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.     

Insulin-like effects of fluoroacetate on lipolysis and lipogenesis in adipose tissue.

Hormone-stimulated lipolysis in adipose tissue was inhibited by fluoroacetate and there was a concomitant decrease in both the basal and hormone-stimulated cyclic AMP levels. Adenylate cyclase (EC 4.6.1.1) activity in membrane preparations was inhibited by fluoroacetate. There was no influence of fluoroacetate on the low Km cyclic AMP phosphodiesterase (EC 3.1.4.17) activity. The rate of glucose conversion to fatty acids was increased when adipose tissue was incubated in the presence of fluoroacetate. The outputs of pyruvate and lactate into the incubation medium were decreased at this time, suggesting decreased tissue pyruvate levels and a site of activation of lipogenesis distal to pyruvate formation. Pyruvate dehydrogenase (EC 1.2.4.1) activity was increased twofold in adipose tissue incubated in the presence of fluoroacetate. This was attributed to a fluoroacetate-induced inhibition of pyruvate dehydrogenase kinase, the enzyme responsible for inactivating the pyruvate dehydrogenase complex. Glucose transport was increased to a small but significant degree by fluoroacetate. In addition, both the tissue content of citrate and its release into the incubation medium were increased, suggesting that fluoroacetate resulted in an inhibition of aconitase (EC 4.2.1.3). The tissue ATP content was unchanged. Because the antilipolytic and lipogenic effects of fluoroacetate parallel those of insulin, they may share a common mechanism.     

Pathway of carbon flow during fatty acid synthesis from lactate and pyruvate in rat adipose tissue

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.     

Interrelations between ureogenesis and gluconeogenesis in isolated hepatocytes. The role of antion transport and the competition for energy.

1. Glucose synthesis from lactate plus pyruvate and from lactate plus alanine was measured in the presence or absence of 1mM-oleate or 2mM-octanoate at low (2mM) or high (8mM) concentrations of NH4Cl. 2. Both fatty acids alone or with 2mM-NH4Cl doubled glucose production from lactate plus pyruvate. Glucose synthesis from lactate plus alanine, in the presence of oleate, was decreased 16% by 2mM-NH4Cl. 3. In the presence of fatty acids, 8mM-NH4Cl decreased gluconeogenesis by 60-65% from both lactate plus pyruvate and lactate plus alanine. This inhibition was correlated with a high accumulation of aspartate and a drastic decrease in 2-oxoglutarate and malate in the cells. 4. In the presence of 2mM- or 8 mM-NH4Cl, oleate and glucogenic precursors, the addition of 2.5mM-ornithine stimulated urea synthesis. 5. This was paralleled by a decrease of 16% in glucose synthesis from lactate plus pyruvate in the presence of 2mM-NH4Cl and had no effect at 8mM-NH4Cl. In the system producing glucose from lactate plus alanine, ornithine completely reversed the inhibition caused by 2mM-NH4Cl and only partly that by 8mM-NH4Cl. 6. Gluconeogenesis from pyruvate was also inhibited by 2mM-NH4Cl in the presence of oleate or ethanol. This way due to the decrease of malate, which is the C4 precursor of glucose in this system. 7. The limitation of gluconeogenesis by 2-oxoglutarate and malate concentrations in the liver cell and the competition for energy between glucose and urea synthesis is discussed.     

Role of sulfhydryl groups in benzoquinone-induced Ca2+ release by rat liver mitochondria

Incubation of rat liver mitochondria with benzoquinone derivatives in the presence of succinate plus rotenone has been shown to cause NAD(P)H oxidation followed by Ca2+ release. Further investigation revealed: (1)p-Benzoquinone-induced Ca2+ release was not initiated by a collapse of the mitochondrial membrane potential. However, Ca2+ release and subsequent Ca2+ cycling caused limited increased membrane permeability. (2) p-Benzoquinone-induced NAD(P)H oxidation and Ca2+ release were prevented by isocitrate, 3-hydroxybutyrate, and glutamate but not by pyruvate or 2-oxoglutarate. (3) Inhibition of pyruvate and 2-oxoglutarate dehydrogenases by p-benzoquinone was attributed to arylation of the SH groups of the cofactors, CoA and lipoic acid. Isocitrate dehydrogenase was also inhibited by p-benzoquinone, but the cofactors NAD(P)H and Mn2+ protected the enzyme. Glutamate dehydrogenase was not inhibited by p-benzoquinone. (4) Arylation of mitochondrial protein thiols by p-benzoquinone was associated with an inhibition of state 3 respiration, which was attributed to the inactivation of the phosphate translocase. In contrast, state 4 respiration, and the F1.F0-ATPase and ATP/ADP translocase activities were not inhibited. It was concluded that inhibition of mitochondrial NAD(P)H dehydrogenases by arylation of critical thiol groups will decrease the NAD(P)+-reducing capacity, and possibly lower the NAD(P)H/NAD(P)+ redox status in favor of Ca2+ release.     

