The effect of acetate on the regulation of the branched chain α-keto acid and the pyruvate dehydrogenase complexes in the perfused rat liver

The regulatory consequences of acetate infusion on the pyruvate and the branched chain α-keto acid dehydrogenase reactions in the isolated, perfused rat liver were investigated. Metabolic flux through these two decarboxylation reactions was monitored by measuring the rate of ¹⁴CO₂ production from infused 1-¹⁴C-labeled substrates. When acetate was presented to the liver as the sole substrate the rate of ketogenesis which resulted was maximal at concentrations of acetate in excess of 10 mm. The increase in hepatic ketogenesis during acetate infusion was not accompanied by an alteration of the mitochondrial oxidation-reduction state as measured by the ratio of β-hydroxybutyrate/ acetoacetate in the effluent perfusate. While acetate infusion did not affect the rate of α-keto[1-¹⁴C]isocaproate decarboxylation, the rate of α-keto[1-¹⁴C]isovalerate decarboxylation was stimulated appreciably upon acetate addition. No change was observed in the amount of extractable branched chain α-keto acid dehydrogenase during acetate infusion. The rate of [1-¹⁴C]pyruvate decarboxylation was stimulated in the presence of acetate at low (<1 mm) but not at high (>1 mm) perfusate pyruvate concentrations. The stimulation of the metabolic flux through the pyruvate dehydrogenase reaction upon acetate infusion was accompanied by an increase in the activation state of the pyruvate dehydrogenase complex from 25.7 to 35.6% in the active form. In a liver perfused in the presence of the pyruvate dehydrogenase kinase inhibitor, dichloroacetate, at a low concentration of pyruvate (0.05 mm) the infusion of acetate did not affect the rate of pyruvate decarboxylation. As the rate of mitochondrial acetoacetate efflux is increased during acetate infusion the stimulation of pyruvate and α-ketoisovalerate decarboxylation is attributed to an accelerated rate of exchange of mitochondrial acetoacetate for cytosolic pyruvate or α-ketoisovalerate on the monocarboxylate transporter.     

Reversal of cerulenin-induced inhibition of phospholipids and sterol synthesis by exogenous fatty acids/sterols in Epidermophyton floccosum

Cerulenin, a specific inhibitor of fatty acids and sterol biosynthesis inhibited the growth of Epidermophyton floccosum, which was reversed when growth medium was supplemented with palmitic acid and sterols. Unsaturated fatty acids partially restored the growth. Cerulenin inhibited both phospholipid and sterol biosynthesis (60-70%) at the minimum inhibitory concentration (0.5 microgram/ml) as demonstrated by [32P]orthophosphoric acid and [14C]acetate incorporation into the respective lipids. Cerulenin-induced inhibition of phospholipid and sterol synthesis was dose dependent up to 0.5 microgram/ml. Exogenously supplied fatty acids and sterols restored the biosynthesis of phospholipids in cerulenin-treated cultures, while that of sterols was enhanced. The biosynthesis of both saturated and unsaturated fatty acids was inhibited by cerulenin.     

Formation of Hydrogen and Formate by Ruminococcus albus

Radioisotopic growth studies with specifically labeled (14)C-glucose confirmed that Ruminococcus albus, strain 7, ferments glucose mainly by the Embden-Myerhof-Parnas pathway to acetate, ethanol, formate, CO(2), H(2), and an unidentified product. Cell suspensions and extracts converted pyruvate to acetate, H(2), CO(2), and a small amount of ethanol. Formate was not produced from pyruvate and was not degraded to H(2) and CO(2), indicating that formate was not an intermediate in the production of H(2) and CO(2) from pyruvate. Cell extract and (14)C-glucose growth studies showed that the H(2)-producing pyruvate lyase reaction is the major route of H(2) and CO(2) production. An active pyruvate-(14)CO(2) exchange reaction was demonstrable with cell extracts. The (14)C-glucose growth studies indicated that formate, as well as CO(2), arises from the 3 and 4 carbon positions of glucose. A formate-producing pyruvate lyase system was not demonstrable either by pyruvate-(14)C-formate exchange or by net formate formation from pyruvate. Growth studies with unlabeled glucose and labeled (14)CO(2) or (14)C-formate suggest that formate arises from the 3 and 4 carbon positions of glucose by an irreversible reduction of CO(2). The results of the studies on the time course of formate production showed that formate production is a late function of growth, and the rate of production, as well as the total amount produced, increases as the glucose concentration available to the organism increases.     

