Effect of fatty acids and ketones on the activity of pyruvate dehydrogenase in skeletal-muscle mitochondria.

The presence of palmitoyl-L-carnitine and acetoacetate (separately) decreased flux through pyruvate dehydrogenase in isolated mitochondria from rat hind-limb muscle. The effect of acetoacetate was dependent on the presence of 2-oxoglutarate and Ca2+. Palmitoylcarnitine, but not acetoacetate, also decreased the mitochondrial content of active dephospho-pyruvate dehydrogenase (PDHA). This effect was large only in the presence of EGTA. Addition of Ca2+-EGTA buffers stabilizing pCa values of 6.48 or lower gave near-maximal values of PDHA content, irrespective of the presence of fatty acids or ketones when mitochondria were incubated under the same conditions used for the flux studies, i.e. at low concentrations of pyruvate. There was, however, a minor decrement in PDHA content in response to palmitoylcarnitine oxidation when the substrate was L-glutamate plus L-malate. Measurement of NAD+, NADH, CoA and acetyl-CoA in mitochondrial extracts in general showed decreases in [NAD+]/[NADH] and [CoA]/[acetyl-CoA] ratios in response to the oxidation of palmitoylcarnitine and acetoacetate, providing a mechanism for both decreased PDHA content and feedback inhibition of the enzyme in the PDHA form. However, only changes in [CoA]/[acetyl-CoA] ratio appear to underlie the decreased PDHA content on addition of palmitoylcarnitine when mitochondria are incubated with L-glutamate plus L-malate (and no pyruvate) as substrate. The effect of palmitoylcarnitine oxidation on flux through pyruvate dehydrogenase and on PDHA content is less marked in skeletal-muscle mitochondria than in cardiac-muscle mitochondria. This may reflect the less active oxidation of palmitoylcarnitine by skeletal-muscle mitochondria, as judged by State-3 rates of O2 uptake. In addition, Ca2+ concentration is of even greater significance in pyruvate dehydrogenase interconversion in skeletal-muscle mitochondria than in cardiac-muscle mitochondria.     

Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes.

1. Previous studies showed that the activation of pyruvate dehydrogenase within intact rat heart mitochondria of pyruvate is much diminished in mitochondria from starved or diabetic animals [see Kerbey, Randle, Cooper, Whitehouse, Pask & Denton (1976) Biochem. J. 154, 327-348]. In the present study, diminished responses to added Ca2+ and ADP were also found in these mitochondria. 2. Starvation or diabetes did not affect the mitochondrial respiratory control ratio of the ATP content. Moreover, starvation and diabetes did not alter the response of the intramitochondrial Ca2+-sensitive enzyme, 2-oxoglutarate dehydrogenase, to changes in the extramitochondrial concentration of Ca2+ and 2-oxoglutarate, thus indicating that there were no appreciable changes in the distribution of Ca2+ and H+ across the mitochondrial inner membrane. 3. Pyruvate, Ca2+ and ADP were found to have synergistic effects on pyruvate dehydrogenase activity, particularly in mitochondria from starved and diabetic rats. 4. The results suggest that the effects of diabetes and starvation on pyruvate dehydrogenase are not brought about by changes in the distribution of these effectors across the mitochondrial inner membrane or by changes in the intrinsic sensitivity of the kinase or phosphatase of the pyruvate dehydrogenase system to pyruvate, Ca2+ or ADP; rather it is probably that there is an increase in the maximum activity of kinase relative to that of the phosphatase. 6. The results also lend further support to the hypothesis that adrenaline may bring about the activation of pyruvate dehydrogenase in the rat heart by an increase in the intramitochondrial concentration of Ca2+.     

Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet.

1. Pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads was measured by using pig heart pyruvate dehydrogenase [32P]phosphate. About 80% was found to be extramitochondrial and therefore probably not directly concerned with the regulation of pyruvate dehydrogenase activity. The extramitochondrial activity was sensitive to activation by Ca2+, but perhaps less sensitive than the mitochondrial activity.     

Role of individual phosphorylation sites in inactivation of pyruvate dehydrogenase complex in rat heart mitochondria

