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PubMed1992

The carboxy terminal 110 amino acid portion of the insulin receptor is important for insulin signalling to pyruvate dehydrogenase.

Insulin-like growth factor 1 (IGF-1) had no detectable effect on pyruvate dehydrogenase (PDH) in NIH 3T3 cells that stably overexpressed normal human IGF-1 receptors. Insulin stimulated PDH activity 3-4--fold in cells that overexpressed normal human insulin receptors, but not in cells expressing TMI receptors, composed of the ligand binding domain of the insulin receptor coupled to the transmembrane and intracellular components of the IGF-1 receptor, or CEX receptors, in which the carboxy terminal 110 amino acid portion of the insulin receptor was exchanged for the corresponding portion of the IGF-1 receptor. In contrast, insulin stimulated glucose uptake in the control cell line and in each of the chimeric receptor-expressing lines with similar dose-response characteristics. These findings suggest the carboxy terminal portion of the IR may play a role in mediating the stimulation of PDH activity.

3T3 Cells↗
PubMed1987

Tissue-specific changes in the ability of insulin and noradrenaline to activate pyruvate dehydrogenase in vivo during lactation in the rat.

Changes are described in the total pyruvate dehydrogenase (PDH) activity, the proportion of PDH in the active state and its control by insulin and noradrenaline in vivo, in white adipose tissue, liver, skeletal muscle and mammary gland with pregnancy, lactation and on weaning. Lactation resulted in a decrease in total PDH in white adipose tissue and an increase in the mammary gland, whereas the proportion in the active state decreased in muscle and increased in the mammary gland. The ability of insulin to activate PDH of white adipose tissue was lost during lactation, whereas it was retained by the other tissues. The ability of noradrenaline to activate PDH was decreased in white adipose tissue but increased in liver during lactation. These various adaptations should limit the use of glucose and lactate carbon by adipose tissue and skeletal muscle during lactation and thereby facilitate their preferential utilization by the mammary gland.

Adipose Tissue↗
PubMed1989

Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase.

A 413-base cDNA insert encoding a portion of the alpha subunit of pyruvate dehydrogenase (E1 alpha; EC 1.2.4.1) from Saccharomyces cerevisiae was isolated from a lambda gt11 cDNA library by immunoscreening and by hybridization with an oligonucleotide probe which corresponded to the amino acid sequence around the phosphorylation site of E1 alpha. This cDNA was subcloned, sequenced and used as a probe to isolate two additional cDNA inserts which were subcloned and sequenced. These overlapping clones comprised the carboxyl-terminal part of E1 alpha. To identify the missing nucleotide sequence, the polymerase chain reaction was used to amplify yeast genomic DNA with synthetic oligonucleotide primers based on the amino-terminal sequence of E1 alpha and the 5' end of one of the cDNA clones. Three DNA fragments were isolated and sequenced. The composite nucleotide sequence has an open reading frame of 1260 nucleotides encoding a putative presequence of 33 amino acids and a mature protein of 387 amino acids (Mr = 42,703). Hybridization analysis showed that the size of the mRNA is about 1.4 kilobases.

Amino Acid Sequence↗
PubMed2005

Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase.

It has been proposed that "Glu238" within the N-box of pyruvate dehydrogenase kinase (PDK) is a base catalyst. The pH dependence of k(cat) of Arabidopsis thaliana PDK indicates that ionizable groups with pK values of 6.2 and 8.4 are necessary for catalysis, and the temperature dependence of these values suggests that the acidic pK is due to a carboxyl- or imidazole-group. The E238 and K241 mutants had elevated K(m,ATP) values. The acidic pK value of the E238A mutant was shifted to 5.5. The H233A, L234H, and L234A mutants had the same pK values as wild-type AtPDK, contrary to the previous proposal of a "Glu-polarizing" His. Instead, we suggest that the conserved Glu, Lys, and Asn residues of the N-box contribute to coordinating Mg2+ in a position critical for formation of the PDK-MgATP-substrate ternary complex.

Adenosine Triphosphate↗
PubMed1995

Hepatic distribution of E2 component of pyruvate dehydrogenase complex after transplantation.

We have examined the distribution of the E2 component of pyruvate dehydrogenase complex in the liver of patients with native primary biliary cirrhosis (PBC), and after transplantation, using affinity-purified anti-E2 antibodies. In the posttransplantation group, we studied biopsy specimens from patients grafted for conditions other than PBC (n = 6) and those grafted for PBC with (n = 6) and without (n = 5) histological features suggestive of PBC recurrence. Features suggestive of PBC recurrence included portal tract granulomas, bile duct damage, ductopenia, and lymphoid aggregates. In the native liver from patients with PBC, there was increased staining of E2 on the biliary epithelial cells compared with hepatocytes, as previously described. However, in liver biopsy specimens from patients after transplantation, the pattern of staining of E2 was similar to that of normal, control liver in all three groups studied. These findings suggest that E2 overexpression on bile duct cells may not be important in the perpetuation of the bile duct damage in PCB, that expression in the allograft may be modified by immunosuppression, or that PBC does not recur in the allograft.

