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PubMed1986

Cyclic AMP-dependent phosphorylation, Ca2+ and calmodulin: possible influences on acyltransferases and pyruvate dehydrogenase activities of rat mammary gland.

Specific activities of diacylglycerol acyltransferase, glycerol 3-phosphate acyltransferase and pyruvate dehydrogenase were studied in virgin, pregnant, lactating and involuting rat mammary glands. An inverse relationship was evident between cAMP binding to protein kinase(s) and the activities of the above enzymes in lactating rat mammary glands. Results suggested that free Ca2+ concentration may also contribute to control of the activity of pyruvate dehydrogenase in these glands. However, no consistent change was observed between the activities of these enzymes and cAMP binding in young, pregnant and involuting rat mammary glands. Calmodulin levels paralleled bound Ca2+ except in lactating rats. Almost all parameters studied peaked on day 8 of lactation.

Acyltransferases↗
PubMed1987

Relation between cytosolic free Ca2+ concentration and the control of pyruvate dehydrogenase in isolated cardiac myocytes.

The proportion of pyruvate dehydrogenase existing in the active form (PDHA) in suspensions of unstimulated cardiac myocytes oxidizing glucose is approx. 30%. Depolarization of the cells with concentrations of K+ above physiological values leads to an increase in the content of PDHA. Overloading of the cells with Na+ by treatment with veratridine and ouabain gives the same result. Each of these interventions is shown in experiments with Quin 2-loaded myocytes to lead to an increase in cytosolic free Ca2+ concentration ([Ca2+]c). Treatment of the cells with Ruthenium Red, an inhibitor of Ca2+ transport into mitochondria, largely prevents an increase in PDHA in response to addition of KCl or of veratridine plus ouabain. Ruthenium Red does not attenuate the increase in [Ca2+]c that occurs under these conditions. By contrast, treatment of the cells with ryanodine, an inhibitor of sarcoplasmic-reticulum Ca2+ transport and therefore of contraction, does not diminish the response of PDHA content to agents which raise [Ca2+]c; nor does loading of the cells with the Ca2+-chelating agent Quin 2, which also prevents contraction, at appropriate concentrations. It is concluded that an increase in [Ca2+]c causes an increase in PDHA content of cardiac myocytes independently of an increase in mechanical work. In the normal physiological situation the activation of dehydrogenases by Ca2+ is thought to help to maintain the balance of energy supply and demand during periods of increased work-load, which are associated with an increased myoplasmic [Ca2+]c.

Animals↗
PubMed1979

Inactivation and disassembly of the pyruvate dehydrogenase multienzyme complex from bovine kidney by limited proteolysis with an enzyme from rat liver.

Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin-sensitive enzyme (termed 'inactivase') obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex. By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high-molecular-weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.

Animals↗
PubMed1999

The inhibitory effects of lipoic compounds on mammalian pyruvate dehydrogenase complex and its catalytic components.

To examine the stereospecific effects of lipoic compounds on pyruvate metabolism, the effects of R-lipoic acid (R-LA), S-lipoic acid (S-LA) and 1,2-diselenolane-3-pentanoic acid (Se-LA) on the activities of the mammalian pyruvate dehydrogenase complex (PDC) and its catalytic components were investigated. Both S-LA and R-LA markedly inhibited PDC activity; whereas Se-LA displayed inhibition only at higher concentrations. Examination of the effects on the individual catalytic components indicated that Se-LA inhibited the pyruvate dehydrogenase component; whereas R-LA and S-LA inhibited the dihydrolipoamide acetyltransferase component. The three lipoic compounds lowered dihydrolipoamide dehydrogrenase (E3) activity in the forward reaction by about 30 to 45%. The kinetic data of E3 showed that both R-LA and Se-LA are used as substrates by E3 for the reverse reaction. Decarboxylation of [1-14C]pyruvate via PDC by cultured HepG2 cells was not affected by R-LA, but moderately decreased with S-LA and Se-LA. These findings indicate that (i) purified PDC and its catalytic components are affected by lipoic compounds based on their stereoselectivity; and (ii) the oxidation of pyruvate by intact HepG2 cells is not inhibited by R-LA. The later finding with the intact cells is in support of therapeutic role of R-LA as an antioxidant.

