Branched-chain Alpha-keto Acid Research Articles

Identification of specific subunits of highly purified bovine liver branched-chain ketoacid dehydrogenase.

Branched-chain alpha-ketoacid dehydrogenase has been purified to homogeneity from bovine liver mitochondria. The isolated complex has a specific activity of 5-8 mumol of reduced nicotinamide adenine dinucleotide min-1 (mg of protein)-1 as isolated and does not require the addition of exogenous lipoamide dehydrogenase for activity. Addition of porcine heart lipoamide dehydrogenase stimulated complex activity by no more than 20%. Four subunits copurify with the complex with molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 55 000, 52 000, 46 500, and 37 500. Here we show that the 52 000-dalton subunit is the lipoyl-containing transacylase component of the complex. Data are presented to support the hypothesis that the branched-chain ketoacid dehydrogenase complex is physically and catalytically similar to, but separate from, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The transacylase of the branched-chain ketoacid dehydrogenase complex has an exposed trypsin-sensitive region. Proteolytic action of trypsin separates a lipoyl-containing component from the remainder of the protein. Data from our laboratory presented here and elsewhere define a specific function for three of the four subunits.

Mechanism of activation of hepatic branched-chain alpha-ketoacid dehydrogenase by a muscle factor.

We recently reported that a skeletal muscle factor specifically activates the branched-chain alpha-ketoacid (BCKA) dehydrogenase in liver mitochondria (Paul, H. S., and Adibi, S. A. (1982) J. Biol. Chem. 257, 12581-12588). The evidence suggested that the muscle factor may be a phosphatase or may stimulate the phosphatase in liver mitochondria, thus converting the inactive BCKA dehydrogenase into an active form. In the present study we have investigated these possibilities. The muscle factor did not increase the activity of BCKA dehydrogenase which was previously activated in vitro by preincubation of mitochondria or by the addition of Triton or Ca2+ to the incubation medium. The muscle factor had little or no stimulatory effect on BCKA dehydrogenase when this enzyme was activated in vivo by treatment of rats with streptozotocin or clofibrate. The addition of NaF (an inhibitor of phosphatase) to the incubation medium entirely abolished the stimulatory effect of the muscle factor on BCKA dehydrogenase. Elimination of phosphatase by partial purification of BCKA dehydrogenase resulted in loss of stimulation of the dehydrogenase by the muscle factor. These results suggest that the muscle factor activates the BCKA dehydrogenase in liver mitochondria by stimulating its associated phosphatase.

Leucine catabolism during the differentiation of 3T3-L1 cells. Expression of a mitochondrial enzyme system.

Leucine can be utilized efficiently as a precursor for lipid biosynthesis by adipose tissue, especially in the presence of glucose or glucose and insulin. During the differentiation of 3T3-L1 fibroblasts to adipocytes, the rate of lipid biosynthesis from L-[U-14C]leucine increases at least 30-fold and lipogenesis, with [U-14C] acetate as the precursor, increases by 10- to 15-fold. The specific activities of two mitochondrial dehydrogenases in the leucine oxidative pathway, the branched chain alpha-ketoacid dehydrogenase and isovaleryl-CoA dehydrogenase, as well as of leucine:alpha-ketoglutarate transaminase, increase at least 20-fold during the adipose conversion. Isovaleryl-CoA dehydrogenase was assayed in crude extracts using a specific fluorimetric method employing electron transfer flavoprotein as the electron acceptor for the flavoprotein dehydrogenase. The specific activity of 3-hydroxy-3-methylglutaryl-CoA lyase, the mitochondrial enzyme catalyzing the terminal reaction in the leucine degradation pathway, increases 4-fold during differentiation. The increases in the specific activities of the mitochondrial enzymes occur without a change in the specific activity of cytochrome oxidase, indicating that the increases do not simply reflect proliferation of mitochondria. The biosynthesis of at least 20 soluble mitochondrial polypeptides is enhanced during the adipose conversion of the fibroblasts as determined by polyacrylamide gel electrophoresis following incubation of the cells with [35S] methionine. The results provide a conservative estimate of the extent of changes in mitochondrial soluble proteins during the adipose conversion. They also establish that differentiated 3T3-L1 adipocytes metabolize leucine like mature adipose tissue and illustrate the roles of the branched chain alpha-ketoacid dehydrogenase and isovaleryl-CoA dehydrogenase in lipogenesis.

