Stimulation Of Pyruvate Dehydrogenase Research Articles

Isolation from Rat Adipocytes of a Chemical Mediator for Insulin Activation of Pyruvate Dehydrogenase

Insulin treatment of adipocytes increased the amount or activity of a low molecular weight, acid-stable material which, when isolated from intact adipocytes by heat extraction and subsequent Sephadex G25 chromatography, yielded a single active fraction that stimulated mitochondrial pyruvate dehydrogenase by activating the phosphatase and not by altering the kinase activity. Phosphatase activation was demonstrated by the ability of the active material to increase pyruvate dehydrogenase activity in the absence of ATP and by the ability of NaF, a phosphatase inhibitor, to this stimulation. Involvement of the kinase in this activation mechanism was eliminated by the fact that, in the presence of ATP, (1) NaF completely blocked the stimulation of pyruvate dehydrogenase by the active fraction, and (2) the stimulation of pyruvate dehydrogenase by dichloroacetic acid, a kinase inhibitor, was additive to the stimulation caused by the active fraction. This active fraction may contain an intracellular chemical mediator or second messenger for insulin.

Differences in the effect of insulin on the generation by adipocytes and IM-9 lymphocytes of a chemical mediator which simulates the action of insulin on pyruvate dehydrogenase

Insulin treatment of adipocytes increased the amount or activity of a low molecular weight, acid stable material which, when isolated and partially purified from intact adipocytes, yielded a single active fraction which stimulated pyruvate dehydrogenase. IM-9 lymphocytes contained a similar active material but in contrast to the adipocyte, insulin treatment of IM-9 lymphocytes caused a reduction in the amount or activity of the material either by decreasing production of the material or by increasing production of an inhibitory substance. These findings are consistent with the reported biological effects of insulin on these two cell types and suggest that the active material from these two cell types is the putative insulin chemical mediator.

Insulin stimulation of pyruvate dehydrogenase in an isolated plasma membrane-mitochondrial mixture occurs by activation of pyruvate dehydrogenase phosphatase.

Insulin stimulation of pyruvate dehydrogenase in an isolated plasma membrane-mitochondrial mixture occurs by activation of pyruvate dehydrogenase phosphatase.

Evidence that insulin activates an intrinsic plasma membrane protease in generating a secondary chemical mediator.

Three lines of evidence are presented which support the hypothesis that the binding of insulin to rat adipocyte plasma membranes activates a membrane protease which results in the production of a soluble factor that stimulates pyruvate dehydrogenase activity when added to mitochondria. First, low concentrations of trypsin (0.01 to 0.1 microgram/ml) mimic the action of insulin on plasma membranes in this system. Second, inhibitors of trypsin-like proteases (soy trypsin inhibitor, ovomucoid) block insulin action on the plasma membrane, as do exogenous protease substrates which are esters of arginine, presumably by preventing the normal interaction of the protease and its endogenous membrane substrate. Third, digestion by proteases (0.1 mg/ml of trypsin, chymotrypsin, or papain) of the soluble material released from plasma membranes after insulin treatment blocks the stimulation of pyruvate dehydrogenase, suggesting that the mediator may be a peptide released by a proteolytic reaction.

HALOTHANE ANAESTHESIA CAN BLOCK INSULIN STIMULATION OF PYRUVATE DEHYDROGENASE ACTIVITY IN MAMMARY GLANDS OF 24-HOUR STARVED LACTATING RATS

The effect of halothane anaesthesia on the activity of the mitochondrial enzyme pyruvate dehydrogenase was studied in starved lactating rats. Extracts of freeze-clamped mammary gland and liver were assayed for pyruvate dehydrogenase activity. The fraction of the enzyme in the phosphorylated inactive form was increased greatly by starvation or by streptozotocin diabetes, and halothane anaesthesia did not disturb this effect. In starved animals not exposed to halothane, injection of insulin led to a rapid increase in the active fraction of the enzyme in mammary gland but not in liver. In animals under halothane anaesthesia this effect of insulin was largely abolished. The combination of starvation and halothane anaesthesia may impair mitochondrial accumulation of calcium which may be involved in the stimulation of pyruvate dehydrogenase by insulin.

Activation of pyruvate dehydrogenase by direct addition of insulin to an isolated plasma membrane/mitochondria mixture: evidence for generated of insulin's second messenger in a subcellular system.

The addition of insulin to a mixture of plasma membrane and mitochondrial fractions from rat adipocytes results in a decrease in the phosphorylation of a mitochondrial protein identified as the alpha subunit of pyruvate dehydrogenase [pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] (Seals, J. R., McDonald, J. M. & Jarett, L. (1979) J. Biol. Chem. 254, 6991-6996). This study confirms the prediction that a corresponding increase in pyruvate dehydrogenase activity can be effected by insulin treatment of this preparation. Incubation of the plasma membrane/mitochondria mixture with ATP inhibited pyruvate dehydrogenase activity as measured in a subsequent enzyme assay. The presence of insulin during this incubation with ATP resulted in a 24.5% stimulation of enzyme activity compared to incubation without insulin (n = 9, P < 0.001). The effect was specific for biologically active insulin and was insulin dose-dependent in the physiological range of insulin. Supermaximal doses of insulin produced reduced effects. An insulin effect of similar magnitude could also be observed when the plasma membrane/mitochondria mixture was incubated without ATP. Two insulin mimickers, concanavalin A and antibody to insulin receptor, stimulated pyruvate dehydrogenase by 30.4% (n = 6, P <0.001) and 28.1% (n = 8, P<0.001), respectively. Both of these agents also produced reduced effects at supermaximal concentrations. The effects of all three agents required plasma membranes and could not be produced by treatment of mitochondria alone. The results suggest that a mechanism common to all three agents is responsible for transmitting the stimulation from the plasma membrane to the mitochondrial components of the mixture.

