying the existence of the urea cycle. Alanine, glycine, proline, asparagine, arginine, aspartate and citrulline were not oxidized at detectable levels. The osmotic optimum for the oxidation of glutamate extends from 600 to 1000 mOsm l-1. The state 3 rate is more profoundly affected by the osmolarity than the state 4 rate. The role of osmolarity in the control of oxidation of amino acids during volume regulation of osmotically stressed M. edulis is suggested. Mitochondrial volume changes correlate with the optimal range of osmolarity for glutamate oxidation based on the RCR.

Pyruvate-Derived Amino Acids in Spinach Chloroplasts

A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K(m) for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.

Decreased insulin-generation of pyruvate dehydrogenase inhibitor in insulin resistant states

Insulin resistance produced in rats by feeding a high fat diet or by dexamethasone administration (50 ug/day, sc for 4 days) resulted in 50–70% decrease in the generation of pyruvate dehydrogenase inhibitor by insulin exposed liver particulate fractions. The inhibition was dose dependent. Treatment of insulin mediator preparations with neuraminidase and B-D-galactosidase resulted in inactivation of the pyruvate dehydrogenase inhibitor. Presence of exogenous enzyme substrates during enzyme digestion partially protected the inhibitor from inactivation. Protease treatment did not affect the inhibitor while the stimulatory activity of the insulin mediator was abolished by trypsin treatment. These results together with the previous report suggest that insulin resistance results in a decrease in the generation of both of the mediators of insulin action. This may result from a decrease in insulin binding, shown earlier, or from a decrease in precursor availability.

Metabolic effects of bacitracin in isolated rat hepatocytes.

Bacitracin is a proteolytic inhibitor which interacts with the intracellular processing of insulin. Its effects on pyruvate, fatty acid and amino acid metabolism were examined in rat hepatocyte suspensions. Bacitracin (0.25-1.0 mM) increased the oxidation of [1-14C]pyruvate by 50-70% and presumably therefore increased the flux through pyruvate dehydrogenase. This was found both in the presence of extracellular Ca2+ and in its absence, but not in the presence of 2 mM-2-chloropropionate, which inhibits pyruvate dehydrogenase kinase. Insulin did not further stimulate [1-14C]pyruvate oxidation in the presence of 1 mM-bacitracin. Bacitracin decreased 14CO2 formation from [2-14C]pyruvate (20-40%) and [U-14C]palmitate (30-70%), suggesting a decreased flux through the tricarboxylic acid cycle. Fatty acid oxidation before acetyl-CoA formation was also decreased. Bacitracin decreased the incorporation of label from [3H]leucine into protein in the absence of insulin, but not in its presence. Bacitracin is commonly used in studies on insulin action. Our results suggest that in such studies the effects noted may be related not only to an interaction of bacitracin with the intracellular processing of insulin but also to direct metabolic effects of bacitracin independent of insulin.