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PubMed1984

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes by increasing the intramitochondrial concentration of Ca2+.

Increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase which result from the perfusion of rat hearts with adrenaline were still evident during the preparation of mitochondria in sucrose-based media containing EGTA (at 0 degrees C) and their subsequent incubation at 30 degrees C in Na+-free KCl-based media containing respiratory substrates and EGTA. The differences from control values gradually diminished with time of incubation, but were still present after 8 min. Similar increases resulting from an increase in the concentration of Ca2+ in the perfusing medium also persisted. However, similar increases caused by 5 mM-pyruvate were only maintained during the preparation of mitochondria, not their incubation. Parallel increases, within incubated mitochondria, were found in the activity of the 2-oxoglutarate dehydrogenase complex assayed at a non-saturating concentration of 2-oxoglutarate. The enhancement of the activities of both of these Ca2+-sensitive enzymes within incubated mitochondria as a result of perfusion with adrenaline or a raised concentration of Ca2+ in the medium could be abolished within 1 min by the presence of 10 mM-NaCl. This effect of Na+ was blocked by 300 microM-diltiazem, which has been shown to inhibit Na+-induced egress of Ca2+ from rabbit heart mitochondria [Vághy, Johnson, Matlib, Wang & Schwartz (1982) J. Biol. Chem. 257, 6000-6002]. The enhancements could also be abolished by increasing the extramitochondrial concentration of Ca2+ to a value where it caused maximal activation of the enzymes within control mitochondria. The results are consistent with the hypothesis that adrenaline activates rat heart pyruvate dehydrogenase by increasing the intramitochondrial concentration of Ca2+ and that this increase persists through to incubated mitochondria. Support for this conclusion was obtained by the yielding of a similar set of results from parallel experiments performed on control mitochondria that had firstly been preincubated (under conditions of steady-state Ca2+ cycling across the inner membrane) with sufficient proportions of Ca-EGTA buffers to achieve a similar degree of Ca2+-activation of pyruvate dehydrogenase (as caused by adrenaline) and had then undergone the isolation procedure again.

Animals↗

PubMed2000

Sequential deletion of C-terminal amino acids of the E(1)alpha component of the pyruvate dehydrogenase (PDH) complex leads to reduced steady-state levels of functional E(1)alpha(2)beta(2) tetramers: implications for patients with PDH deficiency.

Human pyruvate dehydrogenase (PDH) complex deficiency is an extremely heterogeneous disease in its presentation and clinical course. We have characterized novel mutations that affect the C-terminal portion of the PDH-E(1)alpha-coding sequence. Although the molecular defects underlying these mutations are different, both effectively produce a stop codon prematurely three amino acids from the C-terminus. The clinical and biochemical consequences of these mutations are unusual in that the affected individuals are very long-term survivors with PDH complex deficiency despite having low (<20%) activity in skin fibroblasts. These findings prompted us to investigate the C-terminus of E(1)alpha in greater detail. We constructed and expressed a series of PDH-E(1)alpha deletion mutants in a cell line with zero PDH complex activity due to a null E(1)alpha allele. Sequential deletion of the C-terminus by one, two, three and four amino acids resulted in PDH complex activities of 100, 60, 36 and 14%, respectively, compared with wild-type E(1)alpha expressed in PDH complex-deficient cells. The immunodetectable protein was decreased by the same amount as the activity, suggesting that the stability and/or assembly of the E(1)alpha(2)beta(2)heterotetramer might depend on the intactness of the PDH-E(1)alpha C-terminus. In addition, we compared the somatic and the testis-specific isoforms of E(1)alphaand concluded that they are biochemically equivalent.

Adult↗

PubMed1987

Hormonal regulation of fluxes through pyruvate dehydrogenase and the citric acid cycle in mammalian tissues.

Three key dehydrogenases in the mitochondria of higher animals have been found to be activated by Ca2+ ions; these are pyruvate dehydrogenase and two enzymes in the citric acid cycle, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. Activation can also be demonstrated within permeabilized and intact mitochondria. Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+, then this results in an increase in the intramitochondrial concentration of Ca2+. In this way, rates of pyruvate oxidation and citric acid cycle flux are increased, and hence there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP. In contrast, the activation of pyruvate dehydrogenase which is observed in adipose and other tissues exposed to insulin is brought about by a Ca2+-independent mechanism.

Animals↗

PubMed2002

Up-regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) protein expression in oxidative skeletal muscle does not require the obligatory participation of peroxisome-proliferator-activated receptor alpha (PPARalpha).

