Stimulation Of Pyruvate Dehydrogenase Activity Research Articles

Effect of adenosine on insulin activation of rat adipocyte pyruvate dehydrogenase

Adenosine and its analogue N 6-phenylisopropyladenosine stimulated pyruvate dehydrogenase activity of isolated rat adipocytes. Maximal stimulation was obtained with concentrations between 50 and 100 μM, with the effect decreasing at higher concentrations. The effects of insulin on this enzyme was modified by adenosine. The concentration of insulin (10 μunits ml ) that produced almost half-maximal stimulation, had little or no effect, when adenosine deaminase was present. Adenosine also enhanced the effect of suboptimal but not optimal concentrations of insulin. Thus, the mechanism of adenosine action on adipocyte pyruvate dehydrogenase could in some way be similar or related to that of insulin.

Effects of high fat and high carbohydrate diets on liver pyruvate dehydrogenase and its activation by a chemical mediator released from insulin-treated liver particulate fraction: effect of neuraminidase treatment on the chemical mediator activity.

Rats were fed a high fat diet or a high glucose diet for 5-7 days. Basal pyruvate dehydrogenase activity (both the active form and the total enzyme activity) was decreased in liver homogenates from fat diet-adapted rats as compared to those fed the glucose diet. Supernatants from insulin-exposed liver particulate fractions from fat-fed rats showed decreased stimulation of pyruvate dehydrogenase activity as compared to those from glucose-fed rats. There was no difference in the response of the mitochondria from the two groups when they were stimulated by supernatants from insulin-treated liver particulate fractions from stock diet-fed rats. Liver particulate fractions from fat-fed rats showed decreased generation of the chemical activator in response to Concanavalin A and trypsin stimulation. This suggests that fat feeding results in a decrease in membrane protease substrate availability. Treatment of the insulin mediator with neuraminidase and beta-D-galactosidase resulted in inactivation of the mediator. Presence of exogenous enzyme substrates during enzyme digestion protected the mediator from inactivation, suggesting that carbohydrate residues are important in the action of the insulin mediator. This fat diet-induced decrease in the generation of a chemical mediator of insulin action may result from 1) a decrease in insulin binding, shown earlier; 2) a decrease in the amount of protease substrate; and 3) an alteration in its carbohydrate composition, which is important in its ability to activate pyruvate dehydrogenase.

Partial purification from hepatoma cells of an intracellular substance which mediates the effects of insulin on pyruvate dehydrogenase and low Km cyclic AMP phosphodiesterase

A substance capable of stimulating the activities of pyruvate dehydrogenase and low K m cyclic AMP phosphodiesterase was prepared from H4-II-EC3′ hepatoma cells by acid extraction and partially purified by molecular exclusion chromatography. The material thus prepared by gel chromatography was found to stimulate the activities of these enzymes in a concentration-dependent manner. The amount or activity of the pyruvate dehydrogenase stimulating factor was increased in cells which had been treated with physiological concentrations of insulin (0.2 mU/ml). Increasing the concentration of insulin increased the amount or activity of the factor generated. High concentrations of insulin did not cause a reversal of the effects of insulin. The stimulation of pyruvate dehydrogenase activity by the factor was eliminated when sodium fluoride (75 m m) was present in the enzyme assay, implying that activation was mediated by the pyruvate dehydrogenase phosphatase. The enzyme-stimulating factor isolated from hepatoma cells shares a number of important characteristics with the putative second messenger of insulin prepared from other cell types: (1) it is heat and acid stable, (2) it has a similar apparent molecular weight, (3) it is generated in an insulin-dependent manner, (4) it stimulates the activity of pyruvate dehydrogenase by a fluoride-sensitive mechanism, and (5) it elutes from the anion-exchange resin AG 1-X8 at an ionic strength of 0.4 m. These findings suggest that the stimulator of pyruvate dehydrogenase and of low K m cyclic AMP phosphodiesterase isolated from hepatoma cells has chemical properties identical with those of the putative second messenger of insulin action isolated from a number of other insulin-sensitive tissues.

