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PubMed1994

Mouse testis Pdha-2 promoter upstream sequences confer tissue-and temporal-specific activity in transgenic mice.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine testis-specific isoform of the E1 alpha subunit of the pyruvate dehydrogenase complex. To elucidate the mechanisms regulating its expression in vivo, we have begun to investigate the Pdha-2 promoter in transgenic mice. In this paper, a construct containing 3.0 kb of promoter and upstream sequences is reported to be sufficient for directing the testis-specific expression of a CAT reporter gene in mice harbouring the transgene. Similarly to the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. However, the 3.0-kb Pdha-2 promoter is not active in somatic tissue suggesting that repressor elements may be present within these sequences.

3T3 Cells↗
PubMed1994

[Specific enzyme diagnosis in mitochondrial myopathies and encephalomyopathies].

Mitochondrial enzyme activities (cytochrome c-oxidase = COX, carnitine acyl-transferase = CAT, citrate synthase = CS, lipoamide dehydrogenase = lipDH from the pyruvate-dehydrogenase complex, lactate dehydrogenase = LDH, and malate-dehydrogenase = MDH) were measured from progressive myopathy/encephalomyopathy. Cytochrome oxidase (COX) deficiency was detected from muscle or liver tissues, adult type of COX defectus had been diagnosed in 1 case and infantile type in further 6 cases. The 3 familial atactic children showed decreased activity of carnitine acetyl-transferase, too.

Adolescent↗
PubMed1978

The effect of insulin on pyruvate dehydrogenase interconversion in heart muscle of alloxan-diabetic rats.

Evidence is presented for regulation by insulin of pyruvate dehydrogenase (pdh) interconversion in rat heart muscle in vivo and in vitro. In the alloxan diabetic rat the active (dephospho) enzyme amounted only to 12% of total PDH and was restored to 42% by insulin. Antilipolytic treatment of the dibetic animals was ineffective, indicating that the action of insulin was independent of a lowering of plasma non-esterified fatty acid concentration. On perfusion of isolated hearts from diabetic rats in the presence of glucose the proportion of pyruvate dehydrogenase in the active form remained low but was fully restored upon addition of insulin (2mU/ml) to the medium. No effect of insulin was obtained in the absence of glucose. The correlation between the rate of pyruvate decarboxylation in the perfused heart and of pyruvate dehydrogenase activity, in vitro, suggests that in the diabetic heart the entry of pyruvate into the citric acid cycle is largely controlled by covalent modification of the pyruvate dehydrogenase complex rather than by feedback inhibition. The possible role of insulin therein is discussed.

Animals↗
PubMed1997

Transfection screening for primary defects in the pyruvate dehydrogenase E1alpha subunit gene.

In a significant number of patients with biochemical evidence of a defect in the E1 (pyruvate dehydrogenase) component of the pyruvate dehydrogenase complex, it has not proved possible to identify a mutation in the gene coding regions. To assess the need for more extensive genetic analysis in these patients and to establish a test system in which to study the biochemical consequences of mutations in the E1alpha subunit gene (which is responsible for the great majority of defined cases of pyruvate dehydrogenase deficiency), we have developed a method to screen for E1alpha gene defects based on complementation of the enzyme deficiency in transformed fibroblast cell lines following transfection and expression of the normal cDNA. Using this system, cell lines from patients with a variety of different defined mutations in the E1alpha gene show restoration of enzyme activity. A number of patients have been identified in whom deficient enzyme activity is not restored by expression of the normal cDNA indicating that an alternative explanation for the enzyme defect must be sought.

Cell Line, Transformed↗
PubMed1996

Autoantibodies to mitochondrial 2-oxo-acid dehydrogenase complexes in localized scleroderma.