In insulin deficiency, increased lipid delivery and oxidation suppress skeletal-muscle glucose oxidation by inhibiting pyruvate dehydrogenase complex (PDC) activity via enhanced protein expression of pyruvate dehydrogenase kinase (PDK) isoform 4, which phosphorylates (and inactivates) PDC. Signalling via peroxisome-proliferator-activated receptor alpha (PPARalpha) is an important component of the mechanism enhancing hepatic and renal PDK4 protein expression. Activation of PPARalpha in gastrocnemius, a predominantly fast glycolytic (FG) muscle, also increases PDK4 expression, an effect that, if extended to all muscles, would be predicted to drastically restrict whole-body glucose disposal. Paradoxically, chronic activation of PPARalpha by WY14,643 treatment improves glucose utilization by muscles of insulin-resistant high-fat-fed rats. In the resting state, oxidative skeletal muscles are quantitatively more important for glucose disposal than FG muscles. We evaluated the participation of PPARalpha in regulating PDK4 protein expression in slow oxidative (SO) skeletal muscle (soleus) and fast oxidative-glycolytic (FOG) skeletal muscle (anterior tibialis) containing a high proportion of oxidative fibres. In the fed state, acute (24 h) activation of PPARalpha by WY14,643 in vivo failed to modify PDK4 protein expression in soleus, but modestly enhanced PDK4 protein expression in anterior tibialis. Starvation enhanced PDK4 protein expression in both muscles, with the greater response in anterior tibialis. WY14,643 treatment in vivo during starvation did not further enhance upregulation of PDK4 protein expression in either muscle type. Enhanced PDK4 protein expression after starvation was retained in SO and FOG skeletal muscles of PPARalpha-deficient mice. Our data indicate that PDK4 protein expression in oxidative skeletal muscle is regulated by a lipid-dependent mechanism that is not obligatorily dependent on signalling via PPARalpha.

Animals↗

PubMed2003

The pyruvate dehydrogenase complex as a target for gene therapy.

Here we review the rationale for considering the pyruvate dehydrogenase multienzyme complex (PDC) as a target for gene therapy for defects in mitochondrial energetics. PDC is entirely nuclear encoded and is situated in the mitochondrial inner membrane. The complex catalyzes the rate-determining step in aerobic carbohydrate metabolism and plays a critical role in the efficient conversion of substrate fuel into energy by cells. PDC activity is regulated in large part by reversible phosphorylation (inactivation) of its E1alpha subunit. Congenital defects in PDC are usually due to mutations in E1alpha and are typified by lactic acidosis, neurodegeneration and early death. Acquired deficiency in PDC has been implicated in the etiopathology of several other metabolic or neurodegenerative disorders. Recently, a vector using recombinant adeno-associated virus (rAAV) that contained a fusion protein of full-length E1alpha and the reporter gene green fluorescent protein was used to deliver wild type E1alpha into mitochondria after injection of the construct in vivo into the central nervous system of rats and in vitro into human cells. Transduction of cultured fibroblasts from a male patient with E1alpha deficiency led to partial restoration of PDC activity, as determined by decarboxylation of 14C-pyruvate. These data indicate that at least partial correction of PDC defects may be feasible by gene transfer. Furthermore, the combination of AAV-mediated delivery of E1alpha with pharmacologic activation (dephosphorylation) of the wild type enzyme subunit may provide an optimal therapeutic strategy for patients with acquired or congenital deficiencies in mitochondrial energy metabolism.

Animals↗

PubMed1996

Antibodies to E1 and E2/Protein X components of pyruvate dehydrogenase complex in sera of patients with primary biliary cirrhosis.

AIMS/METHODS: Using purified E1 component of pyruvate dehydrogenase complex (PDC) from bovine heart, we measured the levels of anti-E1 antibodies in PBC sera using ELISA and determined the degree of inhibition that these antibodies exerted on E1 enzyme activity. We also estimated levels of anti-E2/Protein X (Pro-X) antibodies in PBC sera using purified E2 and Pro-X of PDC which were copurified with E1. RESULTS/CONCLUSIONS: Anti-E1 antibodies were detected in 87.5% (35/40) of PBC sera. Some of these sera inhibited E1 enzyme activity but inhibition did not correlate with levels of anti-E1 antibodies. A high positive correlation (r = 0.918) was found between levels of anti-E1 and anti-E2/Pro-X antibodies, suggesting that anti-PDC antibody production was stimulated by PDC itself. Levels of IgG class anti-E2/Pro-X antibodies were significantly higher in sera of symptomatic PBC patients than in those of asymptomatic PBC patients. It was also found that patients who were positive for only IgM class anti-E2/Pro-X antibodies had early-stage PBC.