Activity state of the branched chain α-ketoacid dehydrogenase complex in heart, liver, and kidney of normal, fasted, diabetic, and protein-starved rats

The proportion of active (unphosphorylated) branched chain alpha-ketoacid dehydrogenase was determined in tissues from rats in different metabolic states. Hearts from normal, high-protein, and low-protein fed rats contained about 45% of the enzyme in the active form. Only 10-20% of the enzyme was active in hearts of fasted and diabetic rats. Virtually all of the liver enzyme was in the active form in fed, fasted, diabetic and high-protein fed animals. Protein starved rats, however, exhibited a dramatic decrease in both the % active form and total amount of liver enzyme. Kidneys from normal, fasted, diabetic and high-protein fed rats contained 70-80% of the enzyme in the active form. The % active form of the kidney enzyme decreased in protein starved rats, but less dramatically than in liver. Covalent modification is concluded to be important for in vivo regulation of the branched chain alpha-ketoacid dehydrogenase complex.

Regulation of the branched chain alpha-keto acid and pyruvate dehydrogenases in the perfused rat heart.

Phosphorylation of the alpha-subunits of the branched chain alpha-keto acid and the pyruvate dehydrogenases was measured in mitochondria isolated from rat hearts perfused in the presence of [32P]phosphate and various substrates. Mitochondrial isolations were accomplished rapidly under conditions which would preclude the interconversion of these two enzyme complexes between their active (dephosphorylated) and inactive (phosphorylated) forms. Inactivation of the branched chain complex and the concomitant incorporation of [32P]phosphate into the alpha-chain of the dehydrogenase subcomponent were observed in hearts perfused with glucose (10 mM) or pyruvate (10 mM). alpha-Ketoisocaproate infusion caused a 15-fold activation and dephosphorylation of the branched chain dehydrogenase, while pyruvate dehydrogenase remained inactive and phosphorylated under these conditions. Both dehydrogenase components were partially activated, and most of the [32P]phosphate was removed from their respective alpha-subunits during substrate-free perfusion. Both enzyme complexes were activated and dephosphorylated completely during infusion of dichloroacetate (1 mM) and glucose (10 mM).

The effects of alpha-adrenergic agonists on the regulation of the branched chain alpha-ketoacid oxidation in the perfused rat liver.

The effects of alpha-adrenergic agonists on the regulation of the branched chain alpha-ketoacid oxidation in the perfused rat liver.

Inhibition of branched chain alpha-ketoacid dehydrogenase kinase activity by alpha-chloroisocaproate.

alpha-Chloroisocaproate has been shown to inhibit phosphorylation and inactivation of rabbit liver branched chain alpha-ketoacid dehydrogenase complex. Phosphorylation of pyruvate dehydrogenase was also inhibited by this alpha-ketoisocaproate analog, but phosphorylation of branched chain alpha-ketoacid dehydrogenase was much more sensitive than phosphorylation of pyruvate dehydrogenase (I50 values of 7.5 and 675 microM, respectively). Phosphorylation of glycogen synthase by six other protein kinases was not inhibited by alpha-chloroisocaproate (1 mM). Although less sensitive than phosphorylation of the complex, branched chain alpha-ketoacid dehydrogenase was also found inhibited by alpha-chloroisocaproate. The latter inhibition was competitive with respect to the alpha-ketoacid substrates of the dehydrogenase (Ki values of approximately 0.5 mM). alpha-Chloroisocaproate greatly stimulated the capacity of the perfused rat heart to decarboxylate [1-14C]leucine and promoted conversion of practically all (99%) of the complex into the active form.

Isolation of rabbit liver branched chain alpha-ketoacid dehydrogenase and regulation by phosphorylation.

Isolation of rabbit liver branched chain alpha-ketoacid dehydrogenase and regulation by phosphorylation.

Activation of hepatic branched chain alpha-keto acid dehydrogenase by a skeletal muscle factor.

