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Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin.

ABSTRACT: 1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35--45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.

SUBMITTER: Hughes WA

PROVIDER: S-EPMC1162361 | biostudies-other | 1980 Nov

REPOSITORIES: biostudies-other

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Incorporation of [32P]phosphate into the pyruvate dehydrogenase complex in rat heart mitochondria.

Project description:1. Evidence is given for three sites of phosphorylation in the alpha-chains of the decarboxylase component of purified rat heart pyruvate dehydrogenase complex, analogous to those established for procine and bovine complexes. Inactivation of rat heart complex was correlated with phosphorylation of site 1. Relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. 2. Methods are described for measurement of incorporation of 32Pi into the complex in rat heart mitochondria oxidizing 2-oxoglutarate + L-malate (total, sites 1, 2 and 3). Inactivation of the complex was related linearly to phosphorylation of site 1 in mitochondria of normal or diabetic rats. The relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. Rates of site-2 and site-3 phosphorylation may have been closer to that of site 1 in mitochondria of diabetic rats than in mitochondria of normal rats. 3. The concentration of inactive (phosphorylated) complex was varied in mitochondria from normal rats by inhibiting the kinase reaction with pyruvate at concentrations ranging from 0.15 to 0.4 mM. The results showed that the concentration of inactive complex is related linearly to incorporation of 32Pi into site 1. Inhibition of 32Pi incorporations with pyruvate at all concentrations over this range was site 3 greater than site 2 greater than site 1. 4. With mitochondria from diabetic rats, pyruvate (0.15-0.4 mM) inhibited incorporation of 32Pi into site 3, but it had no effect on the concentration of inactive complex or on incorporations of 32Pi into site 1 or site 2. It is concluded that site-3 phosphorylation is not required for inactivation of the complex in rat heart mitochondria. 5. Evidence is given that phosphorylation of sites 2 and 3 may inhibit reactivation of the complex by dephosphorylation in rat heart mitochondria.

| S-EPMC1161884 | biostudies-other

Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet.

Project description:1. Pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads was measured by using pig heart pyruvate dehydrogenase [32P]phosphate. About 80% was found to be extramitochondrial and therefore probably not directly concerned with the regulation of pyruvate dehydrogenase activity. The extramitochondrial activity was sensitive to activation by Ca2+, but perhaps less sensitive than the mitochondrial activity.

| S-EPMC1172695 | biostudies-other

Sensitivity of pyruvate dehydrogenase phosphate phosphatase to magnesium ions. Similar effects of spermine and insulin.

Project description:The effects of Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within intact mitochondria prepared from control and insulin-treated rat epididymal adipose tissue was explored by incubating the mitochondria in medium containing the ionophore A23187. The apparent Ka for Mg2+ was approximately halved in the mitochondria derived from insulin-treated tissue in both the absence and the presence of Ca2+. In this system, the major effect of Ca2+ was also to decrease the apparent Ka for Mg2+, rather than to change the Vmax. of the phosphatase. Damuni, Humphreys & Reed [(1984) Biochem. Biophys. Res. Commun. 124, 95-99] have reported that spermine activates ox kidney pyruvate dehydrogenase phosphate phosphatase. Studies were carried out on phosphatase from pig heart and rat epididymal adipose tissue which confirm and extend this observation. The major effect of spermine is shown to be a decrease in the Ka for Mg2+, which is apparent in both the presence and the absence of Ca2+. Spermine did not affect the sensitivity of the phosphatase to Ca2+ at saturating concentrations of Mg2+. Other polyamines tested were not as effective as spermine. No alteration in the maximum activity or Mg2+-sensitivity of pyruvate dehydrogenase phosphate phosphatase was apparent in extracts of mitochondria from insulin-treated tissue. The close similarity of the effects of spermine and the changes in kinetic properties of pyruvate dehydrogenase phosphate phosphatase within mitochondria from insulin-treated adipose tissue suggests that insulin may activate pyruvate dehydrogenase by increasing the concentration of spermine within the mitochondria. However, it is concluded that insulin is more likely to alter the interaction of the pyruvate dehydrogenase system with some other polybasic intramitochondrial component whose action can be mimicked by spermine.

| S-EPMC1147100 | biostudies-other

Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase.

Project description:1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.

| S-EPMC1165530 | biostudies-other

Effects of high-fat diet and physical activity on pyruvate dehydrogenase kinase-4 in mouse skeletal muscle.

Project description:BACKGROUND:The expression of PDK4 is elevated by diabetes, fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. It is previously shown that peroxisome proliferator-activated receptor ? coactivator 1? (PGC-1?), a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4) gene expression via the estrogen-related receptor ? (ERR?). We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1? and ERR? and the amount and function of mitochondria in skeletal muscle. METHODS:Insulin resistance was induced by a high-fat (HF) diet for 19?weeks in C57BL/6?J mice, which were either sedentary or with access to running wheels. The skeletal muscle expression levels of PDK4, PGC-1? and ERR? were measured and the quality and quantity of mitochondrial function was assessed. RESULTS:The HF mice were more insulin-resistant than the low-fat (LF) -fed mice. Upregulation of PDK4 and ERR? mRNA and protein levels were seen after the HF diet, and when combined with running even more profound effects on the mRNA expression levels were observed. Chronic HF feeding and voluntary running did not have significant effects on PGC-1? mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria. CONCLUSIONS:Our results support the view that insulin resistance is not mediated by the decreased qualitative or quantitative properties of mitochondria. Instead, the role of PDK4 should be contemplated as a possible contributor to high-fat diet-induced insulin resistance.

| S-EPMC3407034 | biostudies-literature

Dephosphorylation of pig heart pyruvate dehydrogenase phosphate complexes by pig heart pyruvate dehydrogenase phosphate phosphatase.

