Increased expression of hepatic pyruvate dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima Fatty rats: induction by elevated levels of free fatty acids

The activity of the pyruvate dehydrogenase complex (PDC) is regulated by covalent modification of its E1 component, which is catalyzed by specific pyruvate dehydrogenase kinases (PDKs) and phosphatases. In the liver, PDK2 and PDK4 are the most abundant PDK isoforms, which are responsible for inactivation of PDC when glucose availability is scarce in the body. In the present study, regulatory mechanisms of hepatic PDC were examined before and after the onset of type 2 diabetes mellitus in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, using Long-Evans Tokushima Otsuka (LETO) rats as controls. Plasma glucose and insulin concentrations were at normal levels in rats aged 8 weeks, but were significantly higher in OLETF than in LETO rats aged 25 weeks, indicating insulin resistance in OLETF rats. Plasma free fatty acids (FFAs) were 1.6-fold concentrated, and the liver PDC activity was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidative decarboxylation in OLETF rats before and after the onset of diabetes. Pyruvate dehydrogenase kinase activity and abundance of PDK2 and PDK4 proteins, as well as mRNAs, were greater in OLETF rats at both ages. These results suggest that persistently elevated levels of circulating free fatty acid in normal and diabetic OLETF rats play an important role in stimulating PDK2 and PDK4 expression in liver.     

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.     

Association between vitamin D status and metabolic syndrome risk among Korean population: based on the Korean National Health and Nutrition Examination Survey IV-2, 2008

Aims

Pyruvate dehydrogenase kinase 4 (PDK4) plays a crucial role in glucose utilization and lipid metabolism by regulating the pyruvate dehydrogenase complex (PDC) and is an emerging therapeutic target for type 2 diabetes. To date, no study has specifically examined the relationship between PDK4 gene polymorphisms and type 2 diabetes or metabolic syndrome.

Methods

The association of common single nucleotide polymorphisms (SNPs) was examined in PDK4 [−208A/G (rs10085637), IVS3 + 192C/T (rs3779478), IVS6 + 31A/G (rs2301630), IVS7 + 514A/G (rs12668651), IVS10 + 75C/T (rs10247649)] with type 2 diabetes and metabolic syndrome in 651 Korean subjects with type 2 diabetes and 350 nondiabetic Korean subjects. The association of these SNPs with clinical parameters related to metabolic syndromes including obesity, hyperglycemia, hypertension, and dyslipidemia was also examined.

Results

No significant association was found between the studied SNPs and type 2 diabetes, metabolic syndrome, or clinical parameters. The PDK4 gene haplotype ACAGC showed a modest association with type 2 diabetes. However, the significance of this association was lost after considering for multiple comparisons.

Conclusions

PDK4 polymorphisms may not be associated with type 2 diabetes or metabolic syndrome. Further studies utilizing a larger study population are required to confirm these results.     

Mitochondrial regulators of fatty acid metabolism reflect metabolic dysfunction in type 2 diabetes mellitus

The delicate homeostatic balance between glucose and fatty acid metabolism in relation to whole-body energy regulation is influenced by mitochondrial function. We determined expression and regulation of mitochondrial enzymes including pyruvate dehydrogenase kinase (PDK) 4, PDK2, carnitine palmitoyltransferase 1b, and malonyl-coenzyme A decarboxylase in skeletal muscle from people with normal glucose tolerance (NGT) or type 2 diabetes mellitus (T2DM). Vastus lateralis biopsies were obtained from NGT (n = 79) or T2DM (n = 33) men and women matched for age and body mass index. A subset of participants participated in a 4-month lifestyle intervention program consisting of an unsupervised walking exercise. Muscle biopsies were analyzed for expression and DNA methylation status. Primary myotubes were derived from biopsies obtained from NGT individuals for metabolic studies. Cultured skeletal muscle was exposed to agents mimicking exercise activation for messenger RNA (mRNA) expression analysis. The mRNA expression of PDK4, PDK2, and malonyl-coenzyme A decarboxylase was increased in skeletal muscle from T2DM patients. Methylation of the PDK4 promoter was reduced in T2DM and inversely correlated with PDK4 expression. Moreover, PDK4 expression was positively correlated with body mass index, blood glucose, insulin, C peptide, and hemoglobin A(1c). A lifestyle intervention program resulted in increased PDK4 mRNA expression in NGT individuals, but not in those with T2DM. Exposure to caffeine or palmitate increased PDK4 mRNA in a cultured skeletal muscle system. Our findings reveal that skeletal muscle expression of PDK4 and related genes regulating mitochondrial function reflects alterations in substrate utilization and clinical features associated with T2DM. Furthermore, hypomethylation of the PDK4 promoter in T2DM coincided with an impaired response of PDK4 mRNA after exercise.     