Acetyltransferases↗
PubMed1995

Deficiency of pyruvate dehydrogenase activity in pancreatic islets of diabetic GK rats.

We investigated the role of islet pyruvate dehydrogenase (PDH) enzyme activity and fatty acid oxidation in the impaired insulin secretion in spontaneously diabetic GK rats. Blood glucose levels were elevated in 2- to 3-month-old GK rats (8.7 +/- 0.5 vs. 6.5 +/- 0.3 mM in control Wistar rats; P < 0.01), whereas serum insulin levels were comparable to those in control rats. Insulin and DNA contents were similar in freshly isolated islets from GK and control rats, whereas insulin responses to 27 mM glucose from GK islets were reduced by 52%. The effect of acetate or pyruvate on insulin responses evoked by succinate monomethylester (SAM) were compared to indirectly assess deficient generation of acetyl-coenzyme A from pyruvate. Acetate potentiated SAM-induced insulin secretion similarly in GK and control islets, whereas 10 mM pyruvate (which supplies acetyl-coenzyme A through PDH enzyme activity) failed to normally potentiate insulin secretion in GK islets (92% of SAM-induced response in GK vs. 154% in control islets). The PDH activity (active form) was decreased in GK islets by 35% (P < 0.001). The proportion of active form PDH to total PDH activity was reduced in GK islets (56% vs. 71% in control islets; P < 0.01). The activity of PDH kinase (which inactivates PDH by phosphorylation) was increased in GK islets, the rate of ATP-dependent inactivation of PDH was -0.29 +/- 0.02 vs. -0.19 +/- 0.02/min in control islets (P < 0.05). Culturing GK islets for 48 h at 5.5 mM glucose failed to correct the impaired insulin response to glucose and the decreased PDH activity. Serum FFA levels and islet triglyceride contents did not differ between GK and control rats. Etomoxir (1.0 and 10 microM), a carnitine palmitoyl transferase I inhibitor, failed to enhance glucose-induced insulin release in GK islets. The following conclusions were reached: 1) a kinase-mediated decrease in PDH activity in islets of GK rats may in part account for the decreased ratio of oxidized to utilized glucose and impaired insulin release in these islets; and 2) impaired insulin release in the GK rats is not linked to an inhibitory influence of islet fatty acid oxidation.

Acetates↗
PubMedDate not supplied

Multiple effects of amobarbital on Ehrlich ascites tumor cells. Inhibition of pyruvate dehydrogenase.

The inhibition of the proliferation of hyperdiploid Ehrlich ascites tumor cells in suspension cultures by amobarbital is coupled to an increased glycolytic activity as shown by lactic acid production and glucose consumption; higher concentrations of amobarbital than 1 mM enhance the ATP/ADP ratio of the total cell. The actual activity of pyruvate dehydrogenase of intact cells is completely inhibited in the presence of 2 mM amobarbital as was shown by the 14CO2 evolution from [114C]pyruvate or the incorporation of 14CO2 into the total lipid fraction of the cells from [U-14C]lactate. The pyruvate dehydrogenase complex from Ehrlich ascites tumor cells is completely inhibited by 1 mM amobarbital in vitro. The activity of alpha-oxoglutarate dehydrogenase is inhibited by amobarbital, too, as well as shown by measuring the 14CO2 evolution from [1-14C]glutamate with intact cells. It is suggested that the inhibition of pyruvate dehydrogenase in the presence of amobarbital is the result of a direct action on the enzyme as well as the consequence of a change in the cellular redox state or its energy charge.

Adenosine Diphosphate↗
PubMed1975

X-ray small-angle studies of the pyruvate dehydrogenase core complex from Escherichia coli K-12. I. Overall structure of the core complex.

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex which can be obtained with a unique polypeptide chain composition, has been investigated in solution with the X-ray small-angle technique. The molecular mass of the core complex of 3.78-10(6) daltons verifies the ratio of polypeptide chains of 16:16:16 of the three enzyme components, pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, present in the complex. In connection with the values obtained for the radius of gyration (156.5A), volume (1.07(7) A3) and amount of solvent associated with the complex (1.03 g/g) a loose packing of subunits in the complex has to be assumed. The maximum diameter of the core complex of 433 A, as determined from the correlation function, corroborates the large extension of the complex. The comparison of experimental and theoretical scattering curves reveals a relatively isometric overall shape of the core complex.