Inhibition of 2-oxoglutarate dehydrogenase in potato tuber suggests the enzyme is limiting for respiration and confirms its importance in nitrogen assimilation,

The 2-oxoglutarate dehydrogenase complex constitutes a mitochondrially localized tricarboxylic acid cycle multienzyme system responsible for the conversion of 2-oxoglutarate to succinyl-coenzyme A concomitant with NAD(+) reduction. Although regulatory mechanisms of plant enzyme complexes have been characterized in vitro, little is known concerning their role in plant metabolism in situ. This issue has recently been addressed at the cellular level in nonplant systems via the use of specific phosphonate inhibitors of the enzyme. Here, we describe the application of these inhibitors for the functional analysis of the potato (Solanum tuberosum) tuber 2-oxoglutarate dehydrogenase complex. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-oxoglutarate dehydrogenase complex, displaying greater inhibitory effects than a diethyl ester of SP, a phosphono ethyl ester of SP, or a triethyl ester of SP. Incubation of potato tuber slices with the inhibitors revealed that they were adequately taken up by the tissue and produced the anticipated effects on the in situ enzyme activity. In order to assess the metabolic consequences of the 2-oxoglutarate dehydrogenase complex inhibition, we evaluated the levels of a broad range of primary metabolites using an established gas chromatography-mass spectrometry method. We additionally analyzed the rate of respiration in both tuber discs and isolated mitochondria. Finally, we evaluated the metabolic fate of radiolabeled acetate, 2-oxoglutarate or glucose, and (13)C-labeled pyruvate and glutamate following incubation of tuber discs in the presence or absence of either SP or the carboxy ethyl ester of SP. The data obtained are discussed in the context of the roles of the 2-oxoglutarate dehydrogenase complex in respiration and carbon-nitrogen interactions.     

Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing.

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria. The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25 degrees C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation. The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32 degrees C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid. The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.     

Glutamate dehydrogenase activity in developing soybean seed: Isolation and characterization of three forms of the enzyme

Three forms of glutamate dehydrogenase have been isolated from developing soybean seed. Their intracellular locations could not be determined directly because organelles and marker enzymes showed abnormal distribution on sucrose density gradient fractionation. By analogy with enzymes from other parts of the plant, glutamate dehydrogenase 2 was shown to be located in chloroplasts and glutamate dehydrogenase 3 in mitochondria. Glutamate dehydrogenase 1 could not be located in this way because it is found only in the seed. The three enzymes are similar in pH optima, molecular weight and substrate specificity with respect to 2-oxoglutarate and l-glutamate. The mitochondrial enzyme is specific for NAD⁺. The chloroplast enzyme shows low activity with NADP⁺ relative to NAD⁺ but uses NADPH readily in the aminating direction. Glutamate dehydrogenase 1 is active with both nucleotides and is the only form to show substantial deaminating activity with NADP⁺. Glutamate dehydrogenases 1 and 2 are activated and stabilized by glutathione and 2-mercaptoethanol whereas enzyme 3 is unaffected. No significant metabolic control of any of the enzymes could be detected. Malate, citrate, adenine nucleotides and long-chain fatty acyl CoA derivatives gave slight inhibition at high concentrations. Amino acids had no effect on activity. A possible role for the enzyme characteristic of the developing seed is discussed in relation to nutrient supply during the accumulation of reserve materials in the seed.     

Acute effects in vivo of anti-insulin serum on rates of fatty acid synthesis and activities of acetyl-coenzyme A carboxylase and pyruvate dehydrogenase in liver and epididymal adipose tissue of fed rats.

Plasma insulin concentrations in fed rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in adipose tissue and liver were estimated from the incorporation of 3H from 3H2O. In the adipose tissue dehydrogenase and acetyl-CoA carboxylase were evident. In liver, although changes in rates of fatty acid synthesis were found, the initial activity of pyruvate dehydrogenase did not alter, but small parallel changes in acetyl-CoA carboxylase activity were observed.     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

Influence of NAD-linked dehydrogenase activity on flux through oxidative phosphorylation.