The pyruvate carboxylase-pyruvate dehydrogenase axis in islet pyruvate metabolism: Going round in circles?

Pyruvate is the major product of glycolysis in pancreatic β-cells, and its ultimate metabolic fate depends on the relative activities of two enzymes. The first, pyruvate carboxylase (PC) replenishes oxaloacetate withdrawn from the tricarboxylic acid (TCA) cycle via the carboxylation of pyruvate to form oxaloacetate. Flux via PC is also involved in the formation of NADPH, one of several important coupling factors for insulin secretion. In most tissues, PC activity is enhanced by increased acetyl-CoA. The alternative fate of pyruvate is its oxidative decarboxylation to form acetyl-CoA via the pyruvate dehydrogenase complex (PDC). The ultimate fate of acetyl-CoA carbon is oxidation to CO₂ via the TCA cycle, and so the PDC reaction results of the irreversible loss of glucose-derived carbon. Thus, PDC activity is stringently regulated. The mechanisms controlling PDC activity include end product inhibition by increased acetyl-CoA, NADH and ATP, and its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDHKs 1–4). Here we review new developments in the regulation of the activities and expression of PC, PDC and the PDHKs in the pancreatic islet in relation to islet pyruvate disposition and glucose-stimulated insulin secretion (GSIS).     

α-Ketoacid dehydrogenase complexes and respiratory fuel utilisation in diabetes

Summary Activity of the pyruvate dehydrogenase complex determines the rate of glucose oxidation in animals including man. The complex is regulated by reversible phosphorylation, phosphorylation resulting in inactivation. Activity is therefore dependent upon the activities of pyruvate dehydrogenase kinase and phosphatase. Activity of the complex is reduced in diabetes and starvation as a result of insulin deficiency. The mechanism involves activation of pyruvate dehydrogenase kinase by short-term effects of products of fatty acid oxidation and by longer term effects involving specific protein synthesis; in hepatocytes the signals may include lipid fuels and glucagon. Activity of the branched chain ketoacid dehydrogenase complex determines the rate of degradation of branched chain aminoacids which is adjusted according to dietary supply The complex is regulated by reversible phosphorylation, phosphorylation being inactivating. In liver and kidney, but not in muscles a protein activator (free El component) may reactivate phosphorylated complex without dephosphorylation and facilitate hepatic oxidation of branched chain ketoacids. Metabolic adjustments induced by diet and diabetes include loss of activator protein, loss of total complex activity in liver but not muscles, and enhanced inactivation by phosphorylation in liver.     

The metabolic effects of dichloroacetate

Dichloroacetate activates the pyruvate dehydrogenase complex of many tissues by inhibiting the kinase responsible for phosphorylation and inactivation of the complex. Dichloroacetate also activates the myocardial branched-chain α-keto acid dehydrogenase complex but apparently not by direct inhibition of the analogous kinase. Oxalate and glyoxylate, metabolites of dichloroacetate, are responsible for some in vitro effects of dichloroacetate. Dichloroacetate stimulates leucine oxidation by isolated hepatocytes because glyoxylate transaminates with leucine. Dichloroacetate inhibits lactate gluconeogenesis by hepatocytes incubated in low bicarbonate buffer because oxalate inhibits pyruvate carboxylase under such conditions. In vivo, dichloroacetate decreases blood glucose by limiting the supply of gluconeogenic precursors to the liver. This effect is a consequence of pyruvate dehydrogenase activation in peripheral tissues. Dichloroacetate lowers blood cholesterol in hyperlipidemic patients by uncertain means. Dichloroacetate has been tried experimentally in treatment of diabetes, hypercholesterolemia, and hyperlactatemia, but it has neurotoxicity, can cause cataracts, and may be mutagenic.     

The pyruvate carboxylase-pyruvate dehydrogenase axis in islet pyruvate metabolism: Going round in circles?