Sera from patients with localized scleroderma frequently produce cytoplasmic staining by indirect immunofluorescence, although the antigen remains to be determined. We studied the prevalence, antigen specificity and associated clinical characteristics of anti-cytoplasmic antibodies in localized scleroderma. Serum samples from 60 patients with localized scleroderma were examined by indirect immunofluorescence analysis and immunoblotting. By immunofluorescence analysis on HEp-2 cell substrate, seven of 60 (12%) patients were shown to be positive for anti-cytoplasmic antibodies. Among these, six patients with generalized morphea had anti-mitochondrial antibodies as shown by immunoblotting: they showed reactivity with the E2 component of pyruvate dehydrogenase complex (PDC), with protein X, and with the E2 component of alpha-oxo-glutarate dehydrogenase complex, while two of them showed reactivity with PDC-E1 alpha. One of these patients who was positive for anti-PDC-E1 alpha antibody showed laboratory abnormalities, suggesting the presence of primary biliary cirrhosis. The age of disease onset was significantly higher in these six patients than in those without anti-mitochondrial antibodies. Furthermore, five of them were classified into generalized morphea with multiple plaque lesions but without linear lesions (multiple plaque type). These observations suggest that major antigens for anti-cytoplasmic antibodies in patients with localized scleroderma are mitochondrial enzymes, 2-oxo-acid dehydrogenase complexes. Patients with anti-mitochondrial antibodies may comprise a unique subset of localized scleroderma designated multiple plaque type of generalized morphea of older onset.

Adolescent↗
PubMed1994

Nonsuppurative cholangitis is induced in neonatally thymectomized mice: a possible animal model for primary biliary cirrhosis.

Neonatally thymectomized mice are unique in that they are prone to organ-specific autoimmune diseases. We investigated whether autoimmune cholangitis could be induced in these mice when they were immunized with biliary antigens. Neonatally thymectomized A/J mice were immunized with porcine intrahepatic bile duct epithelial cells (group 1), porcine gallbladder epithelial cells (group 2), porcine splenocytes (group 3) or Freund's adjuvant (group 4). Nonthymectomized mice were immunized with bile duct epithelial cells (group 5) or Freund's adjuvant (group 6). The cell suspensions were injected intraperitoneally with Freund's adjuvant once a week for 8 wk. In group 1 varying amounts of mononuclear cells infiltrated around the bile duct in 14 of 22 mice, whereas little or no accumulation was noted in other groups. Ultrastructural observations revealed that the inflammatory cells consisted of lymphocytes, plasma cells and macrophages. The bile duct showed degenerative changes and some lymphocytes infiltrated between bile duct epithelial cells. An immunohistochemical study showed that the accumulated lymphocytes consisted of CD4+ and CD8+ T cells, as well as B cells. Both major histocompatibility complex class I and class II antigens were expressed on bile duct epithelial cells. Antimitochondrial antibody was demonstrated in some mice in groups 1 (9 of 17), 2 (2 of 3) and 5 (4 of 5) by immunofluorescence; the antibody reacted with the 68, 52 and 47 kD polypeptides of the pyruvate dehydrogenase complex on Western blotting. These findings suggest that autoimmune cholangitis can be induced in neonatally thymectomized mice stimulated with biliary antigens and that these mice could be a suitable animal model for primary biliary cirrhosis.

Animals↗
PubMed1997

Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.

3T3 Cells↗
PubMed2002

Automated enzymatic mitochondrial antibody assay for the diagnosis of primary biliary cirrhosis: applications of a routine diagnostic tool for the detection of antimitochondrial antibodies.

BACKGROUND AND AIMS: An automated enzymatic mitochondrial antibody assay (EMA) kit for the diagnosis of primary biliary cirrhosis (PBC) has become commercially available recently. The aim of this study was to assess the clinical utility of the enzyme inhibition assay using this EMA kit for the diagnosis of PBC. METHODS: We tested the immunoreactivity of sera from 54 histologically confirmed Japanese PBC patients to the 2-oxo-acid dehydrogenase complex (2-OADC) enzymes by enzyme inhibition assay using commercially available TRACE (EMA) assay kit, and compared the results with those of indirect immunofluorescence, commercial enzyme-linked immunosorbent assay (ELISA) using MESACUP Mitochondria M2 kit, and immunoblotting on bovine heart mitochondria. RESULTS: Of the 54 sera, 43 (80%) were positive for antimitochondrial antibodies (AMA) by immunofluorescence, 39 (72%) for enzymatic inhibitory antibody to pyruvate dehydrogenase complex (PDC) by EMA, 33 (61%) for immunoglobulin G (IgG) class anti-PDC antibody by ELISA, and 53 (98%) for IgG, IgM, or IgA class antibodies against at least one of the 2-OADC enzymes by immunoblotting. Of these, 43 (80%) were positive for IgG, IgM, or IgA class antibodies against the E2 subunit of PDC (PDC-E2) by immunoblotting. Thirty-six of the 54 sera (67%) showed identical results in all of the four assays, and 40 (74%) were all negative or positive by EMA, ELISA, and immunoblotting in PDC-relevant reactivity. There was a significant correlation between the number of detected immunoglobulin classes of anti-PDC-E2 by immunoblotting and anti-PDC by EMA (P < 0.0001), and a significant inverse correlation between IgG class anti-PDC by ELISA and units of PDC activity by EMA (r = -0.87, P < 0.0001). CONCLUSIONS: Although EMA had lower sensitivity compared with immunofluorescence and immunoblotting, this assay should be included among the routine diagnostic tools for the detection of AMA specific to PBC in clinical laboratories because of its high specificity, objective read-out, and rapid turnaround time.