These experiments were designed to determine whether there are tissue factors which affect the activity of branched chain alpha-keto acid (BCKA) dehydrogenase in the mitochondria of the same or different tissues. The activity of BCKA dehydrogenase was increased by the addition of the cytoplasmic fraction of the gastro-cnemius muscle to liver mitochondria. The specificity of the effect of the muscle factor was established by the fact that it only increased the BCKA dehydrogenase activity in liver mitochondria. The activity of this enzyme in mitochondria from skeletal muscle, heart, kidney, or brain was either not affected or inhibited by the muscle factor. Muscle factor also increased the activity of alpha-ketoglutarate dehydrogenase but decreased that of pyruvate dehydrogenase in liver mitochondria. This factor was found in a variety of skeletal muscles but not in smooth muscle such as uterus. A factor similar to that of muscle was not found in liver or kidney cytoplasm but was detected in plasma. There were similarities between the effects of muscle and plasma factors on the BCKA dehydrogenase activity in liver, kidney, heart, and skeletal muscle mitochondria. Studies concerning the properties and partial characterization of this factor revealed that it (a) is nondialyzable, (b) is a protein, (c) is heat labile, and (d) increases the activity of BCKA dehydrogenase by increasing the Vmax without a change in the apparent Km.

Evidence for the regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex by a phosphorylation/dephosphorylation mechanism.

The regulation of the branched chain alpha-keto acid dehydrogenase complex by covalent modification was investigated in rat liver mitochondria. Depletion of intramitochondrial calcium and magnesium caused an inactivation of the branched chain alpha-keto acid dehydrogenase complex. Following inactivation of the branched chain complex, addition of calcium or magnesium ions separately to incubations of mitochondria only partially reactivated the enzyme complex. However, simultaneous addition of calcium and magnesium activated the branched chain enzyme complex rapidly and nearly completely. Mitochondrial incubations were performed in the presence of [32P]phosphate under conditions known to activate or to inactivate the branched chain alpha-keto acid dehydrogenase complex. Evidence demonstrating that [32P]-phosphate was incorporated into two major protein bands separated in sodium dodecyl sulfate-polyacrylamide gels of the mitochondrial incubations is presented. Migration of the labeled mitochondrial protein bands in the gel system corresponded exactly to the migration of the alpha subunit of the purified heart-derived pyruvate dehydrogenase (decarboxylase, E1) and the alpha subunit of the purified kidney-derived branched chain alpha-keto acid dehydrogenase (decarboxylase, E1). Furthermore, when the measured activity of the branched chain complex was minimized, the amount of [32P]phosphate incorporated into the alpha chain of the branched chain enzyme was maximal. Conversely, incubation conditions which activated maximally the enzyme complex minimized the [32P]phosphate incorporation into the alpha subunit of the branched chain dehydrogenase.

Regulation of the branched chain alpha-ketoacid pathway in liver.

Regulation of the branched chain alpha-ketoacid pathway in liver.

Thiamin-responsive maple-syrup-urine disease: decreased affinity of the mutant branched-chain alpha-keto acid dehydrogenase for alpha-ketoisovalerate and thiamin pyrophosphate.

The biochemical basis for the therapeutic effects of thiamin in thiamin-responsive maple-syrup-urine disease (MSUD) was investigated in intact and disrupted fibroblast cultures from normals and patients with various forms of MSUD. Decarboxylation of alpha-keto[1-14C]isovalerate (KIV) by intact cells from a thiamin-responsive MSUD patient was at 30-40% of the normal rate with or without thiamin in the incubation medium. Under similar conditions, intact classical MSUD fibroblasts failed to decarboxylate KIV. Branched-chain alpha-keto acid (BCKA) dehydrogenase activity measured in disrupted cells from the thiamin-responsive subject showed sigmoidal kinetics in the absence of thiamin pyrophosphate (TPP), with an increased concentration of substrate needed for half-maximal velocity (K0.5 for KIV = 7 mM vs. 0.05 mM in normal cells). When assayed with 0.2 mM TPP present, the mutant enzyme showed (i) a shift in kinetics to near Michaelis-Menten type as observed with the normal BCKA dehydrogenase and (ii) a lower K0.5 value of 4 mM for KIV, suggesting a TPP-mediated increase in the mutant enzyme's affinity for substrate. By contrast, TPP increased only the Vmax and was without effect on the apparent Km for KIV of the BCKA dehydrogenase from cells of normals and patients with classical MSUD and variant thiamin-responsive MSUD (grade 3). Measurement of the apparent Km for TPP of the BCKA dehydrogenase from thiamin-responsive mutant MSUd cells showed a 16-fold increase in the constant to 25 microM compared to enzymes from normal or classical MSUD cells. These findings demonstrate that the primary defect in the thiamin-responsive MSUD patient is a reduced affinity of the mutant BCKA dehydrogenase for TPP that results in impaired oxidative decarboxylation of BCKA.

