scholarly journals Calcium and magnesium ions as effectors of adipose-tissue pyruvate dehydrogenase phosphate phosphatase

1974 ◽  
Vol 140 (2) ◽  
pp. 225-237 ◽  
Author(s):  
David L. Severson ◽  
Richard M. Denton ◽  
Helen T. Pask ◽  
Philip J. Randle
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Metal Ion ◽  
Ruthenium Red ◽  
Atp Depletion ◽  
Cell Uptake ◽  
Ionophore A23187 ◽  
Uptake System ◽  
Fat Cell ◽  
Epididymal Fat ◽  
Fat Pads

The metal-ion requirement of extracted and partially purified pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat-pads was investigated with pig heart pyruvate dehydrogenase [32P]phosphate as substrate. The enzyme required Mg2+ (Km 0.5mm) and was activated additionally by Ca2+ (Km 1μm) or Sr2+ and inhibited by Ni2+. Isolated fat-cell mitochondria, like liver mitochondria, possess a respiration- or ATP-linked Ca2+-uptake system which is inhibited by Ruthenium Red, by uncouplers when linked to respiration, and by oligomycin when linked to ATP. Depletion of fat-cell mitochondria of 75% of their total magnesium content and of 94% of their total calcium content by incubation with the bivalent-metal ionophore A23187 leads to complete loss of pyruvate dehydrogenase phosphate phosphatase activity. Restoration of full activity required addition of both MgCl2 and CaCl2. SrCl2 could replace CaCl2 (but not MgCl2) and NiCl2 was inhibitory. The metal-ion requirement of the phosphatase within mitochondria was thus equivalent to that of the extracted enzyme. Insulin activation of pyruvate dehydrogenase in rat epididymal fat-pads was not accompanied by any measurable increase in the activity of the phosphatase in extracts of the tissue when either endogenous substrate or 32P-labelled pig heart substrate was used for assay. The activation of pyruvate dehydrogenase in fat-pads by insulin was inhibited by Ruthenium Red (which may inhibit cell and mitochondrial uptake of Ca2+) and by MnCl2 and NiCl2 (which may inhibit cell uptake of Ca2+). It is concluded that Mg2+ and Ca2+ are cofactors for pyruvate dehydrogenase phosphate phosphatase and that an increased mitochondrial uptake of Ca2+ might contribute to the activation of pyruvate dehydrogenase by insulin.

scholarly journals Mechanisms regulating adipose-tissue pyruvate dehydrogenase

1972 ◽  
Vol 129 (3) ◽  
pp. 763-773 ◽  
Author(s):  
B. R. Martin ◽  
R. M. Denton ◽  
H. T. Pask ◽  
P. J. Randle
Keyword(s):  
Cyclic Amp ◽  
Pyruvate Dehydrogenase ◽  
Prostaglandin E1 ◽  
Competitive Inhibition ◽  
Tissue Concentration ◽  
Pyruvate Dehydrogenase Kinase ◽  
Fat Cell ◽  
Epididymal Fat ◽  
Fat Pads

1. Isolated rat epididymal fat-cell mitochondria showed an inverse relationship between ATP content and pyruvate dehydrogenase activity consistent with competitive inhibition of pyruvate dehydrogenase kinase by ADP. At constant ATP concentration pyruvate rapidly activated pyruvate dehydrogenase in fat-cell mitochondria, an observation consistent with inhibition of fat-cell pyruvate dehydrogenase kinase by pyruvate. Pyruvate dehydrogenase in fat-cell mitochondria was also activated by nicotinate (100μm) and by extramitochondrial Na+ (replacing K+) but not by ouabain or insulin. 2. In rat epididymal fat-pads incubated in vitro pyruvate dehydrogenase was activated by addition of insulin in the absence of substrate or in the presence of glucose (10mm) or fructose (10mm). Glucose and fructose activated the dehydrogenase in the absence or in the presence of insulin, and pyruvate also activated in the absence of insulin. It is concluded that extracellular glucose, fructose and pyruvate may activate the dehydrogenase by raising intracellular pyruvate and that insulin may activate the dehydrogenase by some other mechanism. 3. Ouabain (300μm) and medium in which K+ was replaced by Na+, activated pyruvate dehydrogenase in epididymal fat-pads. Prostaglandin E1 (1μg/ml), 5-methylpyrazole-3-carboxylate (10μm) and nicotinate (10μm), which are as effective as insulin as inhibitors of lipolysis and which like insulin lower tissue concentration of cyclic AMP (adenosine 3′:5′-cyclic monophosphate), did not activate pyruvate dehydrogenase. Higher concentrations of prostaglandin E1 (10μg/ml) and nicotinate (100μm) produced some activation of the dehydrogenase. 4. It is concluded that the activation of pyruvate dehydrogenase by insulin is not due to the antilipolytic effect of the hormone and that the action of insulin in lowering adipose-cell concentrations of cyclic AMP does not afford an obvious explanation for the effect of the hormone on pyruvate dehydrogenase. The possibility that the effects of insulin, ouabain and K+-free medium may be mediated by Ca2+ is discussed.

scholarly journals Inactivation of rat adipocyte pyruvate dehydrogenase by palmitate. Protection against this effect by insulin in the presence of glucose

