Decreased insulin-generation of pyruvate dehydrogenase inhibitor in insulin resistant states

Insulin resistance produced in rats by feeding a high fat diet or by dexamethasone administration (50 micrograms/day, sc for 4 days) resulted in 50-70% decrease in the generation of pyruvate dehydrogenase inhibitor by insulin exposed liver particulate fractions. The inhibition was dose dependent. Treatment of insulin mediator preparations with neuraminidase and B-D-galactosidase resulted in inactivation of the pyruvate dehydrogenase inhibitor. Presence of exogenous enzyme substrates during enzyme digestion partially protected the inhibitor from inactivation. Protease treatment did not affect the inhibitor while the stimulatory activity of the insulin mediator was abolished by trypsin treatment. These results together with the previous report suggest that insulin resistance results in a decrease in the generation of both of the mediators of insulin action. This may result from a decrease in insulin binding, shown earlier, or from a decrease in precursor availability.     

Insulin stimulates the release from liver plasma membranes of a chemical modulator of pyruvate dehydrogenase

Incubation of a rat liver particulate fraction with physiological concentrations of insulin enhances the production of a small molecular weight substance which modulates adipocyte as well as liver mitochondrial pyruvate dehydrogenase. While low concentrations of insulin enhance production of this activity, levels of greater than 10^?9M produce significantly less. Similarly, while increasing concentrations of mediator cause increased stimulation of pyruvate dehydrogenase activity, higher concentrations no longer exhibit this effect. The putative insulin mediator was partially purified on HPLC and Sephadex G-25 columns. Its molecular weight was about 1000–2000. These results indicate the presence of a chemical mediator of insulin action in liver similar to that observed in other insulin target tissues.     

Enhanced dissociation of pyruvate dehydrogenase from the pyruvate dehydrogenase complex following phosphorylation and regulatory implications

We have shown that the active form of the pyruvate dehydrogenase (PDH_a) component exhibits at least a 9-fold greater affinity for sites on the dihydrolipoyl transacetylase core of the pyruvate dehydrogenase complex than does the inactive (phosphorylated) form of pyruvate dehydrogenase (PDH_b). Consistent with a higher rate of dissociation for PDH_b than for PDH_a, free PDH_a rapidly replaces PDH_b whereas, even at high levels, free PDH_b only slowly replaces PDH_a. Dissociation of newly formed PDH_b, during phosphorylation by the immobile PDH_a kinase, leads to an increased association of free PDH_a as observed by protection against inactivation of the complex, even though PDH_a kinase activity is increased.     

Pyruvate dehydrogenase kinases (PDKs): an overview toward clinical applications

Pyruvate dehydrogenase kinase (PDK) can regulate the catalytic activity of pyruvate decarboxylation oxidation via the mitochondrial pyruvate dehydrogenase complex, and it further links glycolysis with the tricarboxylic acid cycle and ATP generation. This review seeks to elucidate the regulation of PDK activity in different species, mainly mammals, and the role of PDK inhibitors in preventing increased blood glucose, reducing injury caused by myocardial ischemia, and inducing apoptosis of tumor cells. Regulations of PDKs expression or activity represent a very promising approach for treatment of metabolic diseases including diabetes, heart failure, and cancer. The future research and development could be more focused on the biochemical understanding of the diseases, which would help understand the cellular energy metabolism and its regulation by pharmacological effectors of PDKs.     

Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: Preparation and characterization

The dihydrolipoyl transacetylase core component of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N, N′-p-phenylenedimaleimide.In one approach, the N, N′-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found the N, N′-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues.Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.     

Malonaldehyde acts as a mitochondrial toxin: Inhibitory effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Malonaldehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines to modify proteins to inactivity enzymes and also modify nucleosides to cause mutagenicity. Mitochondrial dysfunction is a major contributor to aging and age-associated diseases. We hypothesize that accumulated MDA due to mitochondrial dysfunction during aging targets mitochondrial enzymes to cause further mitochondrial dysfunction and contribute to aging and age-associated diseases. We investigated the effects of MDA on mitochondrial respiration and enzymes (membrane complexes I, II, III and IV, and dehydrogenases, including alpha-ketoglutaric dehydrogenase (KGDH), pyruvate dehydrogenase (PDH), malate dehydrogenase (MDH)) in isolated rat liver mitochondria. MDA showed a dose-dependent inhibition on mitochondrial NADH-linked respiratory control ratio (RCR) and ADP/O ratio declined from the concentrations of 0.2 and 0.8 micromol/mg protein, respectively, and succinate-linked mitochondrial RCR and ADP/O ratio declined from 1.6 and 0.8 micromol/mg protein. MDA also showed dose-dependent inhibition on the activity of PDH, KGDH and MDH significantly from 0.1, 0.2 and 2 micromol/mg protein, respectively. Activity of the complexes I and II was depressed by MDA at 0.8 and 1.6 micromol/mg protein. However, MDA did not affect activity of complexes III and IV in the concentration range studied (0-6.4 micromol/mg protein). These results suggest that MDA can cause mitochondrial dysfunction by inhibiting mitochondrial respiration and enzyme activity, and the sensitivity of the enzymes examined to MDA is in the order of PDH>KGDH>complexes I and II>MDH>complexes III and IV.     