Metabolic changes in Saccharomyces cerevisiae strains lacking citrate synthases

The yeast, Saccharomyces cerevisiae, contains two citrate synthase isoenzymes, mitochondrial (CS1) and cytosolic (CS2). In this study, we have examined the metabolic consequences of the absence of CS1, CS2, and both isoenzymes in the respective mutant strains CS1-, CS2-, and CS1-CS2-. No significant differences were found in the growth rates of the parental, CS1-, or CS2- strains when grown in the single carbon sources galactose, glycerol, lactate, pyruvate, or glutamate. However, in nonfermentable carbon sources, the lag period in growth of CS1- was approximately 4 times that of the parental strain and the CS2- mutant. This difference was found even in glutamate. The CS1- mutant failed to grow on acetate in either complete or minimal liquid medium. Total cellular citrate concentration in the CS1- compared to the parental strain was higher when the cells were grown in lactate or pyruvate. On these same substrates, the malate concentration was 2-fold higher in the CS1-mutant when compared to the parental or CS2- strains. The production of 14CO2 by CS1- from [1-14C]acetate was 36% and that from [2-14C]acetate was 9.2% of the amount from the parental or CS2- strains. The 14CO2 production from [1-14C]glutamate was 28% and 20% in CS1- and CS1-CS2-, respectively, compared to the parental strain. Since these results are not easily explained solely by the absence of mitochondrial citrate synthase enzyme, we also determined the activity of some other enzymes of the citric acid cycle and electron transport chain. We found decreased activity of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and aconitase, while the rest of the citric acid cycle enzymes and oxidative enzymes did not change significantly. The same changes in enzyme activities were found in two different yeast strains carrying the same citrate synthase mutations.     

Effect of propionate on pyruvate metabolism in adipose tissue

Glyceride-glycerol formation in rat adipose tissue from pyruvate-2-(14)C is increased by fasting, while fatty acid synthesis is markedly depressed. In tissues of fasted animals glyceride-glycerol formation is maximal with concentrations of pyruvate exceeding 2.5 mM. With 0.25 mM pyruvate-2-(14)C, glyceride-glycerol formation is increased severalfold by the addition of 0.25 mM propionate. No further increase in synthesis is caused by propionate when pyruvate is supplied in optimal amounts. Addition of equimolar concentrations of acetate or pyruvate does not replace propionate. The effect of propionate on glyceride-glycerol synthesis from pyruvate is also given by a series of even-chain fatty acids. However, only propionate promotes fatty acid synthesis in tissues of fasted and fed animals. Fixation of (14)CO(2) in glyceride-glycerol is dependent on the presence of propionate and is maximal in tissues of fasted rats and when pyruvate is also added. Succinate has no significant effect. Actinomycin treatment blocks glyceride-glycerol synthesis in tissues of fed and fasted animals, in the presence and absence of propionate. At the same time, fatty acid synthesis in tissues of fasted rats is markedly increased.     