Role of ATP in the regulation of branched-chain alpha-keto acid dehydrogenase activity in liver and muscle mitochondria of fed, fasted, and diabetic rats.

The activity of branched-chain alpha-keto acid (BCKA) dehydrogenase was increased after preincubation of liver and muscle mitochondria of control rats. Preincubation depleted mitochondrial ATP. Addition of ATP prevented the activation of BCKA dehydrogenase as well as reversed the activity of a fully activated enzyme to normal. Inhibition of phosphatase blocked the activation of BCKA dehydrogenase. There was a small or no increase in BCKA dehydrogenase activity when mitochondria from tissues of fasted, diabetic, and clofibrate-treated rats were preincubated. In fasted and diabetic rats, ATP was either less effective or failed to reverse the increased dehydrogenase activity in preincubated mitochondria. The concentration of ATP in liver and muscle mitochondria of diabetic rats was approximately one-half that of the control rats. We conclude that (a) in the fed state approximately 30-40% of BCKA dehydrogenase exists in the active form. The enzyme can be fully activated by preincubation of mitochondria which causes the depletion of ATP. Phosphatase is necessary for this activation. (b) In fasted, diabetic, and clofibrate-treated rats, approximately 70-100% of the enzyme exists in the active form which may be related to the mitochondrial depletion of ATP in vivo, and (c) while ATP can reverse the activation in control rats, it fails to do so in diabetic rats suggesting that other metabolic alterations may be involved in the regulation of BCKA dehydrogenase in diabetes.

Studies on the regulation of the branched chain alpha-keto acid dehydrogenase in the perfused rat liver.

The regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex was investigated in the isolated, perfused rat liver. The metabolic flux through the branched chain alpha-keto acid dehydrogenase was monitored by measuring the production of 14CO2 from infused 1-14C-labeled branched chain alpha-keto acid substrates. The rate of decarboxylation of alpha-keto[1-14C]isocaproate exceeded that of alpha-keto[1-14C]isovalerate at all concentrations of the substrates infused. Coinfusion of either alpha-ketoisovalerate or alpha-keto-beta-methylvalerate inhibited the rate of alpha-keto[1-14C]isocaproate decarboxylation. The rate of alpha-keto[1-14C]isovalerate decarboxylation ws enhanced during coinfusion of L(--)carnitine, while alpha-keto[1-14C]isocaproate decarboxylation was unaffected. The presence of pyruvate in the perfusion medium resulted in an inhibition of the flux through the branched chain complex with either alpha-ketoisocaproate or alpha-ketoisovalerate as the substrate. DL-beta-hydroxybutyrate infusion inhibited alpha-keto[1-14C]isocaproate decarboxylation by 18% but resulted in nearly a 100% stimulation of alpha-keto[1-14C]isovalerate decarboxylation. The evidence presented indicates that (alpha) the metabolic flux through the branched chain alpha-keto acid dehydrogenase complex can be monitored effectively in a continuous fashion in the perfused liver by following the release of 14CO2 from infused 1-14C-labeled substrates and (b) the changes observed in the metabolic flux through the branched chain complex during coinfusion of alternative substrates and other compounds may be entirely different depending upon which branched chain alpha-keto acid substrate is utilized to monitor this reaction.

L-Leucine activates branched chain alpha-keto acid dehydrogenase in rat adipose tissue.

The activity of branched chain alpha-keto acid dehydrogenase in extracts of adipose tissue was elevated after homogenization of tissue segments which had been incubated in buffer containing 0.3 mM leucine. A maximum increase (4-fold) was observed in extracts of tissues incubated in buffer containing 2.5 mM leucine, alpha-Ketoisocaproate and leucine caused maximum increases which were of similar magnitude and which required the same length of incubation of the tissue segments (5 to 15 min). The effect of leucine on branched chain alpha-keto acid dehydrogenase activity was observed both in the presence and absence of insulin, which also increased the activity of the enzyme in tissue extracts. Intact adipose tissue segments oxidized [I-14C]leucine at a maximum rate approximately 4 times that of [1-(14)C]valine. The rate of valine oxidation by intact tissue segments was doubled by addition of 0.2 to 0.5 mM unlabeled leucine, but not isoleucine, to medium containing 2 mM [1-(14)C]valine. Leucine, but not valine, also stimulated the rate of oxidation of 2 mM [U-14C]isoleucine by intact tissue segments. These results suggest that branched chain alpha-keto acid dehydrogenase activity, which is thought to limit the rate of branched chain amino acid oxidation in adipose tissue, may be sensitive to changes in the concentration of leucine in rat blood.