Insulin resistance in the liver in fasting and diabetes mellitus: the failure of insulin to stimulate the release of a chemical modulator of pyruvate dehydrogenase

To further define the mechanism(s) of insulin resistance in the liver associated with diabetes and fasting, we evaluated the ability of insulin to release an activator of pyruvate dehydrogenase activity from a liver particulate fraction. Insulin reproduceably and significantly enhanced the release of mediator from the liver particulate fraction of control animals. The particulate fractions from fasted and diabetic animals were resistant to this effect of insulin. Refeeding and insulin treatment, respectively, restored responsiveness to insulin. These data support the concept that alterations at or near the plasma membrane can be responsible for or accompany the insulin resistance observed in the liver in fasting and diabetes mellitus.     

Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts

The pyruvate dehydrogenase complex (PDC) in pea (Pisum sativum L., cv. Little Marvel) was studied immunologically using antibodies to specific subunits of mammalian PDC. Pea mitochondria and chloroplasts were both found to contain PDC, but distinct differences were noted in the subunit relative molecular mass (M_r) values of the individual enzymes in the mitochondrial and chloroplast PDC complexes. In particular, the mitochondrial E3 enzyme (dihydrolipoamide dehydrogenase; EC 1.8.1.4) has a high subunit M_r value of 67 000, while the chloroplast E3 enzyme has a subunit M_r value of 52 000, similar in size to the prokaryotic, yeast ad mammalian E3 enzymes. In addition, component X (not previously noted in plant PDC) was also found to be present in two distinct forms in pea mitochondrial and chloroplast complexes. As in the case of E3, mitochondrial component X has a higher subunit M_r value (67 000) than component X from chloroplasts (48 000), which is similar in size to its mammalian counterpart. The subunit M_r value of E2 (dihydrolipoamide acetyltransferase; EC 2.3.1.12) in both mitochondria and chloroplasts (50 000) is lower than that of mammalian E2 (74 000) but similar to that of yeast E2 (58 000), and is consistent with the presence of only a single lipoyl domain. Neither mitochondria nor chloroplasts showed any appreciable cross-reactivity with antiserum to mammalian E1 (pyruvate dehydrogenase; EC 1.2.4.1). However, mitochondria cross-reacted strongly with antiserum to yeast E1, giving a single band (M_r 41 000) which is thought to be E1_a. Chloroplasts showed no cross-reactivity with yeast E1, indicating that the mitochondrial E1_a subunit and its chloroplast equivalent are antigenically distinct polypeptides.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - M_r relative molecular mass - PDC pyruvate dehydrogenase multienzyme complex - SDS sodium dodecyl sulphate The financial support of the Agricultural and Food Research Council is gratefully acknowledged. We thank Steve Hill (Department of Botany, University of Edinburgh, UK) for advice on mitochondrial isolation, and James Neagle (Department of Biochemistry, University of Glasgow) and Ailsa Carmichael for helpful discussion.     

Kaempferol Glycosides in the Seed-Coat of Ophiopogon jaburan (Kunth) Lodd.

Blue seed-coats of Ophiopogon jaburan have been found to contain kaempferol, kaempferol 3-glucoside (astragalin), two new glucosides of kaempferol, and a trace amount of an unknown flavonol-like compound. One of the new glucosides was determined to be kaempferol 4′-glucoside and the other to be kaempferol 3, 4′-diglucoside by means of paper-chro-matographic and spectral analyses.     