Biopsy↗
PubMed1984

Pyruvate dehydrogenase activity in regions of the rat brain during postnatal development.

Activity of the pyruvate dehydrogenase complex (PDHC) was measured in seven brain regions of the male rat at various times during the postnatal period using an arylamine acetyltransferase coupled assay. Three days after birth, PDHC activity was found to be less than 15% of adult values in all brain regions with the exception of hypothalamus and medulla-pons (30% of adult values in each case). Activity of the enzyme complex in these latter regions attained adult levels by 21 days postnatally, some 5-15 days ahead of that found in cerebral cortex, striatum, hippocampus, and cerebellum. Such differences in PDHC maturation reflect the greater degree of early maturity of the phylogenetically older brain structures. Cerebellar PDHC developed more slowly than in other brain regions to attain only 40% of adult levels by the time of weaning. The pattern of maturation of cerebellar PDHC is paralleled by increased incorporation of glucose into cerebral amino acids and by the pattern of development of parallel fiber synaptogenesis. These findings suggest that PDHC may play a key role in the regional development of metabolic compartmentation and the associated maturation of cerebral function in the rat.

Animals↗
PubMed1992

Transcriptional expression of a testis-specific variant of the mouse pyruvate dehydrogenase E1 alpha subunit.

An isogene of the E1 alpha subunit of the mouse pyruvate dehydrogenase complex was shown in a previous study to map to chromosome 19. Here we demonstrate using Northern blot analysis that this gene is expressed in a testis-specific fashion. Two testis-specific E1 alpha transcripts were detected: (1) a 2.0-kb transcript that is abundant in pachytene cells but also detectable during earlier stages of spermatogenesis, and (2) a shorter 1.7-kb transcript detectable only in round spermatids. Analysis of both transcripts following RNase H digestion revealed that the smaller message is not derived from the shortening of the poly(A) tail and most likely results from the alternate use of two polyadenylation signals. Finally, analysis of polysomes isolated from 20- and 80-day-old mouse testes demonstrated that the 2.0-kb transcript is associated with the polysomal fraction, suggesting that this message is transcribed and actively translated at the same time. We conclude from these results that expression of the testis-specific E1 alpha is initially expressed during the meiotic prophase stage of spermatogenesis and not under any apparent post-transcriptional regulation.

Animals↗
PubMed1987

Light microscopic immunocytochemical localization of pyruvate dehydrogenase complex in rat brain: topographical distribution and relation to cholinergic and catecholaminergic nuclei.

Pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) includes 3 catalytically active mitochondrial enzymes involved in the formation of cellular energy through the tricarboxylic acid cycle and in the synthesis of ACh. We sought to determine whether immunocytochemically detected PDHC was enriched in neurons of the rat CNS, and, if so, whether the perikarya containing higher levels of PDHC immunoreactivity were differentially distributed with respect to their size or location within nuclear groups containing ACh, catecholamines or other unidentified transmitters. Under the labeling conditions used in this study, the peroxidase-antiperoxidase immunoreaction product for PDHC was detectable principally in neuronal perikarya. The intensity of immunoreactivity within perikarya was variable as judged visually and by cellular, computer-assisted densitometry. In the forebrain, the most intensely labeled perikarya were seen in the medial septal nuclei, the nuclei of the diagonal band, the nuclei basalis, the dorsal and ventral striatum, and the entorhinal cortex. More caudally, intense immunoreactivity was detected in perikarya in the supraoptic hypothalamic nuclei, reticular thalamic nuclei, lateral substantia nigra, most of the tegmental nuclei, lateral nuclei of the trapezoid body, raphe pontis and obscuris, and the caudal part of the lateral reticular nuclei. In addition, many of the motor nuclei of the cranial nerves, including the dorsal motor nuclei of the vagus and the hypoglossal nuclei, and the nucleus ambiguus contained perikarya with intense PDHC labeling. Densitometry revealed no differences in intensity of immunoreactivity in soma of varying sizes. However, the intensity of neuronal labeling for PDHC was significantly greater in several nuclear groups that were shown in adjacent sections to contain cholinergic, but not catecholaminergic, enzymes. In contrast, the primary olfactory cortex, pyramidal cell layer of the regio inferior of hippocampus, and the Purkinje cell layer of the cerebellum were regions having perikarya with intense PDHC immunoreactivity but lacking both the synthetic and the degradative enzymes for ACh. These results provide the first morphological evidence that PDHC, a general metabolic enzyme complex, is enriched in selective perikarya that are heterogeneously distributed in brain and are especially abundant in many of the regions containing cholinergic neurons. The heterogeneity of PDHC immunoreactivity suggests that certain cholinergic as well as noncholinergic nuclei may be selectively vulnerable to mitochondrial diseases involving pyruvate utilization.