Adult↗
PubMed1998

Autoantibodies against subunits of pyruvate dehydrogenase and citrate synthase in a case of paediatric biliary cirrhosis.

In a newborn girl with a history of connatal liver damage, histological examination of a liver biopsy sample taken during the seventh week of life revealed incipient destruction of bile ducts. Very high titres of antimitochondrial antibodies were later detected in the plasma. As the hepatic injury tended towards fibrosis, the histological diagnosis became primary biliary cirrhosis. Autoantibodies against E1 alpha, E2, and E3 subunits and protein X component of pyruvate dehydrogenase complex, and against citrate synthase were detected on western immunoblotting in a 1 in 1000 dilution of the patient's serum. The patient died of her illness at 11 years of age. In liver specimens obtained at autopsy human immunoglobulin deposition was detected on the surface of almost all hepatic cells by immunohistology. As there is a physical and functional interaction between pyruvate dehydrogenase and citrate synthase within the mitochondria, the presence of autoantibodies against certain proteins in the patient suggests that in this form of the disease the molecular recognition and then the autoimmunisation process could be directed against a mitochondrial enzyme cluster containing both pyruvate dehydrogenase and citrate synthase.

Autoantibodies↗
PubMed1998

The pyruvate dehydrogenase multi-enzyme complex from Gram-negative bacteria.

Pyruvate dehydrogenase multi-enzyme complexes from Gram-negative bacteria consists of three enzymes, pyruvate dehydrogenase/decarboxylase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3). The acetyltransferase harbors all properties required for multi-enzyme catalysis: it forms a large core of 24 subunits, it contains multiple binding sites for the E1p and E3 components, the acetyltransferase catalytic site and mobile substrate carrying lipoyl domains that visit the active sites. Today, the Azotobacter vinelandii complex is the best understood oxo acid dehydrogenase complex with respect to structural details. A description of multi-enzyme catalysis starts with the structural and catalytic properties of the individual components of the complex. Integration of the individual properties is obtained by a description of how the many copies of the individual enzymes are arranged in the complex and how the lipoyl domains couple the activities of the respective active sites by way of flexible linkers. These latter aspects are the most difficult to study and future research need to be aimed at these properties.

Acetyltransferases↗
PubMed2002

Exercise with low muscle glycogen augments TCA cycle anaplerosis but impairs oxidative energy provision in humans.

