Bovine heart pyruvate dehydrogenase kinase stimulation by alpha-ketoisovalerate.

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and alpha-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic non-competitive or hyperbolic uncompetitive inhibition at various K+ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 microM over the range of K+ concentrations tested. In the absence of K+, 1 mM alpha-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K+, alpha-ketoisovalerate stimulated PDH kinase activity almost 2-fold over the range of 0-80 microM thiamin pyrophosphate. Half-maximal stimulation by alpha-ketoisovalerate occurred at about 200 microM in the presence of 100 microM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 microM thiamin pyrophosphate and 1 mM NH4+. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM alpha-ketoisovalerate were mixed noncompetitive, but alpha-ketoisovalerate increased the Vm and Km for adenosine 5'-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove alpha-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by alpha-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.

Bovine heart pyruvate dehydrogenase kinase stimulation by monovalent ions.

The effects of monovalent ions on endogenous pyruvate dehydrogenase (PDH) kinase activity in purified bovine heart pyruvate dehydrogenase complex were investigated. Activity of PDH kinase was stimulated 1.9-, 1.95-, 1.65-, and 1.4-fold by 10 mM K+, Rb+, NH+4, and Cs+, respectively, whereas Na+ and Li+ had no effect on PDH kinase activity. The crystal radii of stimulatory ions were in the range of 1.33 to 1.69 A while the crystal radii of nonstimulatory ions were in the range of 0.6 to 0.94 A. Stimulation of PDH kinase by monovalent ions was not pH dependent. Protein dilution studies showed that monovalent ion stimulation was measurable within 10 s after protein addition to PDH kinase assays. Furthermore, stimulation occurred at all protein concentrations tested. At ATP concentrations from 12.5 to 25 microM, K+ and NH+4 stimulation was constant from 0 to 110 and 0 to 30 mM, respectively. At higher ATP concentrations, from 50 to 500 microM, K+ and NH+4 stimulation peaked at approximately 30 and 3 mM, respectively, and thereafter declined as the ion concentration increased. Maximal PDH kinase stimulation by K+ or NH+4 also declined as Na+ was increased from 0 to 120 mM, but at a fixed salt concentration of 120 mM, both K+ and NH+4 stimulated PDH kinase activity. Phosphopeptide analysis demonstrated that K+ and NH+4 stimulated phosphorylation at sites 1 and 2, but that site 3 phosphorylation was relatively constant under all conditions. Thiamin pyrophosphate and 5,5'-dithiobis-(2-nitrobenzoate) blocked monovalent ion stimulation half-maximally at 4 and 6 microM, respectively. However, neither thiamin pyrophosphate nor 5,5'-dithiobis-(2-nitrobenzoate) significantly inhibited PDH kinase activity in the absence of monovalent ions. The results indicate that heart PDH kinase stimulation by monovalent ions does not occur by changing the binding equilibrium between PDH and dihydrolipoyl transacetylase core. Instead, monovalent ions bind and exert their regulatory effects at or near the active site of PDH kinase.

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase.

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.

Stimulation of the glycine cleavage system by short-chain fatty acids in isolated rat liver mitochondria.

The effect of short-chain fatty acids on glycine catabolism by the glycine cleavage system was investigated in isolated rat liver mitochondria. Metabolic flux through the glycine cleavage system was monitored by measuring the production of 14CO2 from [1-14C]glycine. Propionate, butyrate, pentanoate, and octanoate, and to a lesser extent acetate, all significantly stimulated 14CO2 production from [1-14C]glycine by isolated rat liver mitochondria maintained in state 4. Concomitant with the stimulation of 14CO2 production was a decrease in the measured intramitochondrial content of NADPH which we have previously demonstrated correlates with the metabolic flux through the glycine cleavage system [Hampson, R.K., Barron, L.L., & Olson, M.S. (1983) J. Biol. Chem. 258, 2993-2999]. The propionate-mediated stimulation of mitochondrial 14CO2 production from [1-14C]glycine was not diminished by the addition of L-carnitine but was abrogated nearly completely by the addition of oligomycin. Incubation of the mitochondria with short-chain fatty acids evoked a large decrease in the measured intramitochondrial ATP content and a large increase in the AMP content. However, manipulation of the intramitochondrial adenine nucleotide profile demonstrated that no direct correlation existed between ATP, ADP, or AMP and 14CO2 production from [1-14C]glycine. These experimental results indicate that short-chain fatty acid oxidation causes the oxidation of the NADP(H) redox couple in isolated rat liver mitochondria, resulting in the stimulation of the metabolic flux through the glycine cleavage system.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulatory effects of fatty acids on decarboxylation of leucine and 4-methyl-2-oxopentanoate in the perfused rat heart.

The regulatory effects of fatty acids on the oxidative decarboxylation of leucine and 4-methyl-2-oxopentanoate were investigated in the isolated rat heart. Infusion of the long-chain fatty acid palmitate resulted in both an inactivation of the branched-chain 2-oxo acid dehydrogenase and an inhibition of the measured metabolic flux through this enzyme complex. Pyruvate addition also caused both an inactivation and an inhibition of the flux through the complex. On the other hand, the medium-chain fatty acid octanoate caused an activation of and a stimulation of flux through the branched-chain 2-oxo acid dehydrogenase when the perfusion conditions before octanoate addition maintained the enzyme complex in its inactive state. When the enzyme complex was activated before octanoate infusion, this fatty acid caused a significant inhibition of the flux through the branched-chain 2-oxo acid dehydrogenase reaction. Inclusion of glucose in the perfusion medium prevented the octanoate-mediated activation of the branched-chain 2-oxo acid dehydrogenase.

The stimulation of hepatic gluconeogenesis by acetoacetate precursors. A role for the monocarboxylate translocator.

The regulation of the gluconeogenic pathway from the 3-carbon precursors pyruvate, lactate, and alanine was investigated in the isolated perfused rat liver. Using pyruvate (less than 1 mM), lactate, or alanine as the gluconeogenic precursor, infusion of the acetoacetate precursors oleate, acetate, or beta-hydroxybutyrate stimulated the rate of glucose production and, in the case of pyruvate (less than 1 mM), the rate of pyruvate decarboxylation. alpha-Cyanocinnamate, an inhibitor of the monocarboxylate transporter, prevented the stimulation of pyruvate decarboxylation and glucose production due to acetate infusion. With lactate as the gluconeogenic precursor, acetate infusion in the presence of L-carnitine stimulated the rate of gluconeogenesis (100%) and ketogenesis (60%) without altering the tissue acetyl-CoA level usually considered a requisite for the stimulation of gluconeogenesis by fatty acids. Hence, our studies suggest that gluconeogenesis from pyruvate or other substrates which are converted to pyruvate prior to glucose synthesis may be limited or controlled by the rate of entry of pyruvate into the mitochondrial compartment on the monocarboxylate translocator.

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

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