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PubMed2006

Concept on the pathogenesis and treatment of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that predominantly affects women and is characterized by chronic, progressive destruction of small intrahepatic bile ducts with portal inflammation and ultimately fibrosis, leading to liver failure in the absence of treatment. Little is known about the etiology of PBC. PBC is characterized by anti-mitochondrial antibodies and destruction of intra-hepatic bile ducts. The serologic hallmark of PBC is the presence of auto-antibodies to mitochondria, especially to the E2 component of the pyruvate dehydrogenase complex (PDC). Current theories on the pathogenesis of PBC favor the hypothesis that the disease develops as a result of an inappropriate immune response following stimulation by an environmental or infectious agent. Some reports suggest that xenobiotics and viral infections may induce PBC. The pathogenetic mechanism is believed to be caused by a defect in immunologic tolerance, resulting in the activation and expansion of self-antigen specific T and B lymphocyte clones and the production of circulating autoantibodies in addition to a myriad of cytokines and other inflammatory mediators. This leads to ductulopenia and persistent cholestasis, by developing end-stage hepatic-cell failure. In this review are given our own and literary data about mechanisms of development of intrahepatic cholestasis and possible ways of its correction.

Autoimmune Diseases↗
PubMed1999

Towards the pathogenesis of autoimmune liver disease.

There have been recent improvements in the clinical understanding and definition of the major types of autoimmune liver disease. However, still lacking is knowledge of their prevalence and pathogenesis. Three areas of study are in progress in our laboratory. First, in type 1 autoimmune hepatitis, the search continues to identify a liver/disease-specific autoantigenic reactant. Using hepatocyte membrane preparations, immunoblotting has underlined the problem of distinguishing, among multiple reactants, those that may be causally rather than consequentially related to hepatocellular damage. Second, in primary biliary cirrhosis (PBC), the need for population screening to ascertain prevalence and detect preclinical cases can be met by a rapid automated procedure for detection, by specific enzyme inhibition in microtitre wells, of antibody (anti-M2) to the pyruvate dehydrogenase complex E2 subunit (PDC-E2). Third, the structure of the conformational epitope within the inner lipoyl domain of PDC-E2 is being investigated by screening random phage-displayed peptide libraries using PBC sera. This has yielded phage clones in which the sequence of the peptide insert portrays the structure of this epitope, as judged by clustering of PBC-derived sequences to particular branches of a guide-tree that shows relatedness of peptides, and by reactivity of selected phage clones with anti-PDC-E2. Thus phage display identifies a peptide 'mimotope' of the antibody epitope in the inner lipoyl domain of PDC-E2.

Amino Acid Sequence↗
PubMed2006

Leigh's disease due to a new mutation in the PDHX gene.

OBJECTIVE: To describe the clinical course, neuroradiological presentation, biochemical and molecular studies of a new patient with pyruvate dehydrogenase complex (PDHc) deficiency. To compare this case with the data on other published cases. METHODS: Brain magnetic resonance imaging (MRI), basal metabolic investigations with lactate measurements in body fluids, PDHc activity assay on cultured skin fibroblasts, immunoblot analysis and molecular studies (polymerase chain reaction [PCR] and sequencing procedures). RESULTS: Our patient accused an unspecific encephalopathy for years and presented at 13 years of age an acute deterioration with basal ganglia necrosis and subcortical white matter involvement. PDHc deficiency was secondary to a large deletion (3913 bp) in the PDHX gene, which encodes E3 binding protein (E3BP) subunit. INTERPRETATION: These data provide an additional case of E3BP deficiency with a unique and previously unreported deletion in the PDHX gene.

Adolescent↗
PubMed1999

Leptin has acute effects on glucose and lipid metabolism in both lean and gold thioglucose-obese mice.