Animals↗

PubMed2002

Evaluation of the role of peroxisome-proliferator-activated receptor alpha in the regulation of cardiac pyruvate dehydrogenase kinase 4 protein expression in response to starvation, high-fat feeding and hyperthyroidism.

Inactivation of cardiac pyruvate dehydrogenase complex (PDC) after prolonged starvation and in response to hyperthyroidism is associated with enhanced protein expression of pyruvate dehydrogenase kinase (PDK) isoform 4. The present study examined the potential role of peroxisome-proliferator-activated receptor alpha (PPARalpha) in adaptive modification of cardiac PDK4 protein expression after starvation and in hyperthyroidism. PDK4 protein expression was analysed by immunoblotting in homogenates of hearts from fed or 48 h-starved rats, rats rendered hyperthyroid by subcutaneous injection of tri-iodothyronine and a subgroup of euthyroid rats maintained on a high-fat/low-carbohydrate diet, with or without treatment with the PPARalpha agonist WY14,643. In addition, PDK4 protein expression was analysed in hearts from fed, 24 h-starved or 6 h-refed wild-type or PPARalpha-null mice. PPARalpha activation by WY14,643 in vivo over the timescale of the response to starvation failed to up-regulate cardiac PDK4 protein expression in rats maintained on standard diet (WY14,643, 1.1-fold increase; starvation, 1.8-fold increase) or influence the cardiac PDK4 response to starvation. By contrast, PPARalpha activation by WY14,643 in vivo significantly enhanced cardiac PDK4 protein expression in rats maintained on a high-fat diet, which itself increased cardiac PDK4 protein expression. PPARalpha deficiency did not abolish up-regulation of cardiac PDK4 protein expression in response to starvation (2.9-fold increases in both wild-type and PPARalpha-null mice). Starvation and hyperthyroidism exerted additive effects on cardiac PDK4 protein expression, but PPARalpha activation by WY14,643 did not influence the response of cardiac PDK4 protein expression to hyperthyroidism in either the fed or starved state. Our data support the hypothesis that cardiac PDK4 protein expression is regulated, at least in part, by a fatty acid-dependent, PPARalpha-independent mechanism and strongly implicate a fall in insulin in either initiating or facilitating the response of cardiac PDK4 protein expression to starvation.

Animals↗

PubMed1964

ELECTRON MICROSCOPIC AND BIOCHEMICAL STUDIES OF PYRUVATE DEHYDROGENASE COMPLEX OF ESCHERICHIA COLI.

Examination with the electron microscope of the pyruvate dehydrogenase complex and its component enzymes from Escherichia coli indicates that the complex has a polyhedral structure with a diameter of about 300 to 350 angstroms and a height of 200 to 250 angstroms. The reconstituted complex closely resembles the native complex in appearance. A tentative model of this multienzyme complex is proposed on the basis of the correlated biochemical and electron-microscopic data

Acetates↗

PubMed1987

The domain structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

Limited proteolysis with trypsin has been used to study the domain structure of the dihydrolipoyltransacetylase (E2) component of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Two stable end products were obtained and identified as the N-terminal lipoyl domain and the C-terminal catalytic domain. By performing proteolysis of E2, which was covalently attached via its lipoyl groups to an activated thiol-Sepharose matrix, a separation was obtained between the catalytic domain and the covalently attached lipoyl domain. The latter was removed from the column after reduction of the S-S bond and purified by ultrafiltration. The lipoyl domain is monomeric with a mass of 32.6 kDa. It is an elongated structure with f/fo = 1.62. Circulair dichroic studies indicates little secondary structure. The catalytic domain is polymeric with S20.w = 17 S and mass = 530 kDa. It is a compact structure with f/fo = 1.24 and shows 40% of the secondary structure of E2. The cubic structure of the native E2 is retained by this fragment as observed by electron microscopy. Ultracentrifugation in 6 M guanidine hydrochloride in the presence of 2 mM dithiothreitol yields a mass of 15.8 kDa. An N-terminal sequence of 36 amino acids is homologous with residues 370-406 of Escherichia coli E2. The catalytic domain possesses the catalytic site, but in contrast to the E. coli subunit binding domain the pyruvate dehydrogenase (E1) and lipoamide dehydrogenase (E3) binding sites are lost during proteolysis. From comparison with the E. coli E2 sequence a model is presented in which the several functions, such as lipoyl domain, the E3 binding site, the catalytic site, the E2/E2 interaction sites, and the E1 binding site, are indicated.