The effects of specific lipogenic substrates and metabolic inhibitors on de Novo fatty acid synthesis in isolated hepatocytes from chow-fed female rats

The effects of glucose (10 mm), glycerol (3 mm), and lactate/pyruvate (10 mm) on the incorporation of ³H from ³H₂O into fatty acids were studied in isolated hepatocytes prepared from chow-fed female rats. Lactate/pyruvate markedly increased lipogenic rates, while glucose and glycerol did not significantly affect rates of lipogenesis. In cells incubated with lactate/pyruvate plus glycerol, the increase in ³H incorporation was greater than observed with lactate/pyruvate alone. In hepatocytes isolated from 24-h starved rats, lactate/pyruvate again increased de novo fatty acid synthesis to a greater extent than either glucose or glycerol. Glycerol significantly increased lipogenesis compared to the endogenous rates and when incubated with lactate/pyruvate produced an increase above lactate/pyruvate alone. (?)-Hydroxycitrate, a potent inhibitor of ATP-citrate lyase (EC 4.1.3.8), and agaric acid, an inhibitor of tricarboxylate anion translocation, were studied in hepatocytes to determine their effects on lipogenesis by measuring ³H₂O, [1-¹⁴C]acetate, and [2-¹⁴C]lactate incorporation into fatty acids. ³H incorporation into fatty acids was markedly inhibited by both inhibitors with agaric acid (60 μm) producing the greater inhibition. (?)-Hydroxycitrate (2 mm) increased acetate incorporation into fatty acids from [1-¹⁴C]acetate and agaric acid produced a strong inhibitory effect. Combined effects of (?)-hydroxycitrate and agaric acid on lipogenesis from [1-¹⁴C]acetate showed an inhibitory response to a lesser extent than with agaric acid alone. With substrate concentrations of acetate present, there was no significant increase in rates of lipogenesis from [1-¹⁴C]acetate and the increase previously observed with (?)-hydroxycitrate alone was minimized. Agaric acid significantly inhibited fatty acid synthesis from acetate in the presence of exogenous substrate, but the effect was decreased in comparison to rates with only endogenous substrate present. With [2-¹⁴C]lactate as the lipogenic precursor, agaric acid and (?)-hydroxycitrate strongly inhibited fatty acid synthesis. However, agaric acid despite its lower concentration (60 μm vs 2 mm) was twice as effective as (?)-hydroxycitrate. A similar pattern was observed when substrate concentrations of lactate/pyruvate (10 mm) were added to the incubations. When (?)-hydroxycitrate and agaric acid were simultaneously incubated in the presence of endogenous substrate, there was an additive effect of the inhibitors on decreasing fatty acid synthesis. Results are discussed in relation to the origin of substrate for hepatic lipogenesis and whether specific metabolites increase lipogenic rates.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.     

Control of the citric acid cycle by glyoxylate. The mechanism of inhibition of oxoglutarate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase

1. The effects of glyoxylate on partially purified preparations of aconitate hydratase, isocitrate dehydrogenase and oxoglutarate dehydrogenase were compared with those of oxalomalate and hydroxyoxoglutarate (obtained by condensation of glyoxylate with oxaloacetate and pyruvate respectively). 2. Glyoxylate (1mm) did not affect aconitate hydratase and isocitrate dehydrogenase, whereas oxalomalate (1mm) inhibited the enzyme activities completely. 3. Glyoxylate (0.025mm) inhibited oxoglutarate dehydrogenase irreversibly, whereas the same concentrations of oxalomalate and hydroxyoxoglutarate were ineffective. This inhibitory effect was prevented if oxoglutarate, pyruvate or oxaloacetate was mixed with the enzyme before the glyoxylate. 4. Incubation of oxoglutarate dehydrogenase with radioactive glyoxylate produced radioactive carbon dioxide; radioactivity was also recovered in the portion of the enzyme identified with thiamin pyrophosphate. 5. The behaviour of glyoxylate in producing multiple inhibitions of the citric acid cycle, either by direct interaction with oxoglutarate dehydrogenase, or by means of its condensation compounds which inhibit aconitate hydratase and isocitrate dehydrogenase, is discussed.     

Effects of the hypocholesteremic agent trifluperidol on the sterol, steryl ester, and fatty acid metabolism of Saccharomyces cerevisiae.