We tested the hypotheses that: (i) exercise with low muscle glycogen would reduce pyruvate flux through the alanine aminotransferase (AAT) reaction and attenuate the increase in tricarboxylic acid (TCA) cycle intermediates, and (ii) attenuation of tricarboxylic acid cycle intermediate (TCAI) pool expansion would limit TCA cycle flux, thereby accelerating phosphocreatine (PCr) degradation. Eight men cycled for 10 min at 70 % of their (VO(2,max) on two occasions: (i) following their normal diet (CON) and (ii) after cycling to exhaustion and consuming a low carbohydrate diet for approximately 2 days (LG). Biopsies (m. vastus lateralis) confirmed that [glycogen] was lower in LG vs. CON at rest (257 +/- 18 vs. 611 +/- 54 mmol (kg dry mass)(-1); P 0.05); however, net glycogenolysis was not different after 1 or 10 min of exercise. PCr degradation from rest to 1 min was approximately 26 % higher in LG vs. CON (38 +/- 4 vs. 28 +/- 4 mmol (kg dry mass)(-1); P< or =0.05). The sum of five measured TCAIs (approximately 90 % of total pool) was not different between trials at rest and after 1 min, but was higher after 10 min in LG vs. CON (5.51 +/- 0.43 vs. 4.45 +/- 0.49 mmol (kg dry mass)(-1); P 0.05). Pyruvate dehydrogenase complex (PDC) activity was lower during exercise in LG vs. CON (2.2 +/- 0.2 vs. 1.4 +/- 0.2 mmol min(-1) (kg wet weight)(-1) after 10 min; P< or =0.05), and acetylcarnitine was approximately threefold less, implying increased pyruvate availability for flux through AAT. Resting muscle [glutamate] was higher in LG vs. CON (16.1 +/- 0.8 vs. 11.8 +/- 0.4 mmol (kg dry mass)(-1); P< or =0.05) and the net decrease in [glutamate] during exercise was approximately 30 % greater in LG vs. CON. These findings suggest that: (i) contrary to our hypotheses, LG increased anaplerosis by decreasing PDC flux and/or increasing the conversion of glutamate carbon to TCAIs, and (ii) accelerating the rate of muscle TCAI expansion did not affect oxidative energy provision during the initial phase of contraction, since changes in [TCAI] were not temporally related to PCr degradation.

Adult↗
PubMed1988

Acute effects of surgery on carbohydrate production and utilization in the fed rat.

1. The work utilized a model of uncomplicated abdominal surgery (laparotomy under ether anaesthesia) to delineate the effects of abdominal trauma on glucose homoeostasis in the fed rat. 2. Regulation of glucose production and utilization was investigated by observing the response to the administration of glucose, insulin plus glucose and 5-methylpyrazole 3-carboxylic acid. 3. Glucose administration suppressed hepatic glucose output as assessed by portal-venous concentration differences in control or surgically stressed rats. In contrast, glycaemia was increased and lactaemia decreased in the latter group. Portal-venous concentrations differences for lactate were unaffected. 4. Surgery increased plasma fatty acid concentrations and the antilipolytic response to glucose or glucose plus insulin was diminished. Post-operative increases in fatty acid concentrations were associated with inhibition of hepatic pyruvate dehydrogenase complex which was reversed by insulin, indicating a differential sensitivity of adipose tissue and liver to the hormone. 5. The model of surgical stress utilized, while affecting extrahepatic glucose disposal, did not elicit depletion of liver glycogen or inactivation of L-pyruvate kinase. 6. It is concluded that the initial response to uncomplicated abdominal surgery involves carbohydrate conservation rather than increased glucose production, with effects to decrease extrahepatic glucose uptake and hepatic glucose oxidation.

Animals↗
PubMed1984

Arginine residues in the active centers of muscle pyruvate dehydrogenase.

The pyruvate dehydrogenase component isolated from the pigeon breast muscle pyruvate dehydrogenase complex is inactivated by the arginine-specific reagents, 2,3-butanedione and phenylglyoxal. The kinetics of inactivation is biphasic. The reaction order with respect to the inhibitor concentration at the fast and slow steps of the inactivation reaction is close to unity. This suggests that complete inactivation of the enzyme results from the interaction of two molecules of 2,3-butanedione or phenylglyoxal with essential groups of pyruvate dehydrogenase. In the presence of thiamine pyrophosphate and Mg2+ pyruvate protects the enzyme against inactivation. The experimental results are suggestive of the presence of two arginine residues in the substrate-binding sites of two active centres of the holoenzyme.

Animals↗
PubMed1983

Pyruvate decarboxylating activity in extracts from pigeon liver acetone powder.

Extracts of pigeon liver acetone powder showed, by a radiochemical method, considerable pyruvate decarboxylating activity. The 10-30% saturated ammonium sulfate fraction of these extracts required the addition of CoA, NAD+, and TPP for maximum pyruvate decarboxylating activity and presumably contained the pyruvate dehydrogenase complex (PDHC). The activity in the 40-60% saturated ammonium sulfate fraction had no requirement for CoA and/or NAD+ but had an absolute requirement for TPP. In similar preparations obtained from fresh pigeon liver, the PDHC activity was found in the 0-30% saturated ammonium sulfate fraction and only a small amount of pyruvate decarboxylating activity (independent of CoA and NAD+) was found in the 40-60% saturated ammonium sulfate fraction. The pyruvate decarboxylating activity which is found in the 40-60% saturated ammonium sulfate fraction of these extracts and has an absolute requirement for TPP, may represent pyruvate dehydrogenase (E1, EC 1.2.4.1), probably derived from PDHC by the acetone treatment at pH 7.8, or aging of the preparation.

