Mitochondrial protein phosphorylation and cardiomyopathy in genetically diabetic mice: the effect of estrone treatment

The alpha-subunit of pyruvate dehydrogenase and succinyl-CoA synthetase are phosphorylated after incubation of cardiac mitochondria from genetically diabetic mice with [gamma-32P]ATP. There is significantly increased incorporation of 32P into pyruvate dehydrogenase from diabetic mice when compared to controls. The enhanced rate of pyruvate dehydrogenase phosphorylation correlates well with the previously reported defective oxidative metabolism and decreased activity of this enzyme from diabetic mice. The relationship between abnormal mitochondrial function and development of cardiomyopathy in the diabetic mice has been studied further by in vivo estrone treatment. The results indicate that ultrastructural alterations of myocardium are closely associated with the defective pyruvate oxidation (via phosphorylation of pyruvate dehydrogenase) and both processes can be prevented by 7-12 weeks estrone treatment.     

Insulin mediator causes dephosphorylation of the alpha subunit of pyruvate dehydrogenase by stimulating phosphatase activity

Insulin treatment of rats results in an increased amount or activity of insulin mediators in liver and skeletal muscle. These mediators stimulated pyruvate dehydrogenase and inhibited adenylate cyclase. The insulin-generated mediators caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in mitochondria prelabeled with [gamma-32P]ATP. An assay was developed which quantitatively measured mediator activity by determining the rate of alpha-subunit dephosphorylation. The dephosphorylation was directly proportional to the amount of mediator added and was directly related to activation of pyruvate dehydrogenase. The decrease of alpha-subunit phosphorylation resulted from stimulation of pyruvate dehydrogenase phosphatase, since it occurred in the absence of ATP and was inhibited by NaF. These data further delineate the mechanism of insulin mediator activation of pyruvate dehydrogenase.     

Characterization of oxalate as a catabolite of dichloroacetate responsible for the inhibition of gluconeogenesis and pyruvate carboxylation in rat liver cells

In isolated hepatocytes, dichloroacetate decreased glucose synthesis from lactate, pyruvate and alanine, but not from substrates which bypass pyruvate carboxylase (propionate, glycerol). It was also found to inhibit pyruvate carboxylation in isolated mitochondria, but only after a preincubation period, and had no effect on partially purified pyruvate carboxylase. Hepatocytes and liver mitochondria metabolized [¹⁴C] dichloroacetate to oxalate which inhibits pyruvate carboxylase and mimics, without preincubation, the effects of dichloroacetate in mitochondrial pyruvate carboxylation. Thus, oxalate appears to be responsible for the inhibition of gluconeogenesis by dichloroacetate at the level of pyruvate carboxylation.     

The regulation of branched-chain 2-oxo acid dehydrogenase of liver, kidney and heart by phosphorylation.

1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.     

Oxidation of branched-chain amino acids in skeletal muscle and liver of rat Effects of octanoate and energy state

The effect of octanoate on the oxidative decarboxylation of ¹⁴C-labeled amino acids has been studied in perfused hindquarter and liver of rat. Regulation of the branched-chain α-keto acid dehydrogenase has been further studied with α-[¹⁴C-1]ketoisovalerate in isolated rat muscle and liver mitochondria. (1) Octanoate has a stimulatory effect on the oxidation of branched-chain amino acids in perfused hindquarter. The oxidative decarboxylation of other amino acids are inhibited. Octanoate inhibits the oxidative decarboxylation of all amino acids in perfused liver. (2) The oxidation of valine is stimulated by octanoate and hexanoate also in isolated muscle mitochondria. The stimulatory effect is probably related to activation of the fatty acids since acyl-carnitines inhibit the oxidation. (3) The oxidation of α-ketoisovalerate in mitochondria is inhibited by competing substrates (pyruvate, α-ketoglutarate and succinate). This inhibition is counteracted by octanoate and ADP. (4) Low concentrations (1–5 μM) of 2,4-dinitrophenol (DNP) activates wheras higher concentrations inactivates the branched-chain α-keto acid dehydrogenase in intact but not in solubilized muscle mitochondria. The inactivation is counteracted by ATP, but is increased by octanoate. (5) The observations seem to suggest that the activation (like the inactivation) of branched-chain α-keto acid dehydrogenase in skeletal muscle is dependent on the mitochondrial energy state which therefore may regulate both activation and inactivation of the dehydrogenase.     

A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver.

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.     