Trifluperidol (TFP), at a concentration of 100 muM, inhibited the 24-h growth of Saccharomyces cerevisiae by about 30%. Effects on lipid metabolism were investigated by monitoring the incorporation of [1-14C]sodium acetate into various lipid fractions after 4 and 24 h of growth in the presence of several concentrations of TFP. Although little effect was noted on the amount of free sterols, 24-h incorporation of label into steryl esters was increased two- to fourfold by 100 muM TFP. Major sterol components of the steryl ester fraction isolated from an untreated culture were zymosterol (48%) and ergosterol (24%), whereas from the TFP-treated culture delta8,24(28)-ergostadienol (66.6%) and delta8-ergostenol (14.7%) were most abundant. Free sterols present in the highest concentration in the untreated culture were ergosterol (78.2%) and lanosterol (13%); whereas delta8,22-ergostadienol (38.5%), delta8-ergostenol (35.4%), and delta8,24(28)-ergostadienol (25.4%) were the most abundant free sterols obtained from the TFP-treated culture. Thus, the major block in the sterol biosynthetic pathway in yeast appears to be delta8 leads to delta7 isomerization. In these same cultures the relative amounts of C12 and C14 acids isolated from both steryl ester and miscellaneous lipid fractions were increased more than threefold over controls.     

Nonfunctional Tricarboxylic Acid Cycle and the Mechanism of Glutamate Biosynthesis in Acetobacter suboxydans

Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated.     

Pyruvate dehydrogenase and 3-fluoropyruvate: chemical competence of 2-acetylthiamin pyrophosphate as an acetyl group donor to dihydrolipoamide

The pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex catalyzes the decomposition of 3-fluoropyruvate to CO2, fluoride anion, and acetate. Acetylthiamin pyrophosphate (acetyl-TPP) is an intermediate in this reaction. Incubation of the pyruvate dehydrogenase complex with 3-fluoro[1,2-14C]pyruvate, TPP, coenzyme A (CoASH), and either NADH or pyruvate as reducing systems leads to the formation of [14C]acetyl-CoA. In this reaction the acetyl group of acetyl-TPP is partitioned by transfer to both CoASH (87 +/- 2%) and water (13 +/- 2%). When the E1 component is incubated with 3-fluoro[1,2-14C]pyruvate, TPP, and dihydrolipoamide, [14C]acetyldihydrolipoamide is produced. The formation of [14C]acetyldihydrolipoamide was examined as a function of dihydrolipoamide concentration (0.25-16 mM). A plot of the extent of acetyl group partitioning to dihydrolipoamide as a function of 1/[dihydrolipoamide] showed 95 +/- 2% acetyl group transfer to dihydrolipoamide when dihydrolipoamide concentration was extrapolated to infinity. It is concluded that acetyl-TPP is chemically competent as an intermediate for the pyruvate dehydrogenase complex catalyzed oxidative decarboxylation of pyruvate.     

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.     

Effect of valine on the control of fatty acid synthesis in white adipose tissue of the rat.

In adipocytes from fed rats, the rate of fatty acid synthesis in the presence of glucose and insulin was inhibited 40% by valine (5 mm). tthis inhibition was largely abolished by the addition to the incubation medium of the transaminase inhibitor aminooxy acetate, and of pyruvate and agents which raise the intracellular pyruvate levels such as N,N,N1,N1-tetramethyl-p-phenylenediamine. Pyruvate output into the incubation medium from fat pads obtained from fed rats and incubated with glucose and insulin was decreased significantly by the addition of valine. When adipocytes were incubated under similar conditions, the final concentration of pyruvate in the incubation medium was 42 +/- 1.6 muM under control conditions and approximately one third of this value in the presence of 2.5 mM valine. Valine had no significant effect on pyruvate dehydrogenase (lipoate) (EC 1.2.4.1) activity when assayed in homogenates prepared from adipose tissue previously incubated for 60 min with the amino acid. Although the ketoacid analogue of valine alpha-ketoisovaleric acid, is a competitive inhibitor of pyruvate dehydrogenase (lipoate) (K1 = 1.4 mM), this cannot solely account for the valine-induced reduced rate of lipogenesis. Rather, the mechanism involves a reduction in pyruvate concentration and thereby a diminished flow through pyruvate dehydrogenase (lipoate). Details of the possible mechanism are discussed.     