Animals↗
PubMed1985

Studies on the possible involvement of prostaglandins in insulin generation of pyruvate dehydrogenase activator.

Insulin-exposed liver particulate fraction supernatants from control rats stimulated mitochondrial pyruvate dehydrogenase (PDH) activity by 26%, while the stimulation by similar preparations from indomethacin-injected rats (5 mg/kg twice daily, i.p., for 2 days) was 4%. In vitro addition of indomethacin to the particulate fraction during insulin exposure also inhibited stimulation of PDH by insulin. This inhibitory effect of indomethacin was completely overcome by the in vitro addition of prostaglandin E2 (PGE2) to the liver particulate incubation mixture. Intact adipocytes showed a similar (62%) decrease in insulin activation of PDH in the presence of indomethacin. In a cell-free adipocyte system (co-incubation of mitochondria and plasma membrane), indomethacin addition resulted in 90% decrease in insulin-stimulated PDH response. PGE2 addition completely reversed this inhibition. In contrast to its effects on PDH activation, indomethacin had no effect on insulin-stimulated glucose oxidation. In vitro incubation of fat cells with dexamethasone (1 microM) also resulted in decreased insulin activation of PDH. Inclusion of arachidonic acid during dexamethasone exposure of fat cells resulted in partial restoration of the insulin effect on PDH in fat cells and in cell-free preparations. However, addition of PGE2 during insulin exposure of plasma membranes from dexamethasone-treated preparations showed no significant restoration of the insulin effect on PDH. These studies suggest that: (1) PG metabolism is involved in insulin's generation of the second messenger, and (2) the mechanism of dexamethasone-induced inhibition of insulin effect on PDH is a complex phenomenon involving the synthesis and action of eicosanoids.

Adipose Tissue↗
PubMed1984

Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes.

BC3H-1 myocytes were cultured with 32PO4 for 3 days to label phospholipids to constant specific activity. Subsequent treatment with physiological concentrations of insulin provoked 40-70% increases in 32PO4 levels (reflecting increases in mass) in phosphatidic acid, phosphatidylinositol, and polyphosphoinositides, and, lesser, 20-25% increases in phosphatidylserine and the combined chromatographic area containing phosphatidylethanolamine plus phosphatidylcholine plus phosphatidylcholine. Insulin-induced increases in phospholipids were significant within 5 min and near-maximal at 15-30 min. Comparable rapid insulin-induced increases in [3H]phosphatidylinositol were observed in myocytes prelabeled with [3H]inositol. These insulin effects (as per prolonged pulse-chase experiments) were due to increase phospholipid synthesis rather than decreased phospholipid degradation. Cycloheximide (and puromycin) pretreatment prevented insulin-induced increases in phospholipids and rapidly reversed ongoing insulin effects on phospholipids and pyruvate dehydrogenase activity. Insulin also rapidly increased diacylglycerol levels. These findings suggest that: (a) insulin provokes rapid increases in de novo synthesis of phosphatidic acid and its derivatives, e.g. phosphoinositides and diacylglycerol; (b) protein synthesis inhibitors diminish phospholipid levels in insulin-treated (but not control) tissues by increasing phospholipid degradation (?phospholipase(s) activation); and (c) changes in phospholipids and diacylglycerol may be important for changes in pyruvate dehydrogenase and other enzymatic activities during treatment with insulin and/or protein synthesis inhibitors.

Animals↗
PubMed1996

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.

Amino Acid Sequence↗
PubMed1986

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.

Animals↗
PubMed1989

Antibodies against an inter-domain segment of polypeptide chain inhibit active-site coupling in the pyruvate dehydrogenase multienzyme complex.

A synthetic peptide, AAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA, proved to be an efficient and specific immunogen in rabbits. The amino acid sequence of the peptide is identical to that of the inter-domain region (PEP3) linking the innermost of the three lipoyl domains to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli. Fab fragments from anti-PEP3 antibodies selectively inhibited active-site coupling in the complex without affecting the individual activities of the three component enzymes, highlighting the role of the inter-domain regions as flexible linkers in catalysis.

Antibodies↗
PubMed2004

Age-related compensatory activation of pyruvate dehydrogenase complex in rat heart.