Escherichia coli↗
PubMed1978

Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries.

1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, alpha-subunit 40600, beta-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the alpha-subunit of the decarboxylase. 5. The molar ratio of phosphate to alpha-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [(32)P]phosphate complex by using methyl [1-(14)C]acetimidate, followed by separation of alpha-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two alpha-chains (i.e. one phosphate group/alpha(2)beta(2) tetramer). Complete phosphorylation corresponded to three phosphate groups per alpha(2)beta(2) tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 alpha-subunits:4 beta-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.

Chemical Phenomena↗
PubMed1983

Effects of high fat and high carbohydrate diets on liver pyruvate dehydrogenase and its activation by a chemical mediator released from insulin-treated liver particulate fraction: effect of neuraminidase treatment on the chemical mediator activity.

Rats were fed a high fat diet or a high glucose diet for 5-7 days. Basal pyruvate dehydrogenase activity (both the active form and the total enzyme activity) was decreased in liver homogenates from fat diet-adapted rats as compared to those fed the glucose diet. Supernatants from insulin-exposed liver particulate fractions from fat-fed rats showed decreased stimulation of pyruvate dehydrogenase activity as compared to those from glucose-fed rats. There was no difference in the response of the mitochondria from the two groups when they were stimulated by supernatants from insulin-treated liver particulate fractions from stock diet-fed rats. Liver particulate fractions from fat-fed rats showed decreased generation of the chemical activator in response to Concanavalin A and trypsin stimulation. This suggests that fat feeding results in a decrease in membrane protease substrate availability. Treatment of the insulin mediator with neuraminidase and beta-D-galactosidase resulted in inactivation of the mediator. Presence of exogenous enzyme substrates during enzyme digestion protected the mediator from inactivation, suggesting that carbohydrate residues are important in the action of the insulin mediator. This fat diet-induced decrease in the generation of a chemical mediator of insulin action may result from 1) a decrease in insulin binding, shown earlier; 2) a decrease in the amount of protease substrate; and 3) an alteration in its carbohydrate composition, which is important in its ability to activate pyruvate dehydrogenase.

Animals↗
PubMed2004

Suicidal dephosphorylation of thiamine pyrophosphate coupled with pyruvate dehydrogenase complex.

Earlier it was noted that purified pyruvate dehydrogenase complex (PDC) produced by "Sigma" usually contains almost saturating amounts of thiamine pyrophosphate (ThPP). In this communication we present the observation that the endogenous ThPP coupled to PDC is dephosphorylated while staying at -10 degrees C, because in the enzyme preparation thiamine monophosphate and un-phosphorylated thiamine appear (HPLC determination). Under the same conditions exogenous ThPP is not dephosphorylated despite contact with the PDC preparation. This may suggest that interactions of some active groups of the enzyme with molecules of endogenous ThPP leads to break-up of the phosphoesters bonds, and destruction of the coenzyme. Decrease of PDC activity during storage is not in proportion with the degree of ThPP dephosphorylation. However the observed instability of PDC activity may be a consequence of the spontaneous process of its coenzyme autodestruction.

Animals↗
PubMed1985

Diagnosis of partial deficiency of the pyruvate dehydrogenase complex in biopsied muscle.

We have measured the total activity of pyruvate dehydrogenase (PDH) complex, by in vitro activation with a broad specificity protein phosphatase, and the basal activity, supposed to be present in vivo, in biopsied muscles from three patients with PDH complex deficiency and 11 patients with lactic acidemia. Results showed that the total PDH complex activity must be determined in biopsied muscles for the diagnosis, because the basal activities of two of three patients with PDH complex deficiency overlapped those of two patients with lactic acidemia whose total activities were within normal range.

Adult↗
PubMed1993

Defect in the lipoyl-bearing protein X subunit of the pyruvate dehydrogenase complex in two patients with encephalomyelopathy.