Sepsis alters pyruvate dehydrogenase kinase activity in skeletal muscle

Chronic sepsis promotes a stable increase in pyruvate dehydrogenase kinase (PDHK) activity in skeletal muscle. PDHK is found tightly bound to the pyruvate dehydrogenase (PDH) complex and as free kinase. We investigated the ability of sepsis to modify the activity of the PDHK intrinsic to the PDH and free PDHK. Sepsis was induced by the intraabdominal introduction of a fecal-agar pellet infected with E. coli and B. fragilis. Five days later, mitochondria were isolated from skeletal muscle and PDHK measured in mitochondrial extracts. Sepsis caused an approximate 2-fold stimulation of PDHK. The mitochondrial extracts from control and septic rats were fractionated by gel chromatography on Sephacryl S-300 to separate PDHK intrinsic to PDH complex and free PDHK. PDH complex eluted at void volume and was assayed for PDHK intrinsic to the complex. The activity of PDHK intrinsic to PDH complex was a significantly increased 3 fold during sepsis. Free PDHK activity eluted after the PDH complex and its activity was enhanced by 70% during sepsis. Incubation of PDHK intrinsic to PDH with dichloroactate, an uncompetitive inhibitor of PDHK, showed the PDHK from septic rats relatively less sensitive to inhibition than controls. These results indicate that sepsis induces stable changes in PDHK in skeletal muscle.     

Pyruvate dehydrogenase complex (PDC) export from the mitochondrial matrix

Studies on mitochondria protein import had revealed in detail molecular mechanisms of how peptides and proteins could be selectively targeted and translocated across membrane bound organelles. The opposite process of mitochondrial export, while known to occur in various aspects of cellular physiology and pathology, is less well understood. Two very recent reports have indicated that a large mitochondrial matrix protein complex, the pyruvate dehydrogenase complex (PDC) (or its component subunits), could be exported to the lysosomes and the nucleus, respectively. In the case of the latter, evidence was presented to suggest that the entire complex of 8–10 MDa could translocate in its entirety from the mitochondrial matrix to the nucleus upon mitogenic or stress stimuli. We discuss these findings in perspective to what is currently known about the processes of transport in and out of the mitochondrion.     

On the origin of mitochondria: a reexamination of the molecular structure and kinetic properties of pyruvate dehydrogenase complex from brewer''s yeast

45Ca2+ incorporated in response to glucose was selectively mobilized from the beta-cell-rich pancreatic islets of ob/ob-mice after raising the intracellular Na+ by removal of K+ or addition of ouabain or veratridine. Also studies of insulin release indicated opposite effects of glucose and Na+ on the intracellular sequestration of calcium. The fact that glucose inhibits insulin release induced by raised intracellular Na+ indicates that this sugar can lower the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+]. The concept of a dual action of glucose on the cytoplasmic [Ca2+] might well explain previous observations of an inhibitory component in the glucose action on the 45Ca2+ efflux.     

Reduction of mitochondrial pyruvate dehydrogenase phosphatase activity in lactating rat mammary gland following starvation or insulin deprivation.

The initial rates of activation of inactivated pyruvate dehydrogenase from lactating rat mammary gland and from pig heart were employed to assay pyruvate dehydrogenase phosphatase activity in mammary gland mitochondrial extracts. 24 h-starvation or 3 h-deprivation of insulin diminished phosphatase activity compared to fed controls. Refeeding and insulin treatment of 24 h starved animals restored in 1 h control levels of phosphatase activity.     

Serine utilization in mouse liver: influence of caloric restriction and aging

The influence of caloric restriction (CR) on the activities of hepatic serine metabolizing enzymes in young (3 months) and old (30 months) mice was studied. Serine dehydratase (SDH) activity increased markedly with age in both diet groups and in old mice was higher in the CR group. No effects of CR were observed in the young. Serine:pyruvate transaminase (SPT) and glycerate kinase activities were unaffected by age and diet. However, glycerate dehydrogenase activity was decreased in old CR mice but not in young CR. The results of this study show that long-term CR influenced serine utilization only in the pathway catalyzed by SDH. This suggests that in mouse liver this pathway is critical for serine utilization in gluconeogenesis, while the SPT pathway plays a minor role. The increase in SDH activity with long-term CR is consistent with sustained increase in gluconeogenesis.     

Thermally Induced Changes of Lipoate Acetyltransferase Inner Core Isolated from the Bacillus stearothermophilus Pyruvate Dehydrogenase Complex

Incubation at 70°C converted the Bacillus stearothermophilus lipoate acetyltransferase inner core into an unidentified active molecular form, X, yielding an inactive aggregate. The core and X showed similar thermostabilities, but they were different in the recovery of enzyme activity after incubation with 1.2-2.0 M guanidine hydrochloride and its subsequent removal; the core was hardly recovered, but X was well recovered.