Pyruvate is the major product of glycolysis in pancreatic β-cells, and its ultimate metabolic fate depends on the relative activities of two enzymes. The first, pyruvate carboxylase (PC) replenishes oxaloacetate withdrawn from the tricarboxylic acid (TCA) cycle via the carboxylation of pyruvate to form oxaloacetate. Flux via PC is also involved in the formation of NADPH, one of several important coupling factors for insulin secretion. In most tissues, PC activity is enhanced by increased acetyl-CoA. The alternative fate of pyruvate is its oxidative decarboxylation to form acetyl-CoA via the pyruvate dehydrogenase complex (PDC). The ultimate fate of acetyl-CoA carbon is oxidation to CO2 via the TCA cycle, and so the PDC reaction results of the irreversible loss of glucose-derived carbon. Thus, PDC activity is stringently regulated. The mechanisms controlling PDC activity include end-product inhibition by increased acetyl-CoA, NADH and ATP, and its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDHKs 1-4). Here we review new developments in the regulation of the activities and expression of PC, PDC and the PDHKs in the pancreatic islet in relation to islet pyruvate disposition and glucose-stimulated insulin secretion (GSIS).     

The orphan nuclear receptor, NOR-1, a target of beta-adrenergic signaling, regulates gene expression that controls oxidative metabolism in skeletal muscle

beta 1-3-Adrenoreceptor (AR)-deficient mice are unable to regulate energy expenditure and develop diet-induced obesity on a high-fat diet. We determined previously that beta2-AR agonist treatment activated expression of the mRNA encoding the orphan nuclear receptor, NOR-1, in muscle cells and plantaris muscle. Here we show that beta2-AR agonist treatment significantly and transiently activated the expression of NOR-1 (and the other members of the NR4A subgroup) in slow-twitch oxidative soleus muscle and fast-twitch glycolytic tibialis anterior muscle. The activation induced by beta-adrenergic signaling is consistent with the involvement of protein kinase A, MAPK, and phosphorylation of cAMP response element-binding protein. Stable cell lines transfected with a silent interfering RNA targeting NOR-1 displayed decreased palmitate oxidation and lactate accumulation. In concordance with these observations, ATP production in the NOR-1 silent interfering RNA (but not control)-transfected cells was resistant to (azide-mediated) inhibition of oxidative metabolism and expressed significantly higher levels of hypoxia inducible factor-1alpha. In addition, we observed the repression of genes that promote fatty acid oxidation (peroxisomal proliferator-activated receptor-gamma coactivator-1alpha/beta and lipin-1alpha) and trichloroacetic acid cycle-mediated carbohydrate (pyruvate) oxidation [pyruvate dehydrogenase phosphatase 1 regulatory and catalytic subunits (pyruvate dehydrogenase phosphatases-1r and -c)]. Furthermore, we observed that beta2-AR agonist administration in mouse skeletal muscle induced the expression of genes that activate fatty acid oxidation and modulate pyruvate use, including PGC-1alpha, lipin-1alpha, FOXO1, and PDK4. Finally, we demonstrate that NOR-1 is recruited to the lipin-1alpha and PDK-4 promoters, and this is consistent with NOR-1-mediated regulation of these genes. In conclusion, NOR-1 is necessary for oxidative metabolism in skeletal muscle.     

Rationale for a metabolic approach in diabetic coronary patients

The incidence of ischaemic heart disease and acute myocardial infarction are greater in people with diabetes than in nondiabetic individuals. Heart disease patients with diabetes have a higher incidence of mortality during and following an acute myocardial infarction and a high risk for progression to heart failure post-infarction. The greater occurrence of ischaemic heart disease is partially due to a poorer coronary artery disease risk factor profile in diabetic patients, and, importantly, due to diabetes-induced abnormalities in the myocardium, termed 'diabetic cardiomyopathy'. The main metabolic abnormalities in the diabetic myocardium are impaired carbohydrate metabolism, specifically reduced pyruvate oxidation in the mitochondria and a greater reliance on fatty acids and ketone bodies as fuels. The healthy heart takes up glucose and lactate and converts them to pyruvate; however, in the diabetic heart there is a reduced capacity to oxidize pyruvate, and thus less glucose and lactate uptake. The defective metabolism is due to high circulating free fatty acids and ketone body concentrations in the plasma, resulting in greater acetyl-Co-enzyme A/Co-enzyme A and reduced nicotinamide adenonine dinucleotide/nicotinamide adenonine dinucleotide+ ratios in the mitochondria, and the subsequent inhibition of pyruvate dehydrogenase. Pharmacological inhibition of fatty acid oxidation during ischaemia increases myocardial pyruvate oxidation and provides clinical benefit to patients with stable angina or ischaemic left ventricular dysfunction. Recent clinical trials with trimetazidine, an inhibitor of the fatty acid beta-oxidation enzyme long chain 3-ketoacylthiolase, showed improvement in cardiac function and exercise performance in diabetic patients with ischaemic heart disease, illustrating the effectiveness of this approach in diabetes.     