1. A method is described using trypsin/formic acid cleavage for unambiguously measuring occupancies of phosphorylation sites in rat heart pyruvate dehydrogenase [³²P]phosphate complexes. 2. In mitochondria oxidizing 2-oxoglutarate+l-malate relative initial rates of phosphorylation were site 1>site 2>site 3. 3. Dephosphorylation and reactivation of fully phosphorylated complex was initiated in mitochondria by inhibiting the kinase reaction. Using dichloroacetate relative rates of dephosphorylation were site 2>(1=3). Using sodium dithionite or sodium pyruvate or uncouplers+sodium arsenite or steady state turnover (³¹P replacing ³²P in inactive complex) relative rates were site 2>site 1>site 3. With dithionite reactivation was faster than site 3 dephosphorylation, i.e. site 3 is apparently not inactivating. 4. The steady state proportion of inactive complex was varied (92–48%) in mitochondria oxidizing 2-oxoglutarate/l-malate by increasing extramitochondrial Ca²⁺ (0–2.6μm). This action of Ca²⁺ induced dephosphorylation (site 3>site 2>site 1). These experiments enable prediction of site occupancies in vivo for given steady state proportions of inactive complexes. 5. The proportion of inactive complex was related linearly to occupancy of site 1. 6. Sodium dithionite (10mm) and Ca²⁺ (0.5μm) together resulted in faster dephosphorylations of each site than either agent alone; relative rates were site 2>(1=3). 7. Dephosphorylation and possibly phosphorylation of sites 1 and 2 was not purely sequential as shown by detection of complexes phosphorylated in site 2 but not in site 1. Estimates of the contribution of site 2 phosphorylation to inactivation ranged from 0.7 to 6.4%. 8. It is concluded that the primary function of site 1 phosphorylation is inactivation, phosphorylation of site 2 is not primarily concerned with inactivation and that phosphorylation of site 3 is non-inactivating.     

Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.     

Influence of starvation and clofibrate administration on oxidative phosphorylation by rat liver mitochondria.

Whole cells, homogenates and mitochondrial obtained from the livers of albino rats which were starved for 6 days or more showed a 50% decrease in oxidative activity. The decrease could be corrected by the addition of cytochrome c in vitro. The phosphorylative activity of mitochondria remained unaffected. The decrease in oxidative rate was not observed when starving animals were given the anti-hypercholesterolaemic drug clofibrate. The total cellular concentration of cytochrome c was not affected by starvation. However, the concentration of the pigment in hepatic mitochondria isolated from starving animals was less than half that in normal mitochondria. Clofibrate-treated animals did not show a decreased concentration of cytochrome c in hepatic mitochondria. Mitochondria isolated from starving animals, though deficient in cytochrome c, did not show any decrease in succinate dehydrogenase activity or in the rate of substrate-dependent reduction of potassium ferricyanide or attendant phosphorylation. In coupled mitochondria, ferricyanide may not accept electrons from the cytochrome c in the respiratory chain. Starvation decreases the concentration of high-affinity binding sites for cytochrome c on the mitochondrial membrane. The dissociation constant increases in magnitude.     

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Phospholipids and the regulation of pyruvate dehydrogenase from rat adipocyte mitochondria

Aqueous dispersions of 4 out of 9 phospholipids added individually to the mitochondrial fraction from rat adipocytes altered the activity of pyruvate dehydrogenase in a dose-dependent manner from 1 to 300 microM. Phosphatidylserine increased and phosphatidylcholine, phosphatidylinositol and phosphatidylinositol-4-phosphate decreased enzyme activity. The stimulation of pyruvate dehydrogenase induced by phosphatidylserine may be reversed to below basal activity by phosphatidylinositol-4-phosphate and to basal activity by NaF, a pyruvate dehydrogenase phosphatase inhibitor. The inhibition of pyruvate dehydrogenase induced by phosphatidylinositol-4-phosphate may be restored to basal levels by the addition of calcium. These results suggest that phosphatidylserine activates pyruvate dehydrogenase activity through activation of the phosphatase, perhaps forming a phosphatidylserine-calcium complex. The inhibition by phosphatidylinositol-4-phosphate may be mediated by disruption of the enzyme complex. The phospholipids may play a physiological role in the regulation of pyruvate dehydrogenase activity.     

Comparative development of the pyruvate dehydrogenase complex and citrate synthase in rat brain mitochondria.

The enzyme activity of the pyruvate dehydrogenase complex (PDHC) was measured in mitochondria prepared from developing rat brain, before and after steady-state dephosphorylation of the E1 alpha subunit. A marked increase in dephosphorylated (fully activated) PDHC activity occurred between days 10 and 15 post partum, which represented approx. 60% of the difference in fully activated PDHC activity measured in foetal and adult rat brain mitochondria. There was no detectable change in the active proportion of the enzyme during mitochondrial preparation nor any qualitative alteration in the detectable catalytic and regulatory components of the complex, which might account for developmental changes in PDHC activity. The PDHC protein content of developing rat brain mitochondria and homogenates was measured by an enzyme-linked immunoadsorbent assay. The development of PDHC protein in both fractions agreed closely with the development of the PDHC activity. The results suggest that the developmental increase in PDHC activity is due to increased synthesis of PDHC protein, which is partly a consequence of an increase in mitochondrial numbers. However, the marked increase in PDHC activity measured between days 10 and 15 post partum is mainly due to an increase in the amount of PDHC per mitochondrion. The development of citrate synthase enzyme activity and protein was measured in rat brain homogenates and mitochondria. As only a small increase in citrate synthase activity and protein was detected in mitochondria between days 10 and 15 post partum, the marked increase in PDHC protein and enzyme activity may represent specific PDHC synthesis. As several indicators of acquired neurological competence become apparent during this period, it is proposed that preferential synthesis of PDHC may be crucial to this process. The results are discussed with respect to the possible roles played by PDHC in changes of respiratory-substrate utilization and the acquisition of neurological competence occurring during the development of the brain of a non-precocial species such as the rat.     