Acetyltransferases↗

PubMed1981

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes from bacterial and mammalian sources.

A simple method was developed for assessing the intramolecular coupling of active sites in the lipoate acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complexes from Escherichia coli, Bacillus stearothermophilus and ox heart and pig heart mitochondria. Samples of enzyme complex were prepared in which the pyruvate decarboxylase (E1) component was selectively and partly inhibited by treatment with increasing amounts of a transition-state analogue, thiamin thio-thiazolone pyrophosphate. The fraction of the E2 component acetylated by incubation with [2-14C] pyruvate, in the absence of CoA, was determined for each sample of partly inhibited enzyme and was found in all cases to exceed the fraction of overall complex activity remaining. This indicated the potential for transacetylation reactions among the lipoic acid residues within the E2 core. A graphic presentation of the data allowed comparison of the active-site coupling in the various enzymes, which may differ in their lipoic acid content (one or two residues per E2 chain). It is clear that active-site coupling is a general property of pyruvate dehydrogenase complexes of octahedral and icosahedral symmetries, the large numbers of subunits in each E2 core enhancing the effect.

Acetylation↗

PubMed1997

Cloning, expression, and properties of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

cDNA encoding the regulatory subunit of bovine mitochondrial pyruvate dehydrogenase phosphatase (PDPr) has been cloned. Overlapping cDNA fragments were generated by the polymerase chain reaction from bovine genomic DNA and from cDNA synthesized from bovine poly(A)+ RNA and total RNA. The complete cDNA (2885 base pairs) contains an open reading frame of 2634 nucleotides encoding a putative presequence of 31 amino acid residues and a mature protein of 847 residues with a calculated Mr of 95,656. This value is in agreement with the molecular mass of native PDPr (95,800 +/- 200 Da) determined by matrix-assisted laser desorption-ionization mass spectrometry. The mature form of PDPr was expressed in Escherichia coli as a maltose-binding protein fusion, and the recombinant protein was purified to near homogeneity. It exhibited properties characteristic of the native PDPr, including recognition by antibodies against native bovine PDPr, ability to decrease the sensitivity of the catalytic subunit to Mg2+, and reversal of this inhibitory effect by the polyamine spermine. A BLAST search of protein data bases revealed that PDPr is distantly related to the mitochondrial flavoprotein dimethylglycine dehydrogenase, which functions in choline degradation.

ATP-Binding Cassette Transporters↗

PubMed2002

Molecular architecture and mechanism of an icosahedral pyruvate dehydrogenase complex: a multifunctional catalytic machine.

Electron cryo-microscopy of 'single particles' is a powerful method to determine the three-dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi-enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl-CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub-complex, obtained by combining a 28 A structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.

Catalysis↗

PubMed1998

Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex. Identification of important amino acid residues.

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mitochondrion, is diminished in animal models of diabetes. Studies with purified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydrolipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdomain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inactivation of the complex. By combining molecular modelling, site-directed mutagenesis and biophysical characterizations, we have also identified two amino acid residues in this subdomain (Ile229 and Phe231) that largely determine the magnitude of this effect.

Acetyltransferases↗

PubMed2006

Direct kinetic evidence for half-of-the-sites reactivity in the E1 component of the human pyruvate dehydrogenase multienzyme complex through alternating sites cofactor activation.