Among the many metabolic encephalomyelopathies caused by deficiencies in the pyruvate dehydrogenase complex (PDHC), nearly all involve its E1 subunit. We describe two new familial cases of PDHC deficiency with encephalomyelopathy, chronic lactic acidemia, and a normal E1 subunit of PDHC but deficiency in another component. Activity of PDHC was measured in cultured skin fibroblasts and skeletal muscle, and immunoblot studies were performed on mitochondrial extracts from skin fibroblasts. Spectra of muscle tissue, obtained in vivo with phosphorus 31 nuclear magnetic resonance, were recorded both at rest and with exercise. The PDHC activity was markedly reduced to 10% to 20% of normal values in both cultured skin fibroblasts and skeletal muscle. Immunoblotting of skin fibroblast mitochondrial extracts showed a specific deficiency in the protein X component of PDHC but normal E1, E2, and E3 components. Spectra obtained with 31P nuclear magnetic resonance showed alterations compatible with those found in mitochondrial myopathies. This is the second description of an encephalomyelopathy associated with a specific absence of the lipoyl-containing protein X component, which has a structural role in the formation of a functional PDHC.

Child, Preschool↗
PubMed1986

Activation of human muscle pyruvate dehydrogenase with activity and immobilization.

The activity of pyruvate dehydrogenase (PDH) was measured in samples of triceps muscle obtained in 15 males, before and after immobilization for 5 weeks and 5 months of strength training carried out in random order with cross-over of treatment. Although the total PDH activity was unchanged at 300 nmol min-1 g-1, the proportion in the active form increased after strength training (62 +/- 10.1%) and decreased after immobilization (12 +/- 2.3%) compared with control (36 +/- 3.4%). In six subjects measurements were repeated after 10 min maximal exercise (arm ergometer). The proportion of PDH in the active form increased least after immobilization (to 52 +/- 13.6%), compared with control (95 +/- 11.7%) and post-training (98 +/- 6.6%). After exercise muscle glycogen fell to the greatest extent and lactate rose the least in the post-training state, with opposite findings post-immobilization, suggesting that PDH activation contributes to the control of lactate formation in muscle during heavy exercise, and that the effects of training and immobilization are mediated at least in part through changes in the activation of this regulatory enzyme.

Adult↗
PubMed2006

Retinoic acids and trichostatin A (TSA), a histone deacetylase inhibitor, induce human pyruvate dehydrogenase kinase 4 (PDK4) gene expression.

Induction of pyruvate dehydrogenase kinase 4 (PDK4) conserves glucose and substrates for gluconeogenesis and thereby helps regulate blood glucose levels during starvation. We report here that retinoic acids (RA) as well as Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), regulate PDK4 gene expression. Two retinoic acid response elements (RAREs) to which retinoid X receptor alpha (RXRalpha) and retinoic acid receptor alpha (RARalpha) bind and activate transcription are present in the human PDK4 (hPDK4) proximal promoter. Sp1 and CCAAT box binding factor (CBF) bind to the region between two RAREs. Mutation of either the Sp1 or the CBF site significantly decreases basal expression, transactivation by RXRalpha/RARalpha/RA, and the ability of TSA to stimulate hPDK4 gene transcription. By the chromatin immunoprecipitation assay, RA and TSA increase acetylation of histones bound to the proximal promoter as well as occupancy of CBP and Sp1. Interaction of p300/CBP with E1A completely prevented hPDK4 gene activation by RXRalpha/RARalpha/RA and TSA. The p300/CBP may enhance acetylation of histones bound to the hPDK4 promoter and cooperate with Sp1 and CBF to stimulate transcription of the hPDK4 gene in response to RA and TSA.

Acetylation↗
PubMed1992

Antimitochondrial (pyruvate dehydrogenase) autoantibodies in autoimmune rheumatic diseases.