High-fat/low-carbohydrate diet reduces insulin-stimulated carbohydrate oxidation but stimulates nonoxidative glucose disposal in humans: An important role for skeletal muscle pyruvate dehydrogenase kinase 4

AIM: The aim of this report was to study the effect of high-fat (HF)/low-carbohydrate (CHO) diet on regulation of substrate metabolism in humans. METHODS: Ten healthy men consumed either a HF (75% energy as fat) or control (35%) diet for 6 d in random order. On d 7, blood glucose disappearance rate (Rd) was determined before and during a hyperinsulinemic euglycemic clamp. Substrate oxidation was determined by indirect calorimetry. Muscle biopsies were obtained prediet, postdiet, and postclamps. RESULTS: Rd was similar under basal conditions but slightly elevated (approximately 10%, P < 0.05) during the last 30 min of the clamp after the HF diet. HF diet reduced CHO oxidation under basal (by approximately 40%, P < 0.05) and clamp conditions (by approximately 20%, P < 0.05), increased insulin-mediated whole-body nonoxidative glucose disposal (by 30%, P < 0.05) and muscle glycogen storage (by approximately 25%, P < 0.05). Muscle pyruvate dehydrogenase complex activity was blunted under basal and clamp conditions after HF compared with control (P < 0.05) and was accompanied by an approximately 2-fold increase (P < 0.05) in pyruvate dehydrogenase kinase 4 (PDK4) mRNA and protein expression. CONCLUSION: Short-term HF/low-CHO dietary intake did not induce whole-body insulin resistance, but caused a shift in im glucose metabolism from oxidation to glycogen storage. Insulin-stimulated CHO oxidation and muscle pyruvate dehydrogenase complex activity were blunted after the HF diet. Up-regulation of muscle PDK4 expression was an early molecular adaptation to these changes, and we showed for the first time in healthy humans, unlike insulin-resistant individuals, that insulin can suppress PDK4 but not PDK2 gene expression in skeletal muscle.     

Reperfusion-induced translocation of deltaPKC to cardiac mitochondria prevents pyruvate dehydrogenase reactivation

Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive delta-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the deltaPKC activator H2O2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of deltaPKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of deltaPKC is associated with phosphorylation of the alphaE1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that deltaPKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of deltaPKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.     

Regulation of pyruvate metabolism in metabolic-related diseases

Pyruvate is an obligatory intermediate in the oxidative disposal of glucose and a major precursor for the synthesis of glucose, glycerol, fatty acids, and non-essential amino acids. Stringent control of the fate of pyruvate is critically important for cellular homeostasis. The regulatory mechanisms for its metabolism are therefore of great interest. Recent advances include the findings that (a) the mitochondrial pyruvate carrier is sensitive to inhibition by thiazolidinediones; (b) pyruvate dehydrogenase kinases induce the Warburg effect in many disease states; and (c) pyruvate carboxylase is an important determinate of the rates of gluconeogenesis in humans with type 2 diabetes. These enzymes are potential therapeutic targets for several diseases.     

Pyruvate dehydrogenase complex in the rabbit adrenal glands: activation by administration of ACTH

The pyruvate dehydrogenase complex (PDC) from the rabbit adrenal mitochondria was partially purified (18-fold) and its main kinetic parameters were determined: Michaelis' constants for pyruvate, CoA and NAD are 16, 10 and 140, respectively. The inhibitor constant for acetyl-CoA is 25 microM. Phosphate ions augment the PDC affinity for the substrate. After ACTH administration to rabbits the active PDC form in the adrenals rises simultaneously with the PDC phosphatase activity growth. The data obtained are indicative of the adrenal PDC involvement in mobilizing metabolism to provide intensive steroidogenesis.     