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.     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

Metabolism of pyruvate by isolated rat mesenteric lymphocytes, lymphocyte mitochondria and isolated mouse macrophages.

1. The activities of pyruvate dehydrogenase in rat lymphocytes and mouse macrophages are much lower than those of the key enzymes of glycolysis and glutaminolysis. However, the rates of utilization of pyruvate (at 2 mM), from the incubation medium, are not markedly lower than the rate of utilization of glucose by incubated lymphocytes or that of glutamine by incubated macrophages. This suggests that the low rate of oxidation of pyruvate produced from either glucose or glutamine in these cells is due to the high capacity of lactate dehydrogenase, which competes with pyruvate dehydrogenase for pyruvate. 2. Incubation of either macrophages or lymphocytes with dichloroacetate had no effect on the activity of subsequently isolated pyruvate dehydrogenase; incubation of mitochondria isolated from lymphocytes with dichloroacetate had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, and the double-reciprocal plot of [1-14C]pyruvate concentration against rate of 14CO2 production was linear. In contrast, ADP or an uncoupling agent increased the rate of 14CO2 production from [1-14C]pyruvate by isolated lymphocyte mitochondria. These data suggest either that pyruvate dehydrogenase is primarily in the a form or that pyruvate dehydrogenase in these cells is not controlled by an interconversion cycle, but by end-product inhibition by NADH and/or acetyl-CoA. 3. The rate of conversion of [3-14C]pyruvate into CO2 was about 15% of that from [1-14C]pyruvate in isolated lymphocytes, but was only 1% in isolated lymphocyte mitochondria. The inhibitor of mitochondrial pyruvate transport, alpha-cyano-4-hydroxycinnamate, inhibited both [1-14C]- and [3-14C]-pyruvate conversion into 14CO2 to the same extent, and by more than 80%. 4. Incubations of rat lymphocytes with concanavalin A had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, but increased the rate of conversion of [3-14C]pyruvate into 14CO2 by about 50%. This suggests that this mitogen causes a stimulation of the activity of pyruvate carboxylase.     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...     

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.     

Lipogenesis from glucose and pyruvate in fat cells from genetically obese rats

Subcutaneous fat cells were isolated from genetically obese rats and from rats with obesity produced by hypothalamic lesions. Insulin did not augment the oxidation of fatty acids or their synthesis from glucose-1-(14)C or glucose-1-(3)H by fat cells from either group. Radioactivity from pyruvate-3-(14)C was incorporated into fatty acids to the same degree by fat cells from these two groups. The presence of 5 mm glucose in the incubation medium containing fat cells and pyruvate-3-(14)C or aspartate-3-(14)C stimulated the synthesis of fatty acids to a greater extent in cells of genetically obese rats. Fasting, in contrast, reduced the incorporation of radioactivity from pyruvate and glucose into fatty acids by fat cells from the genetically obese animals. In all experiments the fat cells from genetically obese rats converted more radioactivity into glyceride-glycerol relative to CO(2) than did fat cells from hypothalamic obese rats. Parabiosis between one thin and one genetically obese litter mate was performed in three pairs of rats without influencing growth of either rat. Thus in the present studies fat cells from genetically obese rats showed two differences from normal fat cells: they channeled more radioactivity from pyruvate into fatty acids in the presence of glucose, and they uniformly converted more radioactivity into glyceride-glycerol.