Anti-pyruvate dehydrogenase (PDH) antibodies were determined in 1451 sera of patients with primary biliary cirrhosis (PBC) and several autoimmune rheumatic conditions by ELISA and immunoblotting. They were detected in sera of 93% of the patients with PBC (179 of 192 patients) in 60 of 277 (22%) patients with Sjogren's syndrome (SjS), 34 of 437 (8%) patients with scleroderma, 33 of 191 patients with SLE (17%), and 5 of 55 (10%) patients with rheumatoid arthritis (RA) but in none of the patients with polymyositis or the antiphospholipid syndrome. The ELISA studies were confirmed by immunoblots showing binding of autoimmune rheumatic sera to the same epitope (74 kd) of mitochondria that the PBC sera reacted with. The identical binding characteristics were also confirmed by protein competition assays with purified PDH. In 4 of 53 patients with SjS who were positive for anti-PDH, high titers as in PBC were detected. The anti-PDH antibodies in Sjogren's patients were associated with deranged liver function tests and extraglandular features but did not correlate with any other non-organ-specific antibody. Follow-up studies confirmed the association of the emergence of anti-PDH antibodies with defects in liver function tests. The antibodies were more prevalent in SLE and RA when they were associated with Sjogren's syndrome (30 and 18.8%, respectively). Among patients with different forms of scleroderma, anti-PDH antibodies were noted in subjects with systemic sclerosis, morphea, and Raynaud's phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Antinuclear↗
PubMed1992

Cyclic AMP and free fatty acids in the longer-term regulation of pyruvate dehydrogenase kinase in rat soleus muscle.

Starvation increased pyruvate dehydrogenase (PDH) kinase activity in extracts of freshly excised rat soleus 2.2-fold (from 0.6 min-1 in fed rats to 1.31 min-1 in 48-h-starved rats). In fed rats, activities were unchanged following 24 h of culture in medium 199, but increased 2.1-fold on 24 h of culture with 50 microM dibutyryl cAMP plus 1 mM n-octanoate and 1.6-1.7-fold with either agent alone. Approx. 70% of the increase in PDH kinase induced by starvation was lost following 24 h of culture in medium 199; the loss was prevented by 50 microM dibutyryl cAMP plus 1 mM n-octanoate. cAMP concentrations in fresh soleus muscle were 1 nmol/g (fed rats) and 1.6 nmol/g (starved rats). After 20-60 min of culture the fed-starved difference disappeared and [cAMP] fell to 0.4 nmol/g. Calcitonin-gene-related peptide (CGRP) increased cAMP 3-fold; the increase was maintained throughout 24 h of culture, but was readily reversed at 30 min or 24 h of culture by 60-min incubation with CGRP-free medium. Starvation of the rat (48 h) had no effect on the sensitivity of soleus towards the [cAMP]-increasing effect of CGRP. It is concluded that culture may reverse effects of starvation on PDH kinase activity by lowering cAMP and by removal from the in vivo effects of circulating free fatty acids; and that starvation and CGRP had no detectable long-term effects on the cAMP system in soleus muscle.

Adenosine Deaminase↗
PubMed1984

Reversible phosphorylation of pyruvate dehydrogenase in rat skeletal-muscle mitochondria. Effects of starvation and diabetes.

The total activity of pyruvate dehydrogenase (PDH) complex in rat hind-limb muscle mitochondria was 76.4 units/g of mitochondrial protein. The proportion of complex in the active form was 34% (as isolated), 8-14% (incubation with respiratory substrates) and greater than 98% (incubation without respiratory substrates). Complex was also inactivated by ATP in the presence of oligomycin B and carbonyl cyanide m-chlorophenylhydrazone. Ca2+ (which activates PDH phosphatase) and pyruvate or dichloroacetate (which inhibit PDH kinase) each increased the concentration of active PDH complex in a concentration-dependent manner in mitochondria oxidizing 2-oxoglutarate/L-malate. Values giving half-maximal activation were 10 nM-Ca2+, 3 mM-pyruvate and 16 microM-dichloroacetate. Activation by Ca2+ was inhibited by Na+ and Mg2+. Mitochondria incubated with [32P]Pi/2-oxoglutarate/L-malate incorporated 32P into three phosphorylation sites in the alpha-chain of PDH; relative rates of phosphorylation were sites 1 greater than 2 greater than 3, and of dephosphorylation, sites 2 greater than 1 greater than 3. Starvation ( 48h ) or induction of alloxan-diabetes had no effect on the total activity of PDH complex in skeletal-muscle mitochondria, but each decreased the concentration of active complex in mitochondria oxidizing 2-oxoglutarate/L-malate and increased the concentrations of Ca2+, pyruvate or dichloracetate required for half-maximal reactivation. In extracts of mitochondria the activity of PDH kinase was increased 2-3-fold by 48 h starvation or alloxan-diabetes, but the activity of PDH phosphatase was unchanged.

Adenosine Triphosphate↗