3D electron microscopy reveals the variable deposition and protein dynamics of the peripheral pyruvate dehydrogenase component about the core

Cryo-electron microscopy was exploited to reveal and study the influence of pyruvate dehydrogenase (E1) occupancy on the conformational states of the Saccharomyces cerevisiae pyruvate dehydrogenase complex (PDC). Structures representative of PDC preparations with approximately 40% and full E1 occupancy were determined after the electron microscopy images from each preparation were classified according to their sizes. The reconstructions derived from two size groups showed that the deposition of the E1 molecules associated with the larger complex is, unexpectedly, not icosahedrally arranged, whereas in the smaller complex the E1 molecules have an arrangement and architecture similar to their more ordered deposition in the WT bovine kidney PDC. This study also shows that the linker of dihydrolipamide acetyltransferase (E2) that tethers E1 to the E2 core increases in length from approximately 50 to 75 A, accounting largely for the size difference of the smaller and larger structures, respectively. Extensive E1 occupancy of its 60 E2 binding sites favors the extended conformation of the linker associated with the larger complex and appears to be related to the loss of icosahedral symmetry of the E1 molecules. However, the presence of a significant fraction of larger molecules also in the WT PDC preparation with low E1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E1. The flexibility of the complex may enhance the catalytic proficiency of this macromolecular machine by promoting the channeling of the intermediates of catalysis between the active sites.     

Acute renal metabolic effect of metformin assessed with hyperpolarised MRI in rats

Aims/hypothesis

Metformin inhibits hepatic mitochondrial glycerol phosphate dehydrogenase, thereby increasing cytosolic lactate and suppressing gluconeogenesis flux in the liver. This inhibition alters cytosolic and mitochondrial reduction–oxidation (redox) potential, which has been reported to protect organ function in several disease states including diabetes. In this study, we investigated the acute metabolic and functional changes induced by metformin in the kidneys of both healthy and insulinopenic Wistar rats used as a model of diabetes.

Methods

Diabetes was induced by intravenous injection of streptozotocin, and kidney metabolism in healthy and diabetic animals was investigated 4 weeks thereafter using hyperpolarised ¹³C-MRI, Clark-type electrodes and biochemical analysis.

Results

Metformin increased renal blood flow, but did not change total kidney oxygen consumption. In healthy rat kidneys, metformin increased [1-¹³C]lactate production and reduced mitochondrial [1-¹³C]pyruvate oxidation (decreased the ¹³C-bicarbonate/[1-¹³C]pyruvate ratio) within 30 min of administration. Corresponding alterations to indices of mitochondrial, cytosolic and whole-cell redox potential were observed. Pyruvate oxidation was maintained in the diabetic rats, suggesting that the diabetic state abrogates metabolic reprogramming caused by metformin.

Conclusions/interpretation

This study demonstrates that metformin-induced acute metabolic alterations in healthy kidneys favoured anaerobic metabolism at the expense of aerobic metabolism. The results suggest that metformin directly alters the renal redox state, with elevated renal cytosolic redox states as well as decreased mitochondrial redox state. These findings suggest redox biology as a novel target to eliminate the renal complications associated with metformin treatment in individuals with impaired renal function.

     

Immunoreactivity of antimitochondrial autoantibodies in Japanese patients with primary biliary cirrhosis

The incidence and prevalence of primary biliary cirrhosis show wide geographic differences. The frequency of this disease in Japan is lower than in Northern Europe. To elucidate the immunoreactivity of serum with enzymes of the 2-oxo-acid dehydrogenase complex (2-OADC) and the M2 mitochondrial antigenic complex in Japanese patients, we examined sera from 107 patients with primary biliary cirrhosis from three geographically different regions of Japan. The sera were assayed by immunofluorescence on frozen tissue sections, immunoblotting on bovine heart mitochondria and recombinant E2 subunit of branched chain oxo-acid dehydrogenase complex (BCOADCE2), ELISA using recombinant E2 subunit of human pyruvate dehydrogenase complex (PDC-E2) and purified porcine 2-oxoglutarate dehydrogenase complex (OGDC), and enzyme inhibition assay using porcine PDC and OGDC. Of the 107 sera, 95 (88%) reacted by immunofluorescence, 102 (95%) by immunoblotting with at least one of the M2 autoantigens, although only 78 (73%) reacted with PDC-E2; 72 (67%) by ELISA with PDC-E2; and 81 (76%) with PDC by the enzyme inhibition assay. Thus, the frequency of reactivity with PDC-E2 by all assays was lower for Japanese than the reported frequency for Caucasian patients with primary biliary cirrhosis, whereas the frequency of reactivity by immunoblotting and ELISA against 2-OADC enzymes other than PDC was relatively higher. The relative frequency of reactivity of autoantibodies to the M2 autoantigens was similar for the three different regions of Japan. The different autoantibody profiles for Japanese and Caucasian patients with primary biliary cirrhosis point to immunogenetic and environmental determinants of this disease, which should provide new insights into its autoimmune origins.     