Mitochondrial uptake and beta-oxidation of long-chain fatty acids are markedly impaired in the aging rat heart. While these alterations would be expected to adversely affect overall pyridine nucleotides, NADH levels do not change significantly with age. This conundrum suggests that specific compensatory mechanisms occur in the aging heart. The comparison of cardiac pyruvate dehydrogenase complex (PDC) kinetics in 4- and 24- to 28-month-old F344 rats revealed a 60% significant increase in V(max) with no change in PDC expression, and a 1.6-fold decrease in the Michaelis constant (K(m)) in old compared to young rats. The observed kinetic adjustments were selective to PDC, as neither the V(max) nor K(m) of citrate synthase changed with age. PDC kinase-4 mRNA levels decreased by 57% in old vs young rat hearts and correlated with a 45% decrease in PDC phosphorylation. We conclude that PDC from old rat hearts catabolizes pyruvate more efficiently due to an adaptive change in phosphorylation.

Aging↗
PubMed1992

Leigh syndrome associated with a deficiency of the pyruvate dehydrogenase complex: results of treatment with a ketogenic diet.

A one-year-old boy suffering from intermittent lactic acidosis, muscular hypotonia, horizontal gaze paralysis and spasticity in both legs had low activity of the pyruvate dehydrogenase complex associated with low amounts of immunoreactive E 1 alpha and E 1 beta. Leigh syndrome was diagnosed on the basis of the clinical and biochemical abnormalities and the typical lesions observed on MRI of the brain. Treatment with a ketogenic diet was associated with clinical and biochemical amelioration. A striking improvement of the cerebral lesions was observed by neuro-imaging.

Brain↗
PubMed1998

Pyruvate augments mechanical function via activation of the pyruvate dehydrogenase complex in reperfused ischemic immature rabbit hearts.

BACKGROUND: Reperfusion of ischemic adult hearts is associated with increased fatty acid oxidation, reduced pyruvate oxidation, and reduced pyruvate dehydrogenase (PDH) activity, leading to a decrease in cardiac efficiency. These effects may be amplified in newborn hearts because of the immaturity of their PDH pathway. We hypothesize that pyruvate can augment mechanical function in the immature heart by activating the PDH complex (PDC) during reperfusion in severely ischemic hearts. MATERIALS AND METHODS: Seven-day old isolated working rabbit hearts (n = 12) were perfused with modified Krebs solution containing 0.4 mM palmitate. Pyruvate (5 mM) was added for a 10-min period either before or after a 30-min period of normothermic global ischemia. Cardiac functional indices before global ischemia and during reperfusion were correlated with active and total PDC activity measured in 28 additional hearts frozen at the various time points throughout the perfusion protocol. RESULTS: Addition of pyruvate before ischemia increased the proportion of active PDC but did not affect any measured functional indices. During early reperfusion, aortic flow, cardiac output, and cardiac work were all significantly depressed compared to preischemic values. Addition of pyruvate significantly increased the proportion of active PDC and was also associated with a significant increase in aortic flow, cardiac work, and developed pressure. Removal of pyruvate from the perfusate resulted in a subsequent significant decrease in PDC activity and these functional parameters. CONCLUSION: During reperfusion of neonatal rabbit hearts, addition of pyruvate improves cardiac performance in association with activation of PDC.

Animals↗
PubMed1988

Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration.

(1) The free Ca2+ concentration of the matrix of rat heart mitochondria ([Ca2+]m) was determined from the fluorescence of internalized indo-1. The value of the Kd of indo-1-Ca2+ in the mitochondrial matrix was determined to be 95 nM, on the basis of equilibration of [Ca2+]m with the extramitochondrial free Ca2+ ([Ca2+]o) in the presence of rotenone, nigericin, valinomycin and Br-A23187. (2) [Ca2+]m responded to energization/de-energization protocols, the inhibition of Ca2+-uptake by Ruthenium Red and the potentiation of Ca2+-efflux by Na+ in a manner which was consistent with the known kinetic properties of the mitochondrial Ca2+-transport processes. (3) The concentration gradient [Ca2+]m/[Ca2+]o was found to be near unity (0.82 +/- 0.18) when mitochondria were incubated in media containing 10 mM-Na+; the additional presence of 1 mM-Mg2+ reduced the gradient to values below unity (0.26 +/- 0.03). The polyamine spermine increased the Ca2+ concentration gradient in the presence of 1 mM-Mg2+. (4) The fraction of pyruvate dehydrogenase in the active form (PDHA) was found to increase with [Ca2+]m, with a K0.5 for activation of approximately 300 nM-Ca2+. This value of the activation constant was not affected by conditions, e.g. addition of Mg2+, which changed the [Ca2+]m/[Ca2+]o concentration gradient, and the presence of different oxidizable substrates, which changed the [NADH/NAD+]m concentration ratio. Thus pyruvate dehydrogenase interconversion responds directly to changes in [Ca2+]m, as inferred in earlier work.

Animals↗