Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet.
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ABSTRACT:
SUBMITTER: Stansbie D
PROVIDER: S-EPMC1172695 | biostudies-other | 1976 Jan
REPOSITORIES: biostudies-other
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Project description:1. Dose-dependent effects of adrenaline on PDHa activity were investigated with both incubated rat epidiymal fat-pads and isolated adipocytes. 2. Adrenaline (10nM- 5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]glucose + insulin (20 munits/ml). Changes in [U-14C]glucose incorporation into fatty acids in these tissues correlated only loosely with changes in PDHa activity. There was a good inverse relationship between adrenaline-induced changes in PDHa activity and increases in lipolysis (glycerol release). 3. Adrenaline (10nM - 0.5 micrometer) decreased PDHa activity in fat-pads incubated with 5 mM-[U-14C]pyruvate + insulin (20 munits/ml), whereas 1 micrometer- and 5 micrometer-adrenaline slightly increased PDHa activity. All concentrations of adrenaline tested decreased [U-14C]pyruvate incorporation into fatty acids. Between 10nM- and 0.5 micrometer-adrenaline percentage decreases in PDHa activity paralleled decreases in faty acid synthesis. 4. Effects of adrenaline on PDHa activity and fatty acid synthesis in fat-pads incubated with 5mM-[U-14C]pyruvate + insulin (20 munits/ml) could not be mimicked by addition of albumin-bound palmitate. 5. The response of PDHa activity to adrenaline (0.1 nM - 1 micrometer) in isolated adipocytes differed with the carbohydrate substrate used in the incubations. With 5 mM-glucose + insulin (20 munits/ml), PDHa activity was significantly increased by 10 nM-adrenaline, but not by 1 micrometer-adrenaline, the response to adrenaline being biphasic. There was some correlation between PDHa activity and accumulation of non-esterified fatty acids. With 5 mM-glucose alone adrenaline (0.1 nM - 1 micrometer) had no effect on PDHa activity even though lipolysis was increased by adrenaline (0.1 micrometer - 1 micrometer). With 5mM-fructose in the presence and absence of insulin, lipolytic doses of adrenaline decreased PDHa activity. No tested concentrations of adrenaline increased PDHa with this substrate. 6. In the presence of 5 mM-fructose, palmitate was significantly more effective than adrenaline with respect to the maximum decrease in PDHa activity that could be elicited. 4. The relationship of changes in PDHa activity to changes in lipogenesis and the likelihood of adrenaline-induced changes in PDHa activity being secondary to changes in non-esterified fatty acid metabolism are discussed.
| S-EPMC1185892 | biostudies-other
Project description:1. Isolated fat-cells and intact epididymal fat-pads were incubated in medium containing 45Ca2+ and the incorporation of 45Ca into mitochondrial and extramitochondrial fractions was studied. Redistribution of 45Ca between these fractions was essentially prevented by the addition of EGTA [ethanedioxybis(ethylamine)tetra-acetate] and Ruthenium Red to the sucrose-based extraction medium. 2. Incorporation of 45Ca into mitochondrial fractions of both fat-cells and fat-pads was found to be complete within 2-5 min, suggesting that mitochondria contain a pool of calcium in rapid isotopic exchange with extracellular Ca2+. This pool was about 20 times larger in mitochondria within fat-cells than within fat-pads. In fat-cells, 45Ca incorporation into the mitochondrial fraction accounted for about 34% of the total 45Ca incorporation into cells after 20 min and about 50% of the total mitochondrial calcium content measured by atomic absorption; values in fat-pads were about 7 and 20% respectively.
| S-EPMC1172694 | biostudies-other
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
Project description:1. The effects of the protein phosphatase inhibitors okadaic acid and microcystin LR on the regulation by insulin of pyruvate dehydrogenase and acetyl-CoA carboxylase have been studied in rat epididymal fat-pads and isolated cells. These inhibitors both completely blocked the phosphatase activity (against phosphorylase a) present in extracts of epididymal fat-pads, with half-maximal effects in the nanomolar range. 2. Okadaic acid treatment of pads and cells lowered the activity of acetyl-CoA carboxylase assayed in tissue extracts, both before and after treatment of the extracts with the activator, citrate. Further, okadaic acid treatment abolished the 2-3-fold difference in activity observed between extracts from control and insulin-treated tissues, assayed without prior treatment with citrate. 3. Incubation of pads with [32P]Pi, sufficient to label the intracellular pool of ATP, demonstrated that okadaic acid increased the overall phosphorylation of acetyl-CoA carboxylase on a number of distinct sites, as judged by two-dimensional mapping of tryptic peptides. These included the 'I-peptide' [Brownsey & Denton (1982) Biochem. J. 202, 77-86], the phosphorylation of which may be associated with the stimulation of the activity of the enzyme by insulin, as well as inhibitory phosphorylation sites. 4. Incubation with 1 microM-okadaic acid had no effect on the basal level of active pyruvate dehydrogenase apparent after tissue extraction, but abolished the 2-3-fold increase in this parameter which was elicited by insulin in the absence of okadaic acid. However, okadaic acid treatment did not affect the persistent increase in active pyruvate dehydrogenase levels which was apparent in mitochondria subsequently isolated from insulin-treated pads and re-incubated with an oxidizable substrate. It is concluded that the effects of okadaic acid are exerted through changes in metabolite concentrations rather than some direct action on the signalling pathway whereby insulin stimulates pyruvate dehydrogenase. 5. Microcystin LR did not mimic the effects of okadaic acid on intact cells and pads described above.