Leptin is reported to have effects in peripheral tissues that are independent of its central effects on food intake and body weight. In this study, the acute effects of a single dose of recombinant mouse leptin on lipid and glucose metabolism in lean and gold thioglucose-injected obese mice were examined. Changes were measured 2 h after leptin injection. In lean mice, liver and white adipose tissue (WAT) lipogenesis was inhibited. The activity of the pyruvate dehydrogenase complex (PDHCa), the rate-determining step for glucose oxidation, was reduced in heart, liver, quadriceps muscle, and both brown and white adipose tissues. Muscle and liver glycogen and liver triglyceride (TG) content was unchanged, but muscle TG was decreased. In obese mice, liver and WAT lipogenesis was inhibited and PDHCa reduced in heart and quadriceps muscle. Muscle and liver glycogen was decreased but not TG. Serum insulin was reduced in obese but not lean mice. These results are consistent with a role for leptin in the maintenance of steady-state energy stores by decreasing lipid synthesis and increasing fat mobilization, with decreased glucose oxidation occurring as a result of increased fatty acid oxidation.

Adipose Tissue↗
PubMed2002

Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pyruvate dehydrogenase complex (PDC-E2)-specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti-PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the Calpha constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6% nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further support of the hypothesis that PDC-E2-specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2-specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC.

Animals↗
PubMed1992

Membrane dihydrolipoamide acetyltransferase (E2) on human biliary epithelial cells in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is associated with serum antibodies that react with the dihydrolipoamide acetyltransferase component (E2) of the mitochondrial pyruvate dehydrogenase complex. We have sought the presence of E2 on the surface of human intrahepatic biliary epithelial cells (BEC). Cultured BECs from PBC but not normal liver were found to have E2 on the membrane after three days' culture. Isolated, viable cells examined by laser-scanning confocal microscopy revealed the pattern of E2 staining on the membrane to be similar to that seen with the membrane glycoprotein marker, HEA-125. By contrast, BECs from normal liver showed membrane staining only with HEA-125. When BECs were fixed before incubation with antibody to E2, cytoplasmic staining was observed. Our results suggest that E2 is present on the surface of biliary epithelial cells in PBC, and support the idea of a pathogenetic association between antimitochondrial antibodies and bileduct damage.

Acetyltransferases↗
PubMed1996

Carnitine and physical exercise.

Carnitine plays a central role in fatty acid (FA) metabolism. It transports long-chain fatty acids into mitochondria for beta-oxidation. Carnitine also modulates the metabolism of coenzyme-A (CoA). It is not surprising that the use of supplementary carnitine to improve physical performance has become widespread in recent years, although there is no unequivocal support to this practice. However, critical reflections and current scientific-based knowledge are important because the implications of reduced or increased carnitine concentrations in vivo are not thoroughly understood. Several rationales have been forwarded in support of the potential ergogenic effects of oral carnitine supplementation. However, the following arguments derived from established scientific observations may be forwarded: (i) carnitine supplementation neither enhances FA oxidation in vivo or spares glycogen or postpones fatigue during exercise. Carnitine supplementation does not unequivocally improve performance of athletes; (ii) carnitine supplementation does not reduce body fat or help to lose weight; (iii) in vivo pyruvate dehydrogenase complex (PDC) is fully active already after a few seconds of intense exercise. Carnitine supplementation induces no further activation of PDC in vivo; (iv) despite an increased acetyl-CoA/free CoA ratio, PDC is not depressed during exercise in vivo and therefore supplementary carnitine has no effect on lactate accumulation; (v) carnitine supplementation per se does not affect the maximal oxygen uptake (VO2max); (vi) during exercise there is a redistribution of free carnitine and acylcarnitines in the muscle but there is no loss of total carnitine. Athletes are not at risk for carnitine deficiency and do not have an increased need for carnitine. Although there are some theoretical points favouring potential ergogenic effects of carnitine supplementation, there is currently no scientific basis for healthy individuals or athletes to use carnitine supplementation to improve exercise performance.

Animals↗
PubMed2003

PPAR alpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.