Project description:1. Pig heart pyruvate dehydrogenase phosphate complex in which all three sites of phosphorylation were completely phosphorylated was re-activated at a slower rate by phosphatase than complex predominantly phosphorylated in site 1. The ratio of initial rates of re-activation was approx. 1:5 with a comparatively crude preparation of phosphatase and with phosphatase purified by gel filtration and ion-exchange chromatography. 2. The ratio of apparent first-order rate constants during dephosphorylation of fully phosphorylated complex averaged 1/3.8/1.3 for site 1/site 2/site 3. Only site-1 dephosphorylation was linearly correlated with re-activation of the complex throughout dephosphorylation. Dephosphorylation of site 3 was linearly correlated with re-activation after an initial burst of dephosphorylation. 3. Because dephosphorylation of site 1 was always associated with dephosphorylation of site 2, it is concluded that dephosphorylation cannot be purely random. 4. The ratio of apparent first-order rate constants for dephosphorylation of site 1 (partially/fully phosphorylated complexes) averaged 1.72. This ratio is smaller than the ratio of approx. 5 for the initial rates of re-activation. Possible mechanisms involved in the diminished rate of re-activation of fully phosphorylated complex are discussed.

| S-EPMC1162854 | biostudies-other

Simple synthesis of 32P-labelled inositol hexakisphosphates for study of phosphate transformations.

Project description:In many soils inositol hexakisphosphate in its various forms is as abundant as inorganic phosphate. The organismal and geochemical processes that exchange phosphate between inositol hexakisphosphate and other pools of soil phosphate are poorly defined, as are the organisms and enzymes involved. We rationalized that simple enzymic synthesis of inositol hexakisphosphate labeled with 32P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates.We employed the enzyme inositol pentakisphosphate 2-kinase, IP5 2-K, to transfer phosphate from [?-32P]ATP to axial hydroxyl(s) of myo-, neo- and 1D-chiro-inositol phosphate substrates.32P-labeled inositol phosphates were separated by anion exchange HPLC with phosphate eluents. Additional HPLC methods were developed to allow facile separation of myo-, neo-, 1D-chiro- and scyllo-inositol hexakisphosphate on acid gradients.We developed enzymic approaches that allow the synthesis of labeled myo-inositol 1,[32P]2,3,4,5,6-hexakisphosphate; neo-inositol 1,[32P]2,3,4,[32P]5,6 - hexakisphosphate and 1D-chiro-inositol [32P]1,2,3,4,5,[32P]6-hexakisphosphate. Additionally, we describe HPLC separations of all inositol hexakisphosphates yet identified in soils, using a collection of soil inositol phosphates described in the seminal historic studies of Cosgrove, Tate and coworkers. Our study will enable others to perform radiotracer experiments to analyze fluxes of phosphate to/from inositol hexakisphosphates in different soils.

| S-EPMC5984642 | biostudies-literature

Histone F1 from rat thymus. Subfractionation and incorporation of [32P] phosphate in vitro.

Project description:Four subfractions of histone F1 from rat thymus were obtained by preparative polyacrylamide-gel electrophoresis. These subfractions are closely related in primary structure but show marked differences in radioactivity when derived from rat thymus nuclei labelled in vitro with [(32)P]phosphate.

| S-EPMC1178954 | biostudies-other

Regulation of pyruvate dehydrogenase by insulin and polyamines within electropermeabilized fat-cells and isolated mitochondria.

Project description:1. Regulation of the mammalian pyruvate dehydrogenase (PDH) complex by insulin and polyamines has been examined by using electropermeabilized rat epididymal fat-cells and isolated mitochondria. The complex could be regulated within the permeabilized cells not only by insulin, but also by certain low-M(r) species, including Ca2+ and the polyamine spermidine. 2. Both spermine and spermidine increased the level of active dephosphorylated PDH (PDHa) in isolated adipose-tissue mitochondria 2-3-fold, with half-maximal effects at 0.9 mM and 1.7 mM respectively. By contrast, PDH activity in rat heart mitochondria was essentially insensitive to the effects of these polyamines. 3. The effects on PDH activity of incubation of adipose-tissue mitochondria with spermine persisted through re-isolation and re-incubation of the mitochondria in the absence of the polyamine. 4. No evidence was found of any increase in the concentration of spermine associated with purified mitochondrial fractions prepared from insulin-treated tissue. 5. Overall, the data provide further evidence against a role for polyamines in the rapid stimulation of PDH by insulin, but suggest that polyamines may be important in mediating longer-term changes in the activity of the complex.

| S-EPMC1132807 | biostudies-other

Activation of the Pseudomonas aeruginosa type III secretion system requires an intact pyruvate dehydrogenase aceAB operon.

Project description:Pseudomonas aeruginosa clinical cystic fibrosis isolate CHA was mutagenized with Tn5Tc to identify new genes involved in type III secretion system (TTSS)-dependent cytotoxicity toward human polymorphonuclear neutrophils. Among 25 mutants affected in TTSS function, 14 contained the insertion at different positions in the aceAB operon encoding the PDH-E1 and -E2 subunits of pyruvate dehydrogenase. In PDH mutants, no transcriptional activation of TTSS genes in response to calcium depletion occurred. Expression in trans of ExsA restored TTSS function and cytotoxicity.

| S-EPMC128050 | biostudies-literature

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