Effects of acute acid-base changes on rat renal pyruvate dehydrogenase. Renal pyruvate dehydrogenase during acid-base alterations

Glutamine, the principal source of urinary ammonia, can be fully oxidized or converted to glucose by the kidney. To be oxidized, the carbon skeleton of glutamine must enter the TCA cycle as acetyl CoA formed by pyruvate dehydrogenase (PDH). The purpose of this study was to measure kidney PDH activity (active and total) following acute acid-base changes in vivo. PDHa activity was elevated after acute metabolic alkalosis and acidosis and unchanged by respiratory acidosis. Kidney ADP/ATP, CoA/acetyl CoA and calculated mitochondrial NAD+/NADH ratios were also determined and revealed an increase in kidney ADP/ATP with alkalosis but no changes during metabolic and respiratory acidosis.     

Effect of streptozotocin-induced diabetes mellitus on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase.

Immunochemical techniques were used to study the effect of streptozotocin-induced diabetes on the amounts of pyruvate carboxylase and pyruvate dehydrogenase and on their rates of synthesis and degradation. Livers from diabetic rats had twice the pyruvate carboxylase activity of livers from normal rats when expressed in terms of DNA or body weight. The changes in catalytic activity closely paralleled changes in immunoprecipitable enzyme protein. Relative rates of synthesis determined by pulse-labelling studies showed that the ratio of synthesis of pyruvate carboxylase to that of average mitochondrial protein was increased 2.0-2.5 times in diabetic animals over that of control animals. Other radioisotopic studies indicated that the rate of degradation of this enzyme was not altered significantly in diabetic rats, suggesting that the increase in this enzyme was due to an increased rate of synthesis. Similar experiments with pyruvate dehydrogenase, the first component of the pyruvate dehydrogenase complex, showed that livers from diabetic rats had approximately the same amount of immunoprecipitable enzyme protein as the control animals, but a larger proportion of the enzyme was in its inactive state. The rates of synthesis and degradation of pyruvate dehydrogenase were not affected significantly by diabetes.     

Occupancy of sites of phosphorylation in inactive rat heart pyruvate dehydrogenase phosphate in vivo.

1. Inactive pyruvate dehydrogenase phosphate complexes were partially purified from hearts of fed, starved or alloxan-diabetic rats by using conditions that prevent phosphorylation or dephosphorylation. 2. Unoccupied sites of phosphorylation were assayed by incorporation of 32P from [gamma-32P]ATP into the complexes. Total sites of phosphorylation were assayed by the same method after complete reactivation, and thus dephosphorylation, of complexes by incubation with pyruvate dehydrogenase phosphate phosphatase. Occupancy is assumed from the difference (total sites--unoccupied sites). Percentage incorporation into individual sites was measured by high-voltage electrophoresis after tryptic digestion. 3. Values (means +/- S.E.M., in nmol of phosphate/unit of inactive complex) for total sites, occupied sites and percentage occupancies, with numbers of observations in parentheses were: fed, 2.1 +/- 0.04, 1.15 +/- 0.04, 54.8 +/- 1.6% (39); starved, 2.05 +/- 0.03, 1.85 +/- 0.03, 90.2 +/- 1.4% (28); alloxan-diabetic, 1.99 +/- 0.03, 1.72 +/- 0.03, 86.4 +/- 1.4% (68%). 4. Values (means +/- S.E.M. for percentage occupancy) for individual sites of phosphorylation in pyruvate dehydrogenase phosphate given in the order sites 1, 2 and 3 were : fed, 100 +/- 2.7, 27.8 +/- 1.6, 33.9 +/- .9; starved, 100 +/- 1.4, 76.2 +/- 2.0, 92.4 +/- 1.5; alloxan-diabetic, 100 +/- 1.2, 64.0 +/- 1.7, 94.6 +/- 1.4. 5. It is concluded that starvation or alloxan-diabetes leads to a 2--3-fold increase in the occupancy of phosphorylation sites 2 and 3 in pyruvate dehydrogenase phosphate in rat heart in vivo.     

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.     

Effects of re-feeding after prolonged starvation on pyruvate dehydrogenase activities in heart, diaphragm and selected skeletal muscles of the rat.

We investigated the capacity for pyruvate oxidation in skeletal muscle, diaphragm and heart after starvation and re-feeding. Starvation for 48 h decreased pyruvate dehydrogenase (PDH) activity in soleus (by 47%), extensor digitorum longus (64%), gastrocnemius (86%), diaphragm (87%), adductor longus (90%), tibialis anterior (92%) and heart (99%). Chow re-feeding increased PDH activity in all muscles to 43-78% of the fed value within 2 h. However, complete re-activation was not observed for at least 4-6 h, during which time hepatic glycogen was replenished. We discuss the importance of muscle PDH activity in relation to sparing carbohydrate for hepatic glycogen synthesis.