Previous studies demonstrated that during cisplatin-induced acute renal failure, there is a significant reduction in proximal tubule fatty acid oxidation. We now report on the effects of peroxisome proliferator-activated receptor-alpha (PPAR alpha) ligand Wy-14643 (WY) on the abnormalities of medium chain fatty acid oxidation and pyruvate dehydrogenase complex (PDC) activity in kidney tissue of cisplatin-treated mice. Cisplatin causes a significant reduction in mRNA levels and enzyme activity of mitochondrial medium chain acyl-CoA dehydrogenase (MCAD). PPAR alpha ligand WY ameliorated cisplatin-induced acute renal failure and prevented cisplatin-induced reduction of mRNA levels and enzyme activity of MCAD. In contrast, in cisplatin-treated PPAR alpha null mice, WY did not protect kidney function and did not reverse cisplatin-induced decreased expression of MCAD. Cisplatin inhibited renal PDC activity before the development of acute tubular necrosis, and PDC inhibition was reversed by pretreatment with PPAR alpha agonist WY. Cisplatin also induced increased mRNA and protein levels of pyruvate dehydrogenase kinase-4 (PDK4), and PPAR alpha ligand WY prevented cisplatin-induced increased expression of PDK4 protein levels in wild-type mice. We conclude that PPAR alpha agonists have therapeutic potential for cisplatin-induced acute renal failure. Use of PPAR alpha ligands prevents acute tubular necrosis by ameliorating cisplatin-induced inhibition of two distinct metabolic processes, MCAD-mediated fatty acid oxidation and PDC activity.

Acute Kidney Injury↗
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↗
PubMed2002

Oral tolerance and pyruvate dehydrogenase in patients with primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the immunological destruction of intralobular bile ducts and serum anti-mitochondrial antibodies (AMA). Based upon previous work of oral tolerance and autoimmunity, we hypothesized that feeding the mitochondrial autoantigens of PBC would alter the clinical course and the level of antimitochondrial antibodies. The bovine pyruvate dehydrogenase complex (PDC) was purified and 5 mg fed in gelatin capsules to 6 patients with early stage PBC for 6 months. Antimitochondrial antibodies and liver biochemistries were measured at every 3 months for 12 months. The clinical trial was completed for all patients except for 1 who showed deterioration of pre-existing skin rash during treatment, which disappeared within 2 weeks after treatment was discontinued. However, after 1 year, neither the titers of AMAs nor liver biochemistries were significantly changed by this treatment. This is the first trial to test the efficacy of oral tolerance induction in PBC. However, the data, which limited in scope, did not demonstrate efficacy and further highlights the difficulties in showing continuing evidence of tolerance induction in autoimmunity.

Administration, Oral↗
PubMed2005

Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.

Acetylation↗
PubMed1984

Pyruvate dehydrogenase phosphate (PDHb) phosphatase activity in fibroblasts from Leigh's disease.

The defective activation of pyruvate dehydrogenase complex (PDHC) in Leigh's disease (subacute necrotizing encephalomyelopathy) could be due to deficiency of pyruvate dehydrogenase phosphate (PDHb) phosphatase (EC 3.1.3.43). This enzyme catalyzes the dephosphorylation and activation of phospho-PDHC. In cultured skin fibroblasts, we assayed this enzyme by measuring the rate of activation of the exogenously added, purified phospho-PDHC (bovine kidney). PDHb phosphatase activity did not differ significantly among normal control cells, Leigh's lines, spinocerebellar ataxias, or other neurologic disorders. The results do not support the idea that PDHb phosphatase is deficient in Leigh's disease.

Animals↗
PubMed1984

A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibroblasts.

A method for measuring the activity of the pyruvate dehydrogenase complex (PDC) by coupling acetyl-CoA production to acetylation of a fluorescent dye is described. Acetylation of cresyl violet acetate by pigeon liver acetyltransferase results in a shift of its fluorescence spectrum from lambda ex max = 575, lambda em max = 620 nm to lambda ex max = 475, lambda em max = 575 nm. The rate of appearance of acetylated dye was followed fluorometrically and was proportional to PDC activity in extracts of cultured human fibroblasts. The assay showed appropriate substrate and cofactor dependence and had a working range between 0.04 and 70 munits. It is 10 times more sensitive than the spectrophotometric assay on which it is based (working range 0.4-31 munits) and is equally convenient. Unactivated PDC activity in fibroblast extracts was 0.75 (0.60-0.92) munits/mg protein (mean and range for six cell lines).

Acetyl Coenzyme A↗
PubMed2004

Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice.

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.

Acetyltransferases↗
PubMed1996

Interaction of carbohydrate and fat fuels in human skeletal muscle: impact of obesity and NIDDM.