Blood and tissue branched-chain amino and α-keto acid concentrations: effect of diet, starvation, and disease

Branched-chain alpha-keto and amino acid (BCKA, BCAA) concentrations were measured in blood, plasma, and tissues of rats fed low protein (8% casein) or high protein (60% casein) diets; and in rats fed a stock diet and subjected to 3 days of starvation of chemically-induced diabetes. Concentrations of these amino and ketoacids were also measured in blood from patients with maple syrup urine disease. Valine, isoleucine, and leucine concentrations in blood from rats fed the stock diet were 124 +/- 7, 58 +/- 4 and 99 +/- 5 microM, respectively. Blood BCAA concentrations of rats fed the high protein diet and diabetic rats were elevated 2- to 3-fold; small increases were observed in blood from starved rats. Changes in blood BCAA concentrations paralleled those in tissues, except in starved rats in which the skeletal muscle free BCAA pool increased proportionately more than the circulating pool. Mean blood BCKA concentrations of rats fed the stock diet were low--7.9 +/- 0.5, 7.1 +/- 0.4 and 12.4 +/- 0.7 microM for alpha-ketoisovaleric, alpha-keto-beta-methylvaleric, and alpha ketoisocaproic acids, respectively. All treatments resulted in increases in blood BCKA concentrations of from 1.4 to 2 fold. In liver and heart, concentrations of BCKA, except for that of alpha-ketoisocaproic acid were near the limits of detection (less than 1 nmole/g). There was significant accumulation of all three BCKA in skeletal muscle which was estimated to contain about 80% of the measured body free BCKA pool. Blood BCKA are well regulated. Only in patients with maple syrup urine disease are plasma concentrations of BCKA useful indicators of altered tissue BCAA metabolism. Skeletal muscle, where oxidation of the BCKA is limited by low BCKA dehydrogenase activity, would seem to be the major source of circulating BCKA.

Studies on the activation and inactivation of the branched chain alpha-keto acid dehydrogenase in the perfused rat heart.

Evidence for a reversible process resulting in stable activated and inactivated states of the mitochondrial branched chain alpha-keto acid dehydrogenase complex in isolated perfused rat heart is presented. The inactivation process is mediated by pyruvate infusion, while activation (up to 18-fold) is facilitated by branched chain alpha-keto acid substrates. The low activity state of the branched chain complex characteristic of freshly excised rat hearts could be maintained by infusion of either pyruvate or glucose. Activation of the complex in the perfused rat heart was achieved slowly by substrate-free perfusion, while rapid activation was accomplished by infusion of branched chain alpha-keto acids. The fully activated enzyme complex resulting from branched chain alpha-keto acid infusion subsequently could be inactivated maximally by infusion of pyruvate alone or intermediate degrees of inactivation could be produced by certain ratios of co-infused pyruvate and branched chain alpha-keto acid. alpha-Ketoisocaproate was an order of magnitude more effective than alpha-keto isovalerate either in preventing inactivation or in stimulating the opposing activation process when co-infused with pyruvate. The mitochondrial pyruvate transport inhibitor, alpha-cyanocinnamate, effectively prevented inactivation of the complex by infused pyruvate. Differential changes in the activation states of the branched chain alpha-keto acid dehydrogenase and pyruvate dehydrogenase complexes were evident when the two complexes were compared in apparently similar flux-inhibited (via octanoate infusion) and flux-stimulated (via dichloroacetate infusion) metabolic conditions. The differential effect of pyruvate concentration on the activity states of the two complexes was also well-defined. The results of the present study suggest distinct systems for the regulation of the activity of the two multienzyme complexes of interest. While our results argue neither for nor against an inactivation of the branched chain alpha-keto acid dehydrogenase complex by a protein kinase, the regulatory properties of such an intramitochondrial protein kinase may not be similar to the pyruvate dehydrogenase kinase. The mechanistic nature of the suggested novel regulatory system concerned with the pyruvate-mediated inactivation of the branched chain alpha-keto acid activation cannot be inferred at the present time.