1. We have examined systematically the relationship between the percentage reduction of cardiac mitochondrial NAD and the flux through oxidative phosphorylation, as measured by O2 uptake. Reduction of NAD was varied by varying the concentration of palmitoyl-L-carnitine, pyruvate, 2-oxoglutarate or glutamate in the presence of malate as the oxidizable substrate. 2. In the presence of ADP (State 3 respiration) there was a substantially linear positive relationship between O2 uptake and the percentage reduction of NAD. Coupled respiration in the absence of ADP also showed an increase with increasing NADH, with the exact shape of the relationship being variable. 3. When pyruvate and 2-oxoglutarate dehydrogenase activity were increased by increasing medium Ca2+ concentration within the range 5 nM to 1.23 microM, at non-saturating substrate concentrations, there was again a positive relationship between O2 uptake and the reduction of NAD; however, rates of O2 uptake tended to be higher at given values of NAD reduction when the incubation medium contained Ca2+. This is taken to indicate an activation by Ca2+ of the enzymes of phosphorylation or of the respiratory chain, in addition to the dehydrogenase activation. 4. When carboxyatractyloside plus ADP were used to generate 50% State 3 rates of O2 uptake with pyruvate or 2-oxoglutarate, sensitivity to Ca2+ was retained. However, when oligomycin plus 1 mM-ADP and 1 mM-ATP were used to generate 50% State 3, no such dependence was seen. 5. The results are interpreted to indicate a substantial role for substrate dehydrogenation in the overall regulation of oxidative phosphorylation when substrates are available at near-physiological concentrations.     

The effects of a thiamine antagonist, pyrithiamine, on levels of selected metabolic intermediates and on activities of thiamine-dependent enzymes in brain and liver

(1) The effects of thiamine deficiency as produced by pyrithiamine injections have been studied in the weanling mouse. Selected metabolites were measured in extracts from brain and liver of quick-frozen animals. Pyruvate and α-oxoglutarate dehydrogenases and transketolase were also measured. (2) In deficient brain, pyruvate and α-oxoglutarate levels were greatly increased. Xylulose-5-P and 6-P-gluconate were more than doubled. Lactate, glucose-6-P, glucose and P-creatine were moderately elevated, and ATP was increased a little. Glutamate was depressed. (3) In deficient liver, α-oxoglutarate was much increased and ATP was twice normal. Glycogen, glucose, glucose-6-P, 6-P-gluconate, pyruvate, and glutamate were not different from the controls. Lactate was depressed. (4) Pyruvate dehydrogenase activity was reduced to 25 per cent or less in brain and liver. Transketolase and α-oxoglutarate dehydrogenase activities were reduced to 50 per cent in both organs. (5) Thiamine treatment, within 5 hr, largely reversed the metabolite changes brought on by pyrithiamine in brain. At the same time pyruvate and α-oxoglutarate dehydrogenase activities were increased 60 per cent or more in both brain and liver. Transketolase activity in liver was only increased 20 per cent at this time, however, and in brain was unchanged. (6) The results are interpreted to indicate that inhibition of pyruvate and α-oxoglutarate dehydrogenases in brain is sufficient to depress in vivo function. The same seems true for the inhibition of α-oxoglutarate dehydrogenase in liver. However, the changes seen in brain 6-P-gluconate and xyluIose-5-P probably depend on factors other than, or in addition to, the decrease in transketolase activity. It seems worthy of emphasis that in spite of the partial metabolic blocks high-energy phosphate stores were actually increased.     

Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria.The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25°C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation.The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32°C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid.The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.     

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.     

The activities of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase in hearts and mammary glands from ruminants and non-ruminants.

1. The activities of 2-oxoglutarate dehydrogenase (EC 1.2.4.2) were measured in hearts and mammary glands of rats, mice, rabbits, guinea pigs, cows, sheep, goats and in the flight muscles of several Hymenoptera. 2. The activity of 2-oxoglutarate dehydrogenase was similar to the maximum flux through the tricarboxylic acid cycle in vivo. Therefore measuring the activity of this enzyme may provide a simple method for estimating the maximum flux through the cycle for comparative investigations. 3. The activities of pyruvate dehydrogenase (EC 1.2.4.1) in mammalian hearts were similar to those of 2-oxoglutarate dehydrogenase, suggesting that in these tissues the tricarboxylic acid cycle can be supplied (under some conditions) by acetyl-CoA derived from pyruvate alone. 4. In the lactating mammary glands of the rat and mouse, the activities of pyruvate dehydrogenase exceeded those of 2-oxoglutarate dehydrogenase, reflecting a flux of pyruvate to acetyl-CoA for fatty acid synthesis in addition to that of oxidation via the tricarboxylic acid cycle. In ruminant mammary glands the activities of pyruvate dehydrogenase were similar to those of 2-oxoglutarate dehydrogenase, reflecting the absence of a significant flux of pyruvate to fatty acids in these tissues.     

Properties of apoglutamate synthase and comparison with glutamate dehydrogenase.