The current study was undertaken to examine the impact that obesity and non-insulin-dependent diabetes mellitus (NIDDM) have on the ability of glucose to stimulate its own uptake and oxidation in muscle. Euglycemic and hyperglycemic clamp experiments were performed with somatostatin infusions so that insulin could be replaced to basal levels or to physiological hyperinsulinemia. Arteriovenous leg balance methods were used to measure the pathways of leg muscle glucose uptake, oxidation, and storage. Percutaneous biopsies of the vastus lateralis muscle were taken to determine the pyruvate dehydrogenase complex or glycogen synthase activities. During basal insulin replacement, obese compared with lean nondiabetic subjects had higher values for glucose uptake, respiratory quotient, and glucose oxidation (all P<0.05) and a higher proportion of leg energy expenditure derived from glucose. Obese NIDD patients had a greater reliance on fat calories than lean diabetics during basal insulin replacement (P< 0.05). Hyperinsulinemia increased leg glucose metabolism (P<0.001) in all groups, but obese NIDD patients were significantly more insulin resistant. Hyperglycemia in NIDDM compensated for insulin resistance to the extent that rates of glucose metabolism were the same as those for nondiabetics studied at euglycemia. When nondiabetics were studied at hyperglycemia matched to the diabetics, the insulin resistance was still readily apparent.

Adult↗
PubMed2000

Selective modification of the pyruvate dehydrogenase kinase isoform profile in skeletal muscle in hyperthyroidism: implications for the regulatory impact of glucose on fatty acid oxidation.

The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P<0.05) but not in the slow-twitch muscle (the soleus). PDK4 protein expression was increased by experimental hyperthyroidism in both muscle types, there being a greater response in the anterior tibialis (4.2-fold increase; P<0.05) than in the soleus (3.2-fold increase; P<0.05). The hyperthyroidism-associated up-regulation of PDK4 expression was observed in conjunction with suppression of skeletal-muscle PDC activity, but not suppression of glucose uptake/phosphorylation, as measured in vivo in conscious unrestrained rats (using the 2-[(3)H]deoxyglucose technique). We propose that increased PDK isoform expression contributes to the pathology of hyperthyroidism and to PDC inactivation by facilitating the operation of the glucose --> lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.

Animals↗
PubMed1995

Subcellular localization of pyruvate dehydrogenase dihydrolipoamide acetyltransferase in human intrahepatic biliary epithelial cells.

In previous histological studies, biliary epithelial cells (BEC) in the liver of patients with primary biliary cirrhosis (PBC), but not controls, reacted strongly with antibodies specific for the major autoantigen associated with PBC, the E2 component of pyruvate dehydrogenase complex (PDC-E2). In this study we have used transmission electron microscopy (TEM) to document the precise subcellular localization of PDC-E2 in BEC. Two antibodies which recognize PDC-E2 were used: affinity-purified anti-PDC-E2 raised in rabbits; and human antibody from the serum of patients with PBC, affinity-purified against human heart PDC. The intracellular localization of antibody binding was determined by laser scanning confocal microscopy and TEM. Both antibodies bound to the inner membrane of mitochondria in BEC isolated from both patients with PBC and controls, but binding to the external aspect of the plasma membrane was observed only in BEC from patients with PBC. Surface antigen expression in PBC may make BEC immunological targets.

Adult↗
PubMed1993

Chromosome localization and RFLP analysis of PDC-E2: the major autoantigen of primary biliary cirrhosis.

Patients with primary biliary cirrhosis are well known for the presence of titer antibodies against dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex. We have taken advantage of a cDNA probe for dihydrolipoamide acetyltransferase to explore the possibility of polymorphism of the E2 subunit by probing genomic DNA from 38 patients with primary biliary cirrhosis and 26 healthy controls. To detect restriction fragment length polymorphism, DNA was digested with ten specific restriction enzymes that often detect polymorphism, including Bam HI, Bgl II, Eco RI, Hind III, Hinf I, Msp I, Pst I, Pvu II, Rsa I and Taq I. A Taq I polymorphism was found in 19 of 38 patients with PBC and 6 of 26 normal controls. In addition, using fluorescence in situ hybridization, the gene for dihydrolipoamide acetyltransferase was mapped on human chromosome 11 band q23.1. Interestingly, this region of the long arm of chromosome 11 is often associated with cytogenetic abnormalities, including translocations.

Autoantigens↗