1979 ◽  
Vol 184 (1) ◽  
pp. 59-62 ◽  
Author(s):  
S R Sooranna ◽  
E D Saggerson
Keyword(s):  
Amino Acids ◽  
Fatty Acids ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Protective Action ◽  
Active Form ◽  
Rat Plasma ◽  
Epididymal Fat ◽  
Fat Pads

1. Adipocytes from rat epididymal fat-pads were incubated for 30 min with 5 mM-glucose and concentrations of lactate, pyruvate and amino acids typical of those found in rat plasma. 2. PDHa (active form of pyruvate dehydrogenase) activity was significantly increased after incubation of the cells with insulin (200 micro-i.u./ml), and decreased by incubation with palmitate (0.5–2 mM). 3. In the presence of insulin, palmitate did not decrease PDHa activity. 4. Dichloroacetate (1 mM) increased PDHa activity in the absence of palmitate to the same extent as did insulin. In the presence of dichloroacetate but the absence of insulin, palmitate decreased PDHa activity. In the presence of dichloroacetate and insulin, palmitate again did not decrease PDHa activity. 5. It is concluded that, in the presence of glucose, insulin has a strong protective action against inactivation of adipocyte PDHa by fatty acids.

scholarly journals Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin

1984 ◽  
Vol 218 (1) ◽  
pp. 249-260 ◽  
Author(s):  
S E Marshall ◽  
J G McCormack ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Active Form ◽  
Ruthenium Red ◽  
Epididymal Adipose Tissue ◽  
High Concentration ◽  
Fat Cell ◽  
Mitochondrial Inner Membrane ◽  
Oxoglutarate Dehydrogenase

The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.

scholarly journals Effects of protein phosphatase inhibitors on the regulation of insulin-sensitive enzymes within rat epididymal fat-pads and cells

1991 ◽  
Vol 276 (3) ◽  
pp. 649-654 ◽  
Author(s):  
G A Rutter ◽  
A C Borthwick ◽  
R M Denton
Keyword(s):  
Okadaic Acid ◽  
Pyruvate Dehydrogenase ◽  
Protein Phosphatase ◽  
Acid Treatment ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Phosphatase Inhibitors ◽  
Epididymal Fat ◽  
Protein Phosphatase Inhibitors ◽  
Fat Pads

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.

scholarly journals Exchangeable and total calcium pools in mitochondria of rat epididymal fat-pads and isolated fat-cells. Role in the regulation of pyruvate dehydrogenase activity

1976 ◽  
Vol 154 (1) ◽  
pp. 209-223 ◽  
Author(s):  
D L Severson ◽  
R M Denton ◽  
B J Bridges ◽  
P J Randle
Keyword(s):  
Isotopic Exchange ◽  
Calcium Content ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Total Calcium ◽  
Epididymal Fat ◽  
Mitochondrial Fractions ◽  
Fat Pads

1. Isolated fat-cells and intact epididymal fat-pads were incubated in medium containing 45+ 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 +. 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.

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

1976 ◽  
Vol 154 (1) ◽  
pp. 225-236 ◽  
Author(s):  
D Stansbie ◽  
R M Denton ◽  
B J Bridges ◽  
H T Pask ◽  
P J Randle
Keyword(s):  
Phosphatase Activity ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
High Fat Diet ◽  
Alloxan Diabetes ◽  
Mitochondrial Activity ◽  
High Fat ◽  
Epididymal Fat ◽  
Fat Pads

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 +, but perhaps less sensitive than the mitochondrial activity.

Effects of Natural Plants Supplementation on Adipocyte Size of the Epididymal Fat Pads in Rats

2007 ◽  
Vol 36 (4) ◽  
pp. 419-423 ◽  
Author(s):  
Hyun-Sook Kim ◽  
Tae-Woo Kim ◽  
Dae-Jung Kim ◽  
Ha-Jin Hwang ◽  
Hyun-Joo Lee ◽  
...  
Keyword(s):  
Adipocyte Size ◽  
Epididymal Fat ◽  
Natural Plants ◽  
Fat Pads

The inhibitory effects of some adenosine nucleolipids on the lipolysis in rat epididymal fat pads in vitro

1979 ◽  
Vol 44 (5) ◽  
pp. 1651-1656 ◽  
Author(s):  
Sixtus Hynie ◽  
Jiří Smrt
Keyword(s):  
Fatty Acids ◽  
Cyclic Amp ◽  
Dibutyryl Cyclic Amp ◽  
Inhibitory Effects ◽  
Epididymal Fat ◽  
Fat Pads

3'-Oleolyl-2,3-dihydroxypropyl-AMP, 3'-stearoyl-2,3-dihydroxypropyl-AMP, octadecyl-AMP and palmitamidoethyl-AMP inhibited in comparison with adenosine or fatty acids much stronger the lipolysis in rat epididymal fat pads in vitro stimulated by isoproterenol, theophylline and dibutyryl cyclic AMP. The inhibition of the effects of the two latter drugs suggest that the described effect is caused not only by the inhibition of the cyclic AMP production but also by the inhibition of its effect on the following steps in process of lipolysis.

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