Stimulation of pyruvate dehydrogenase activity in adipocytes by oxytocin: Evidence for mediation of the insulin-like effect by endogenous hydrogen peroxide independent of glucose transport

Oxytocin, a nonapeptide posterior pituitary hormone, which is known to increase glucose oxidation in fat cells like insulin, is shown here to stimulate pyruvate dehydrogenase activity in these cells. The process appears to involve the activation of preexisting molecules since there was no change in the total enzyme content after full activation. The effect of oxytocin, as well as of insulin, appears to be mediated by endogenous H 2O 2 formation, as evident from (i) the enhanced [ 14C]formate oxidation and its greater inhibition by 3-amino-1,2,4-triazole in the hormone-treated cells than in the control. This is a measure of the catalase:H 2O 2 complex, and the dose dependence of this response is found to be identical with that of glucose oxidation via the hexose monophosphate shunt pathway and of pyruvate dehydrogenase activity; and (ii) treatment of the cells with low concentration of exogenous H 2O 2 causes the activation of pyruvate dehydrogenase to the extent which is comparable with the effect of the hormones. The ED 50 of oxytocin was 7 × 10 −9 m, whereas the ED 50 of insulin was 5 × 10 −11 m. The reduced, inactive (SH) derivatives of the hormones had the same dose-response relationship, but considerably lower effect (10 to 20% of the native molecules of the hormones), indicating the significant role of the disulfide bridge(s) in eliciting these metabolic responses. The stimulation of PDH by oxytocin or insulin is found to be essentially independent of medium glucose which, however, can sustain the response apparently by recycling the intracellular oxidation-reduction state. However, unlike insulin, oxytocin fails to stimulate the rapid uptake of 3- O-[ 3H]methyl- d-glucose in these cells. The data illustrate that the major metabolic actions of insulin, viz., glucose utilization and lipogenesis, are shared by another heterologous polypeptide hormone, e.g., oxytocin, through a common effector, H 2O 2. It is suggested that (i) oxytocin may play a limited surrogate role for insulin in these cells; and (ii) H 2O 2 production may be the general basis of oxytocin's action.

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.

Effect of dichloroacetate on gluconeogenesis in vivo in the presence of a fixed gluconeogenic substrate supply to the liver

Administration of dichloroacetate (DCA) to conscious 48 h fasted dogs causes a reduction in the conversion of circulating alanine and lactate to glucose coincident with both a reduction in the load of alanine and lactate delivered to the liver and a decrease in the plasma insulin-glucagon ( I G ) molar ratio. To determine whether DCA inhibits gluconeogenesis independent of its effect on alanine and lactate levels, the drug was infused concurrently with alanine and lactate in order to fix the load of these gluconeogenic precursors reaching the liver. When the circulating levels of alanine and lactate were fixed, DCA increased the net hepatic uptake of alanine markedly (98 ± 26%) but increased lactate uptake only slightly (8 ± 7%). However, the fraction of these gluconeogenic precursors which were converted to 14C-glucose decreased. Since the net effect of these changes was to increase the overall conversion of alanine and lactate to glucose, it is evident that the increase in precursor uptake overrode the inhibition of the gluconeogenic process per se. This increase in hepatic precursor uptake may have been due to DCA stimulation of pyruvate dehydrogenase activity or the observed decline in the I G molar ratio. The observed increase in overall glucose production (33 ± 10%) was probably attributable to the increase in gluconeogenesis as well as an increase in glycogenolysis since 48 h fasted dogs still have some hepatic glycogen (15 mg/gm). By overcoming the peripheral effects of DCA on gluconeogenic precursor supply it becomes evident that DCA has: (1) a direct inhibitory action on intrahepatic gluconeogenesis, and (2) an effect on the pancreas which by virtue of a change in the I G molar ratio may offset the direct intrahepatic effect of the drug.

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.

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.

Stimulation of pyruvate dehydrogenase activity by insulin-dextran complex in mouse adipose tissue

Soluble and stable insulin-dextran complex was prepared. Pyruvate dehydrogenase activity, as assayed by 14CO 2 formation from [1- 14C]-pyruvate in crude mitochondria of mouse adipose tissue, was increased after incubation of fat pads with native insulin or insulin-dextran. The direct addition of insulin or insulin-dextran to mitochondria was without effect. At submaximal stimulation, insulin-dextran was 10 times less effective than native insulin but the degree of maximal stimulation and the time course of activation by insulin and insulin-dextran were similar. The results favor the concept that the activation of pyruvate dehydrogenase in fat cells does not need the entry of insulin into cells.

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.