Biological Activity of 1,N6-Ethenothiamin, a Fluorescent Analog of Thiamin, in Rats

The ability of the recently synthesized fluorescent analog of thiamin, 1,N6-ethenothiamin, to substitute for thiamin in rats in vivo has been studied. 50 micrograms of ethenothiamin/100 g body weight/day injected in rats allowed about one-half the weight gain with 50 micrograms/100g/day of thiamin. The activities of the pyruvate and alpha-keto-glutarate dehydrogenases of liver mitochondria were partially restored by 50 micrograms/100g/day of ethenothiamin. Ethenothiamin at this level did not appear to restore the branched-chain alpha-keto acid dehydrogenase activities to any extent. When 150 micrograms of ethenothiamin/100g body weight/day was injected, growth was optimum, but these enzyme activities still did not achieve normal levels.

Insulin regulation of branched chain alpha-keto acid dehydrogenase in adipose tissue.

The enzyme which oxidizes alpha-keto[1-14C]isocaproate to 14CO2 is activated by incubation of adipose tissue segments with insulin. A 3-fold reduction in the apparent Km of the enzyme for alpha-ketoisocaproate was observed when homogenates of adipose tissue segments treated with insulin were compared to homogenates of control tissues. The enzyme was assayed at various times after homogenization of adipose tissue segments. Relatively small changes were observed in the activity from control or insulin-treated tissues for 30 min after homogenization. The persistence of the insulin effect after homogenization suggests that insulin may cause a covalent modification of the enzyme. The possibility that alpha-ketoisocaproate is oxidized by pyruvate dehydrogenase, which is also stimulated by insulin, is unlikely since the enzyme responsible for oxidation of 14C-labeled branched chain alpha-keto acids can be inactivated by heat at a rate distinct from that of pyruvate dehydrogenase. Moreover, unlabeled branched chain alpha-keto acids inhibit the oxidation of alpha-keto[1-14C]isocaproate but not that of [1-14C]pyruvate. Branched chain alpha-keto acid hydrogenase can be activated by incubation of adipose tissue homogenates in the presence of magnesium chloride and in the absence of ATP. The addition of ATP plus an ATP-regenerating system reverses the activation of the enzyme. The apparent Km of the enzyme is reduced and the Vmax is increased by incubation of tissue extracts under appropriate conditions.

Effects of branched chain alpha-ketoacids on the metabolism of isolated rat liver cells. I. Regulation of branched chain alpha-ketoacid metabolism.

alpha-Ketoisocaproate (ketoleucine) is shown to be metabolized to ketone bodies rapidly by isolated rat liver cells. Acetoacetate is the major end product and maximum rates were observed with 2 mM substrate. Studies with 2-tetradecylglycidic acid (an inhibitor of long chain fatty acid oxidation) showed that ketogenesis from alpha-ketoisocaproate and from endogenous fatty acids were additive. With alpha-ketoisocaproate present as soole substrate at 2 mM, leucine production was less than 10% of alpha-ketoisocaproate uptake and only 30% of the acetyl coenzyme A generated was oxidized in the citric acid cycle. Metabolism of alpha-ketoisocaproate was inhibited by fatty acids, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and pyruvate. Oxidation of acetyl-CoA generated from alpha-ketoisocaproate was suppressed by oleate and by pyruvate, but was enhanced by lactate. Metabolism between the different branched chain alpha-ketoacids was mutually competitive. When alpha-ketoisocaproate (2 mM) was added in the presence of high pyruvate concentrations (4.4 mM), flux through pyruvate dehydrogenase was decreased, and the proportion of total pyruvate dehydrogenase in the active form (PDHa) also fell. With lactate as substrate, PDHa was only 25% of total activity and was little affected by addition of alpha-ketoisocaproate. These data suggest that enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis. However, acetyl-CoA generation from pyruvate is apparently insufficiently inhibited by alpha-ketoisocaproate to cause a diversion of acetyl-CoA formed during alpha-ketoisocaproate metabolism from ketone body formation to oxidation in the citric acid cycle. Measurements of the cell contents of CoASH, acetyl-CoA, acid-soluble acyl-CoA, and acid-insoluble fatty acyl-CoA indicated that when the branched chain alpha-ketoacids were added as sole substrate, their oxidation was limited at a step distal to the branched chain alpha-ketoacid dehydrogenase. Acid-soluble acyl-CoA derivatives were depleted after oleate addition in the presence of alpha-ketoisocaproate, suggesting an inhibition of the branched chain alpha-ketoacid dehydrogenase by the elevation of the mitochondrial NADH/NAD+ ratio observed during fatty acid oxidation. This effect was not observed in the presence of oleate and 2-tetradecylglycidic acid.