Recent kinetic and structural studies on various thiamin-dependent enzymes, including the bacterial E1 component of the pyruvate dehydrogenase complex (PDHc), suggested an active center communication between the cofactors in these multimeric enzymes. This regulatory mode has been inferred from the dissymmetry of active sites in proteolytic patterns and X-ray structures and from a complex macroscopic kinetic behavior not being consistent with independently working active sites. Here, direct microscopic kinetic evidence for this hypothesis is presented for the alpha2beta2-type E1 component of the human pyruvate dehydrogenase complex. Only one of the two thiamin molecules bound to the two active sites is in a chemically activated state exhibiting an apparent C2 ionization rate constant of approximately 50 s(-1) at pH 7.6 and 30 degrees C, whereas the thiamin in the "inactive site" ionizes with a rate that is at least 3 orders of magnitude smaller. The chemical nonequivalence is also exhibited in the ability to bind the substrate analogue methyl acetylphosphonate and in the catalytic turnover of the substrate pyruvate in the E1-only reaction. In the activated active site, pyruvate is rapidly bound and decarboxylated with apparent forward rate constants of covalent pyruvate binding of 2 s(-1) and decarboxylation of the formed 2-lactyl-thiamin intermediate of 5 s(-1). In the dormant site, these steps are as slow as 0.03 s(-1). Under the conditions that were used, only the heterotetramer can be detected by analytical ultracentrifugation, thus ruling out the possibility that multiple oligomeric species with different reactivities cause the observed kinetic effects. The results are consistent with the recently suggested model of an active site synchronization in PDHc-E1 via a proton wire that keeps the two active sites in an alternating activation state [Frank, R. A., et al. (2004) Science 306, 872]. Kinetic studies on the related thiamin enzymes transketolase, pyruvate oxidase, and bacterial pyruvate decarboxylase are not consistent with a chemical and/or functional nonequivalence of the active sites as observed in the E1 component of hsPDHc. We hypothesize that the alternating sites reaction in PDHc-E1 aids in the synchronized acyl transfer to the E2 component in the highly organized multienzyme complex.

Amino Acid Sequence↗

PubMed1996

Pyruvate dehydrogenase activity and malonyl CoA levels in normal and ischemic swine myocardium: effects of dichloroacetate.

The purposes of this study were to: (1) assess myocardial pyruvate dehydrogenase (PDH) activity and substrate exchange under well-perfused and ischemic conditions; (2) determine the metabolic effects of an intra-coronary infusion of the PDH activator, dichloroacetate (DCA); and (3) measure the effects of ischemia and DCA on malonyl CoA levels. Experiments were performed in anesthetised open-chest swine under non-ischemic conditions, followed by 40 min with a 60% reduction in left anterior descending coronary artery (LAD) blood flow. Myocardial needle biopsies for measurement of PDH activity were taken after an intracoronary infusion of either saline or DCA (1 mM in LAD blood) under aerobic conditions, and after 37 min of ischemia. Pyruvate dehydrogenase activity was measured with and without maximal activation by swine PDH phosphatase. Malonyl CoA and acetyl CoA were measured after 40 min of LAD ischemia in myocardium from the ischemic DCA- or saline-treated LAD bed, and the non-ischemic untreated left circumflex coronary artery (CFX) perfusion bed. Net glucose, lactate and free fatty acid (FFA) uptakes were measured across the LAD perfusion bed throughout the study. Dichloroacetate treatment increased the amount of active dephosphorylated PDH to 88% of the total activity under aerobic conditions, compared to 55% with saline (P < 0.01). Ischemia did not significantly change PDH activation state in either group. Acetyl CoA and malonyl CoA contents were significantly elevated in ischemic DCA-treated myocardium compared to saline-treated ischemic myocardium. Dichloroacetate treatment significantly lowered rates of myocardial FFA uptake under both aerobic and ischemic conditions, but did not effect glucose uptake or lactate exchange. Free fatty acid uptake was negatively correlated to malonyl CoA levels (r = -0.68) during ischemia. It is proposed that the inhibition of FFA uptake observed with DCA in ischemic myocardium is due to malonyl CoA inhibition of carnitine palmitoyl transferase I.

Animals↗

PubMed1994

Expression in Escherichia coli of genes encoding the E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex of Bacillus stearothermophilus and assembly of a functional E1 component (alpha 2 beta 2) in vitro.

The E1 alpha and E1 beta subunits of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus were produced from two genes overexpressed separately in Escherichia coli. A functional E1 enzyme was generated from disrupted mixtures of cells containing the separately overexpressed E1 alpha and E1 beta genes. The purified E1 enzyme exhibited an apparent molecular mass of 150,000 Da, consistent with an alpha 2 beta 2 structure. The Km for pyruvate and kcat (30 degrees C) were found to be 0.9 +/- 0.2 microM and 0.47 +/- 0.03 s-1, respectively. The purified E1 alpha subunit existed as a monomer (42,000 Da), whereas the E1 beta subunit existed mainly (95%) in a tetrameric form (145,000 Da). Mixing equimolar amounts of the pure recombinant E1 alpha and E1 beta subunits in vitro generated a functional E1 enzyme with a molecular mass and an E1 activity similar to those of the E1(alpha 2 beta 2) enzyme purified from disrupted mixtures of cells containing individually expressed subunits. Mixing individual subunits in vitro with one of the subunits in excess resulted in complete assembly of the lesser subunit into the intact E1 (alpha 2 beta 2) enzyme. Thus, no chaperonin is needed in vitro to promote the assembly of the separate subunits to form the E1 component of the pyruvate dehydrogenase multienzyme complex of B. stearothermophilus.