Acetyltransferases↗
PubMed2001

An essential role of Glu-243 and His-239 in the phosphotransfer reaction catalyzed by pyruvate dehydrogenase kinase.

This study was undertaken to examine the mechanistic significance of two highly conserved residues positioned in the active site of pyruvate dehydrogenase kinase, Glu-243 and His-239. We used site-directed mutagenesis to convert Glu-243 to Ala, Asp, or Gln and His-239 to Ala. The resulting mutant kinases demonstrated a greatly reduced capacity for phosphorylation of pyruvate dehydrogenase. The Glu-243 to Asp mutant had approximately 2% residual activity, whereas the Glu-243 to Ala or Gln mutants exhibited less than 0.5 and 0.1% residual activity, respectively. Activity of the His-239 to Ala mutant was decreased by approximately 90%. Active-site titration with [alpha-(32)P]ATP revealed that neither Glu-243 nor His-239 mutations affected nucleotide binding. All mutant kinases showed similar or even somewhat greater affinity than the wild-type kinase toward the protein substrate, pyruvate dehydrogenase complex. Furthermore, neither of the mutations affected the inter-subunit interactions. Finally, pyruvate dehydrogenase kinase was found to possess a weak ATP hydrolytic activity, which required Glu-243 and His-239 similar to the kinase activity. Based on these observations, we propose a mechanism according to which the invariant glutamate residue (Glu-243) acts as a general base catalyst, which activates the hydroxyl group on a serine residue of the protein substrate for direct attack on the gamma phosphate. The glutamate residue in turn might be further polarized through interaction with the neighboring histidine residue (His-239).

Catalysis↗
PubMed1977

Deficiency of dihydrolipoyl dehydrogenase (a component of the pyruvate and alpha-ketoglutarate dehydrogenase complexes): a cause of congenital chronic lactic acidosis in infancy.

A male child died at 7 months of age with progressive neurologic deterioration and persistent metabolic acidosis. Investigations during life showed this child to have elevated blood pyruvate, lactate, and alpha-ketoglutarate as well as elevation of branched chain amino acids and occasional hypoglycemia. Cofactor therapy using either thiamine-HCl (2 g/kg/24 hr) or thiamine tetrahydrofurfuryl disulfide had no measurable effect on the clinical or biochemical status of the patient. Tissue taken postmortem showed normal levels of key gluconeogenic enzymes but a deficiency in the activity of pyruvate dehydrogenase in all tissues tested (liver, brain, kidney, skeletal muscle, and heart). Examination of the individual activities of pyruvate dehydrogenase complex showed pyruvate decarboxylase (E1) to be normal in liver and other tissues. Dihydrolipoyl dehydrogenase (E3), on the other hand, was deficient in all tissues tested. alpha-Ketoglutarate dehydrogenase complex, which depends of E3 for its total activity, was also deficient in all tissues tested. The absence of this enzyme id discussed in relation to the clinical and biochemical status of the patient.

Acidosis↗
PubMed1975

Characteristics of a glycerol utilization mutant of Neurospora crassa.

A mutant of Neurospora crassa able to grow on liquid minimal glycerol medium without evidence of conidiation and with high cell yields has been isolated and shown to be allelic to ff-1. The glycerol-specific induction of glycerokinase and glycerol-3-phosphate dehydrogenase was similar in both wild-type and mutant cells, although higher specific activities as well as higher glycerokinase cross-reacting material levels were found in fully induced mutant cells. After growth in minimal glycerol medium there is a significant reduction in wild-type cells of the activities of both pyruvate dehydrogenase and dihydrolipoyl transacetylase. This evidence indicates a relationship between the conditional acetate requirement by wild-type cells grown on glycerol medium and the levels of the pyruvate dehydrogenase complex.

Acetates↗