| S-EPMC1151054 | biostudies-other
Project description:Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4+ T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the Pdp2 promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4+ cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4+ T cells from lupus-prone MRL/lpr mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level.
| S-EPMC6140513 | biostudies-literature
Project description:The phosphorylation of sites additional to an inactivating site inhibits the formation of active pig heart pyruvate dehydrogenase complex from inactive pyruvate dehydrogenase phosphate complex by pig heart pyruvate dehydrogenase phosphate phosphatase.
| S-EPMC1184185 | biostudies-other
Project description:BackgroundThe 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 γ coactivator 1α (PGC-1α), a master regulator of energy metabolism, coactivates in cell lines pyruvate dehydrogenase kinase-4 (PDK4) gene expression via the estrogen-related receptor α (ERRα). We investigated the effects of long-term high-fat diet and physical activity on the expression of PDK4, PGC-1α and ERRα and the amount and function of mitochondria in skeletal muscle.MethodsInsulin 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-1α and ERRα were measured and the quality and quantity of mitochondrial function was assessed.ResultsThe HF mice were more insulin-resistant than the low-fat (LF) -fed mice. Upregulation of PDK4 and ERRα 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-1α mRNA or protein levels. No remarkable difference was found in the amount or function of mitochondria.ConclusionsOur 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
Project description:Congenital lactic acidosis due to pyruvate dehydrogenase phosphatase (PDP) deficiency is very rare. PDP regulates pyruvate dehydrogenase complex (PDC) and defective PDP leads to PDC deficiency. We report a case with functional PDC deficiency with low activated (+dichloroacetate) and inactivated (+fluoride) PDC activities in lymphocytes and fibroblasts, normal activity of other mitochondrial enzymes in fibroblasts, and novel biallelic frameshift mutation in the PDP1 gene, c.575dupT (p.L192FfsX5), with absent PDP1 product in fibroblasts. Unexpectedly, the patient also had low branched-chain 2-ketoacid dehydrogenase (BCKDH) activity in fibroblasts with slight elevation of branched-chain amino acids in plasma and ketoacids in urine but with no pathogenic mutations in the enzymes of BCKDH, which could suggest shared regulatory function of PDC and BCKDH in fibroblasts, potentially in other tissues or cell types as well, but this remains to be determined. The clinical presentation of this patient overlaps that of other patients with primary-specific PDC deficiency, with neonatal/infantile and childhood lactic acidosis, normal lactate to pyruvate ratio, elevated plasma alanine, delayed psychomotor development, epileptic encephalopathy, feeding difficulties, and hypotonia. This patient exhibited marked improvement of overall development following initiation of ketogenic diet at 31 months of age. To the best of our knowledge, this is the fourth case of functional PDC deficiency with a defined mutation in PDP1.SynopsisPyruvate dehydrogenase phosphatase (PDP) regulates pyruvate dehydrogenase complex (PDC) and defective PDP due to PDP1 mutations leads to PDC deficiency and congenital lactic acidosis.
| S-EPMC6606986 | biostudies-literature
Project description:Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.8 A resolution. The structure reveals that PDP1 belongs to the PPM family of protein serine/threonine phosphatases, which, in spite of a low level of sequence identity, share the structural core consisting of the central beta-sandwich flanked on both sides by loops and alpha-helices. Consistent with the previous studies, two well-fixed magnesium ions are coordinated by five active site residues and five water molecules in the PDP1c catalytic center. Structural analysis indicates that, while the central portion of the PDP1c molecule is highly conserved among the members of the PPM protein family, a number of structural insertions and deletions located at the periphery of PDP1c likely define its functional specificity towards the PDC. One notable feature of PDP1c is a long insertion (residues 98-151) forming a unique hydrophobic pocket on the surface that likely accommodates the lipoyl moiety of the E2 domain in a fashion similar to that of PDHKs. The cavity, however, appears more open than in PDHK, suggesting that its closure may be required to achieve tight, specific binding of the lipoic acid. We propose a mechanism in which the closure of the lipoic acid binding site is triggered by the formation of the intermolecular (PDP1c/L2) Ca2+ binding site in a manner reminiscent of the Ca2+ -induced closure of the regulatory domain of troponin C.
| S-EPMC1994205 | biostudies-literature
Project description:Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Å resolution is reported. The crystals belonged to space group P3221, with unit-cell parameters a = b = 75.3, c = 173.2 Å. The structure was solved by molecular-replacement methods and refined to a final R factor of 21.9% (Rfree = 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn2+ ions in the active site, an additional Mn2+ ion coordinated by His410 and His414, two MnSO4 ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.
| S-EPMC7336360 | biostudies-literature