Base Sequence↗

PubMed2004

Downregulation of the skeletal muscle pyruvate dehydrogenase complex in the Otsuka Long-Evans Tokushima Fatty rat both before and after the onset of diabetes mellitus.

The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidative decarboxylation of pyruvate in mitochondria. The PDC activity is regulated by a phosphorylation/dephosphorylation cycle catalyzed by specific kinases (PDK) and phosphatases (PDP). In this study, the regulatory mechanisms of PDC were examined in skeletal muscle of the spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rat before and after the onset of diabetes. The Long-Evans Tokushima Otsuka (LETO) rat was used as control. Plasma glucose and insulin concentrations were at normal levels in both groups at 8 weeks of age but were significantly higher in OLETF than in LETO rats at 25 weeks of age (1.2-fold for glucose and 15-fold for insulin), indicating development of diabetes in the former. Plasma free fatty acids were 1.6-fold concentrated and the skeletal muscle PDC activity state was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidation in OLETF rats even before the onset of diabetes. The PDK activity and the abundance of the PDK isoform 4 protein as well as mRNA were greater in OLETF rats at both ages. Conversely, the abundance of the PDP isoform 1 protein and mRNA was less in OLETF than in LETO rats at both ages. These results suggest that concomitant greater PDK4 and less PDP1 expression in skeletal muscle of OLETF rats before the onset of diabetes are responsible for the lowering of the PDC activity and may be related with the development of diabetes mellitus.

Age Factors↗

PubMed1985

Modification of bovine kidney pyruvate dehydrogenase kinase activity by CoA esters and their mechanism of action.

The activation of pyruvate dehydrogenasea kinase activity by CoA esters has been further characterized. Half-maximal activation of kinase activity was achieved with about 1.0 microM acetyl-CoA after a 20-s preincubation in the presence of NADH. More than 80% of the acetyl-CoA was consumed during this period in acetylating sites in the pyruvate dehydrogenase complex as a result of the transacetylation reaction proceeding to equilibrium. At 1.0 microM acetyl-CoA, this resulted in more than a 4-fold higher level of CoA than residual acetyl-CoA. Activation of kinase activity could result either from acetylation of specific sites in the complex or tight binding of acetyl-CoA. Removal of CoA enhanced both acetylation and activation, suggesting acetylation mediates activation. For allosteric binding of acetyl-CoA to elicit activation, an activation constant, Ka, less than 50 nM would be required. To further distinguish between those mechanisms, the effects of other CoA esters as well as the reactivity of most of the effective CoA esters were characterized. Several short-chain CoA esters enhanced kinase activity including (in decreasing order of effectiveness) malonyl-CoA, acetoacetyl-CoA, propionyl-CoA, and methylmalonyl-CoA. Butyryl-CoA inhibited kinase activity as did high concentrations of long-chain acyl-CoAs. Inhibition by long-chain acyl-CoAs may result, in part, from detergent-like properties of those esters. Malonyl-CoA, propionyl-CoA, butyryl-CoA, and methylmalonyl-CoA, obtained with radiolabeled acyl groups, were shown to acylate sites in the complex. Propionyl-CoA and butyryl-CoA were tested, in competition with acetyl-CoA or pyruvate, as alternative substrates for acylation of sites in the complex and as competitive effectors of kinase activity. Propionyl-CoA alone rapidly acylated sites in the complex at low concentrations, and low concentrations of propionyl-CoA were effective in activating kinase activity although only a relatively small activation was observed. When an equivalent level (20 microM) of acetyl-CoA and propionyl-CoA was used, marked activation of kinase activity due to a dominant effect of acetyl-CoA was associated with acetylation of a major portion of sites in the complex and with a small portion undergoing acylation with propionyl-CoA. Those results were rapidly achieved in a manner independent of the order of addition of the two CoA esters. That indicates that tight slowly reversible binding of acetyl-CoA is not involved in kinase activation. High levels of propionyl-CoA greatly reduced acetylation by acetyl-CoA and nearly prevented activation of kinase activity by acetyl-CoA.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyl Coenzyme A↗