Occurrence and metabolic role of the pyruvate dehydrogenase complex in the lower termite Coptotermes formosanus (Shiraki)

The activities of the pyruvate dehydrogenase complex in extracts of the gutted body, head, foregut/midgut and hindgut (hindgut epithelium and microorganisms) tissues of the lower termite Coptotermes formosanus (Shiraki) were determined by measuring the [¹⁴C]-acetyl-CoA produced from [2-¹⁴C]-pyruvate and the ¹⁴CO₂ produced from [1-¹⁴C]-pyruvate. The activities of pyruvate dehydrogenase, l-lactate dehydrogenase, acetyl-CoA synthetase, malate dehydrogenase (decarboxylating), and acetate kinase in the termite tissues and the hindgut also were determined. The sum (7.1 nmol/termite/h) of the pyruvate dehydrogenase complex activities in the termite tissues other than the hindgut was five times higher than the activity in the hindgut. Significant amounts of l-lactate dehydrogenase activity were found in all of the tissues. All of the tissues other than the hindgut had significant amounts of acetyl-CoA synthetase activity. Malate dehydrogenase (decarboxylating) activity was about ten times higher in the hindgut extract than in the gutted body and head extracts and the activity in the foregut/midgut extract was very low. These results indicate that acetyl-CoA for the TCA cycle is produced effectively in the tissues of the termite itself, both from pyruvate by the pyruvate dehydrogenase complex and from acetate by acetyl-CoA synthetase.     

The role of enzymes of pyruvate and citrate metabolism in control of gluconeogenesis in oocytes and embryos of the loach,Misgurnus fossilis L.

Summary Cessation of gluconeogenesis during oocyte maturation inMisgurnus fossilis L. is accompanied by an increase of pyruvate dehydrogenase activity (EC 1.2.4.1). The activity of other enzymes of citrate and pyruvate metabolism (citrate synthetase, EC 4.1.3.7, pyruvate carboxylase, EC 6.4.1.1., malate dehydrogenase, EC 1.1.1.37) remains constant during oocyte maturation and early embryogenesis.In the course of oocyte maturation the levels of acetyl-CoA, pyruvate and citrate remained unchanged, but the level of malate and oxaloacetate underwent drastic increase. The level of phosphoenolpyruvate increased about two-fold. The mitochondrial (NAD⁺)/(NADH) ratio was calculated by measurement of intermediates of the glutamate dehydrogenase reaction and it was found to increase six-fold during oocyte maturation. The lower mitochondrial (NAD⁺)/(NADH) ratio in oocytes compared to that in the embryos is likely to be responsible for the transfer of reducing equivalents from mitochondria to cytoplasm, while in embryos transfer in the opposite direction takes place.     

Effect of thiamine on the activity of the pyruvate dehydrogenase complex in rat liver following stimulation of lipogenesis

It is shown that thiamine administration to rats (250 micrograms per 100 g of mass) who were given high-carbohydrate diet (lipogenesis intensification) after fasting inhibits an increase in the pyruvate dehydrogenase activity in the liver homogenate and mitochondria usual under these conditions. This is observed when determining total activity of the pyruvate dehydrogenase complex and activity of its first component--pyruvate dehydrogenase estimated from the ferricyanide reduction and [1-14C] CO2 formation from [1-14C] pyruvate. Fasting animals and animals whom thiamine was administered against a background of lipogenesis intensification revealed a higher ability of the liver tissue to synthesize acetoin as compared with the control group and animals with the intensified lipogenesis without thiamine administration.     

Determination of pyruvate dehydrogenase and acetyl-CoA synthetase activities using citrate synthase

Methods are described for the assay of pyruvate dehydrogenase and acetyl-CoA synthetase activities in rat brain subcellular fractions. Citrate synthase and oxaloacetate serve as a trapping system in these assays. The methods permit the determination of a large number of samples of different turbidity with satisfactory precision. Highest activities of pyruvate dehydrogenase and acetyl-CoA synthetase (117.7 and 7.29 nmol/min/mg of protein, respectively) were found in rat brain mitochondria. A three times lower activity of acetyl-CoA synthetase and negligible of pyruvate dehydrogenase was found in brain cytosol.     

Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis.

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.     

Regulation of pyruvate oxidation in mitochondria isolated from fetal and adult rat liver

The effect of ATP on the velocity of oxygen uptake during the oxidation of pyruvate plus malate, in the presence of oligomycin, 2,4-dinitrophenol and fluorocitrate, was studied in mitochondria, isolated from the livers of adult and fetal rats.It was found that the addition of ATP caused an inhibition in the rate of oxygen uptake of 21 ± 6% in mitochondria from adult rat liver and 49 ± 8% in mitochondria from fetal rat liver. Measurements of the velocity of oxygen uptake during the oxidation of pyruvate plus malate and of palmitoylcarnitine in adult rat liver mitochondria in the presence of ATP showed that the activity of pyruvate dehydrogenase was lower than the activity of citrate synthase.In fetal mitochondria, addition of ATP resulted in an increase in the CoASH/acetyl-CoA ratio, indicating that pyruvate dehydrogenase was rate limiting here as well.It is concluded that ATP inhibited pyruvate oxidation by phosphorylation of the pyruvate dehydrogenase complex, rather than by inhibiting citrate synthase under these conditions.     