Glutamate synthase from Escherichia coli K-12 exhibits NH3-dependent activity. NH3-dependent activity is increased approximately 5-fold in apoglutamate synthase lacking flavin and non-heme iron. Whereas glutamine plus 2-oxoglutarate have the capacity to reoxidize the chemically reduced flavoenzyme, no such reoxidation is obtained with 2-oxoglutarate plus NH3. These results establish that the glutamine- and NH3-dependent syntheses of glutamate occur by different pathways of electron transfer from NADPH. The NH3-dependent activity of native and apoglutamate synthase exhibits similar catalytic properties. Some properties of apoglutamate synthase are similar to those of glutamate dehydrogenase. These properties include pH optima for synthesis and oxidative deamination of glutamate, inactivation by alkylating reagents and p-mercuribenzoate, an enhanced rate of inactivation by alkylating reagents and p-mercuribenzoate at low pH, 2-oxoglutarate protection against inactivation by p-mercuribenzoate, and reactivation of p-mercuribenzoate-treated enzyme by 2-mercaptoethanol. 2-Oxoglutarate protects against alkylation of glutamate synthase by iodo [1-14C]acetamide and reduces incorporation of methyl [1-14C]carboxamide into the small subunit of the enzyme.     

Effects of fasting on the control of fatty-acid synthesis in hepatoma 7777 and host liver. Role of long-chain fatty acyl-CoA,, the mitochondrial citrate transporter and pyruvate dehydrogenase activity.

The effects of fasting on the rate of fatty acid synthesis, the properties of the mitochondrial citrate transporter and on pyruvate dehydrogenase activity were investigated in "poorly-differentiated" tmorris hepatoma 7777 and in host liver preparations. The properties of the citrate transporter from hepatoma mitochondria were similar to those of host liver mitochondria, with the exception that the Km for the liver mitochondrial citrate transporter was 248 plus or minus 20 mu M while that in hepatoma mitochondria was less than 75 mu M. The acid-insoluble CoA content was 180 plus or minus 20 pmol/mg protein in the hepatoma and remained essentially unchanged in the fasted state, while the acid-insoluble CoA levels in livers from fed rats was 720 plus or minus 80 pmol/mg protein and were increased to 1050 plus or minus 50 pmol/mg protein during fasting. After a 36-h fast, the rate of lipogenesis and the percentage of pyruvate dehydrogenase present in the active form were each decreased by approximately 80% in host liver preparations. In contrast, the rate of lipogenesis by hepatoma slices did not decrease during fasting, and essentially all pyruvate dehydrogenase present was in the active form of hepatomas obtained from either fed or fasted animals. Implications concerning the identification of possible regulatory sites in the control of lipogenesis were discussed in relation to the above observations.     

Inhibition of lactate glucogneogenesis in rat kidney by dichloroacetate.

1. Sodium dichloroacetate (1mM) inhibited glucose production from L-lactate in kidney-cortex slices from fed, starved or alloxan-diabetic rates. In general gluconeogenesis from other substrates was no inhibited. 2. Sodium dichloracetate inhibited glucose production from L-lactate but no from pyruvate in perfused isolated kidneys from normal or alloxan-diabetic rats. 3. Sodium dichloroacetate is an inhibitor of the pyruvate dehydrogenase kinase reaction and it effected conversion of pyruvate dehydrogenase into its its active (dephosphorylated) form in kidney in vivo. In general, pyruvate dehydrogenase was mainly in the active form in kidneys perfused or incubated with L-lactate and the inhibitory effect of dichloroacetate on glucose production was not dependent on activation of pyruvate dehydrogenase. 4. Balance data from kidney slices showed that dichloroacetate inhibits lactate uptake, glucose and pyruvate production from lactate, but no oxidation of lactate. 5. The mechanism of this effect of dichloroactetate on glucose production from lactate has not been fully defined, but evidence suggests that it may involve a fall in tissue pyruvate concentration and inhibition of pyruvate carboxylation.     

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin.

The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.     

Regulation of flux through pyruvate dehydrogenase and pyruvate carboxylase in rat hepatocytes. Effects of fatty acids and glucagon

The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions. The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1-14C]pyruvate into 14CO2 plus [14C]glucose. The flux through PDH was determined from the rate of formation of 14CO2 from [1-14C]pyruvate corrected for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by subtracting the flux through PDH from the total pyruvate metabolized. With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h-starved rats. In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59-76%) and PC (80-83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats. In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC. 2[5(4-Chlorophenyl)pentyl]oxirane-2-carboxylate (100 microM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate-stimulated but not the octanoate-stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the beta-oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation. It is concluded that the effects of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.