Pyruvate dehydrogenase activation in adipocyte mitochondria by an insulin-generated mediator from muscle.

Material in a chromatographic fraction from an extract of insulin-treated muscle stimulated pyruvate dehydrogenase activity in addipocyte mitochondria. This action was similar to insulin's activation of the enzyme in a plasma membrane-mitochondria mixture. Neither the chromatographic fraction nor insulin required adenosine triphosphate or magnesium ion (Mg2+), suggesting that both agents acted through a calcium-sensitive phosphatase. This fraction may contain a chemical mediator of insulin action.

Energy Metabolism of Spermatozoa. VII. Interactions Between Lactate, Pyruvate and Malate as Oxidative Substrates for Rabbit Sperm Mitochondria

Rabbit spermatozoa, like other mammalian spermatozoa, have part of their lactate dehydrogenase in the mitochondrial matrix. This enables lactate to be utilized directly as a NAD-linked oxidizable substrate by the mitochondria, but it also enables pyruvate to act as an oxidant of intramitochondrial NADH which should inhibit 02 uptake. These reactions have been investigated in hypotonically treated rabbit epididymal sperm (HTRES) by means of 0, uptake rate determinations and fluorimetric measurements of the reduction level of intramitochondrial NAD. Pyruvate is a potent inhibitor of lactate oxidation; the inhibition is overcome by malate. Pyruvate alone is oxidized slowly but is oxidized rapidly when malate is present. Lactate alone is oxidized rapidly but malate is required for maximal 02 uptake. Fluorimetric measurements show a high reduction level for intramitochondrial NAD with lactate as substrate; this level is increased by malate. Pyruvate reoxidizes intramitochondrial NAD reduced by lactate and inhibits 0, uptake; reversal of this inhibition by malate partially restores the NAD reduction level. Malate plus pyruvate maintain a NAD reduction level sufficient for rapid 02 uptake. We conclude that malate is essential to pyruvate and lactate oxidation by rabbit sperm mitochondria and functions by 2 mechanisms: a) The NADH generated from malate dehydrogenase does not have access to LDH and is made directly available to the respiratory chain. b) The oxaloacetate from malate oxidation stimulates pyruvate dehydrogenase by reaction with the product acetyl C0A. These mechanisms appear to act in concert to prevent inhibition of respiration by pyruvate oxidation of intramitochondrial NADH catalyzed by LDH.

Heart mitochondrial metabolism after feeding herring oil to rats and monkeys

Heart mitochondrial oxidation of palmityl CoA and pyruvic acid was studied in rats and in the monkey Macaca fascicularis to determine the effects of feeding partially hydrogenated herring oil. Herring oil glycerides contain cetoleic acid (cis-11-docosenoic) which could have a similar effect to erucic acid (cis-13-docosenoic) in causing a rat cardiomyopathy. The initial rat heart mitochondrial response to dietary cetoleic acid (67% cis, 33% trans) was an in vitro decrease in palmityl CoA oxidation. Pronlonged feeding of cetoleic acid mixture was associated with a significant metabolic adaptation, increasing pyruvate and palmityl CoA oxidation above control levels. In vitro addition of cetoleyl CoA (pure cis isomer) stimulated pyruvate dehydrogenase activity, a possible response to decreased B-oxidation. There was no significant adaptive change in pyruvate or palmityl CoA use in monkeys after prolonged feeding of partially hydrogenated herring oil. Cetoleyl CoA was a good substrate for monkey heart carnitine acyl transferase even in the presence of palmityl CoA. These observations suggest that C22 fatty acids may be metabolized more rapidly in monkey heart than in rat heart. Metabolic differences argue against using the rat as an experimental model for studying possible cardiotoxic effects of docosenoic acids in primates.

The Regulation of Pyruvate Dehydrogenase in Isolated Beef Heart Mitochondria: THE ROLE OF CALCIUM, MAGNESIUM, AND PERMEANT ANIONS

Factors affecting regulation of the pyruvate dehydrogenase activity of isolated beef heart mitochondria were investigated. It was demonstrated that both calcium and magnesium caused a time-dependent enhancement of the activity of mitochondrial pyruvate dehydrogenase. Permeant anion addition either in presence or absence of exogenous divalent metal cations also caused an activation of this enzyme. Energization of the mitochondrial membranes in the absence of permeant anions resulted in an almost complete prevention of the metal cation-associated stimulation of pyruvate dehydrogenase. Permeant anion addition to the energized mitochondria re-established the ability of calcium and magnesium to activate the pyruvate dehydrogenase. Incubation of mitochondria with ATP plus magnesium resulted in a time-dependent inactivation of the pyruvate dehydrogenase which was intensified by energization of the mitochondrial system. The results of this study indicate the importance of the intramitochondrial location of divalent metal cations in the regulation of the pyruvate dehydrogenase multienzyme complex. The location of the divalent metal cations in the mitochondrial system may be strongly influenced by the energetic state of the mitochondria and the flux of anionic species across the membrane.