Expression of key genes of fatty acid oxidation,including adiponectin receptors,in skeletal muscle of Type 2 diabetic patients

Aims/hypothesis Defective oxidation of long-chain fatty acids is a feature of insulin resistance and Type 2 diabetes. Our aim was to compare the expression levels of the genes encoding the major proteins and enzymes of this pathway in skeletal muscle of healthy subjects and Type 2 diabetic patients.Methods The basal and insulin-regulated mRNA concentration of 16 genes was quantified using real-time PCR in skeletal muscle biopsies taken before and at the end of a 3-hour hyperinsulinaemic–euglycaemic clamp in healthy lean subjects and in insulin-resistant obese patients with manifest Type 2 diabetes.Results Acetyl CoA carboxylase-2 mRNA expression was increased 2.5-fold in the muscle of the diabetic patients. The expression of carnitine palmitoyl transferase-1, of the two adiponectin receptors and of genes involved in fatty acid transport and activation was not altered in diabetic patients. Hyperinsulinaemia for 3 hours increased the expression of several genes of fatty acid oxidation, including adiponectin receptor-1 and peroxisome proliferator-activated receptor coactivator-1. It also reduced pyruvate dehydrogenase 4 mRNA levels. The effects of insulin on gene expression were markedly altered in the muscle of Type 2 diabetic patients except for adiponectin receptor-1 and pyruvate dehydrogenase 4 mRNAs.Conclusions/interpretation The expression of adiponectin receptors was not altered in the muscle of Type 2 diabetic patients. The observed overexpression of acetyl CoA carboxylase-2 is consistent with the hypothesis that increased skeletal muscle malonyl CoA concentrations in Type 2 diabetes may contribute to the inhibition of long-chain fatty acid oxidation.Abbreviations ACC2 acetyl CoA carboxylase-2 - AdipoR1 adiponectin receptor 1 - AdipoR2 adiponectin receptor 2 - CACT carnitine acyl carnitine translocase - CPT1 carnitine palmitoyl CoA transferase 1 - CPT2 carnitine palmitoyl CoA transferase 2 - FABP3 fatty acid binding protein 3 - FABPpm plasma membrane fatty acid binding protein - FACL fatty acid CoA ligase - FAT/CD36 fatty acid translocase - LCACoA long-chain acyl CoAs - LCFA long-chain fatty acid - LCFACoA long-chain fatty acid coenzyme A - Mfn-2 mitofusin-2 - PDK4 pyruvate dehydrogenase kinase 4 - PGC1 PPAR coactivator-1 - PPARs peroxisome proliferator-activated receptors     

A computer model analysis of the active-site coupling mechanism in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

A computer modeling system developed to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) accompanying release of lipoyl moieties by lipoamidase and by trypsin [Hackert, M.L., Oliver, R.M. & Reed, L.J. (1983) Proc. Natl. Acad. Sci. USA 80, 2226-2230] was used to analyze analogous data for the E. coli pyruvate dehydrogenase complex (PDC). The model studies indicate that the activity of PDC, as found for KGDC, is influenced by redundancies and random processes, which we describe as a multiple random coupling mechanism. In both complexes more than one lipoyl moiety services each pyruvate dehydrogenase (EC 1.2.4.1) or alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) (E1) subunit, and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data for PDC was obtained with a model that has four lipoyl domains with four or, more probably, eight lipoyl moieties servicing each E1 subunit. The lipoyl-lipoyl interaction network for PDC has lipoyl domain interactions similar to those found for KGDC plus the additional possibility of interaction of a lipoyl moiety and its paired mate on each dihydrolipoamide acetyltransferase (EC 2.3.1.12) (E2) subunit. The two lipoyl moieties on an E2 subunit in PDC appear to be functionally indistinguishable, each servicing the acetyltransferase site of that E2 subunit and a dihydrolipoamide dehydrogenase (EC 1.6.4.3) (E3) subunit if the latter is bound to that particular E2 subunit. The observed difference between inactivation of PDC by lipoamidase and by trypsin appears to be due to dead-end competitive inhibition by lipoyl domains that have been modified by excision of lipoyl moieties by lipoamidase.