Pyruvate metabolism after in vivo exposure to oral arsenic

This study investigated altered pyruvate metabolism after prolonged oral arsenic exposure. Male rats were given access to deionized drinking water containing 0, 40 or 85 ppm sodium arsenate (As⁵⁺) for 3 weeks. Respiration studies with mitochondria isolated from treated animals indicated decreased state 3 respiration (with ADP) and decreased respiratory control ratios (RCR) for pyruvate/malate-mediated respiration, but not for succinate-mediated respiration, as compared to control respiration values. In addition, pyruvate dehydrogenase activity was measured, in both liver and intestine, before and after Mg-activation in vitro. After 3 weeks, the effects of arsenic at the highest dose level were pronounced on the basal pyruvate dehydrogenase activity (before activation) as well as the total pyruvate dehydrogenase (after activation). The inhibition of pyruvate dehydrogenase activity both before and after Mg-activation suggests an arsenic effect on mitochondrial pyruvate metabolism which, in part, involves inhibition of pyruvate decarboxylase. Evidence is also presented which may indicate an arsenic effect on the kinase and/or phosphatase which regulate pyruvate dehydrogenase activity.     

Mitochondrial compartmentation of metabolic CO2 resulting from its site of origin in relation to urea synthesis

In isolated hepatocytes the entry into urea of metabolic ¹⁴CO₂; derived from [¹⁴C] formate is modified by the addition of dichloroacetate and hydroxypyruvate. An explanation is that this results from changes in the cytoplasmic/mitochondrial pH gradient. ¹⁴CO₂, derived from [1-¹⁴C]alanine enters into urea more readily than ¹⁴CO₂ arising from [1-¹⁴C]glutamate. It is proposed that the difference, which is more than 4-fold, is indicative of a preferred pathway for metabolic CO₂ in liver mitochondria from pyruvate dehydrogenase to carbamoylphosphate synthetase than form oxoglutarate dehydrogenase. Acetazolamide inhibition of carbonic anhydrase is without effect on this observed incorporation into urea.     

The pyruvate branch point in fish liver mitochondria: Effects of acylcarnitine oxidation on pyruvate dehydrogenase and pyruvate carboxylase activities

Summary Palmitoyldl-carnitine inhibits¹⁴CO₂ production from 1-[¹⁴C]-pyruvate and from 1-[¹⁴C]-alanine by mitochondria from rainbow trout liver. The inhibitory effect occurs in both respiratory states III and IV. Fixation of H¹⁴CO ₃ ^– into acid-stable products by intact mitochondria requires pyruvate and ATP and is inhibited by sodium arsenite. This inhibitory effect is completely abolished by acetyldl-carnitine. It is proposed that under these conditions, oxidation of palmitoyldl-carnitine results in inhibition of pyruvate dehydrogenase while oxidation of acetyldl-carnitine results in activation of pyruvate carboxylase in intact rainbow trout liver mitochondria.     

Studies on the regulation of pyruvate dehydrogenase in isolated beef heart mitochondria.

The regulation of the pyruvate dehydrogenase multienzyme complex of isolated beef heart mitochondria by a phosphorylation-dephosphorylation mechanism was investigated. From mitochondria incubated under conditions favoring either a protein kinasemediated inactivation or a phosphatase-mediated reactivation, the pyruvate dehydrogenase complex was extracted and partially purified. Incorporation of ³²P from [γ-³²P]ATP into the pyruvate dehydrogenase complex corresponded to the loss of enzymatic activity. Upon incubation of the mitochondria that were preincubated with [γ-³²P]ATP under metabolic conditions favoring the phosphatase reaction, the amount of radioactivity in the ³²P-labeled fraction decreased significantly with a concomitant increase in the pyruvate dehydrogenase activity. The estimated molecular weight of the ³²P-labeled fraction derived from the mitochondrial incubation was 41,000, corresponding to the reported molecular weight of the α-subunit of the pyruvate dehydrogenase portion of the multienzyme complex.     

The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebro-cortical mitochondria

The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxyburyrate on the flux of PDHC (as determined by [1-¹⁴C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-¹⁴C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-¹⁴C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861–1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.     

Activation of pyruvate dehydrogenase by addition of insulin to a mixture of cell membranes and mitochondria from the brain of normal and alloxan treated rats. (Preliminary note)

The effect of insulin and ATP on pyruvate dehydrogenase activity has been studied in a mixture of plasma membranes/mitochondria from normal and diabetic rat brain. In both enzymatic preparations ATP inhibits almost completely pyruvate dehydrogenase activity; insulin on the contrary induces a conspicuous activation and removes the inhibiting action of ATP.     

Fasting induced alterations in mitochondrial palmitoyl-CoA metabolism may inhibit adipocyte pyruvate dehydrogenase activity.

1. Adipocytes from fed and fasted (24 hr) groups of rats were fractionated into mitochondria, microsomes and plasma membranes. 2. Fasting significantly decreased the mitochondrial activity of palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, beta-oxidation and pyruvate dehydrogenase. 3. Fasting elevated intramitochondrial long-chain acyl-CoA. 4. Pyruvate dehydrogenase was inhibited 50% by addition of 30 microM palmitoyl-CoA. 5. Fasting-induced changes in palmitoyl-CoA metabolism may modulate pyruvate dehydrogenase activity in adipocyte mitochondria.     

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.