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R M Denton

Publications and source records attributed to R M Denton.

At least 145 records · Page 8Linked to original sources
PubMed1977

Demonstration of the phosphorylation of acetyl-coenzyme A carboxylase within intact rat epididymal fat-cells.

Intact rat epididymal fat-cells were incubated with 32Pi and the intracellular proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the phosphorylated proteins has the same RF value as [14C]biotin-labelled acetyl-CoA carboxylase purified from fat-cells and is specifically precipitated after incubation with antiserum raised against acetyl-CoA carboxylase. No significant changes in the extent of phosphorylation of acetyl-CoA carboxylase were detected after exposure of the cells to insulin.

Acetyl-CoA Carboxylase↗
PubMed1977

Evidence that fatty acid synthesis in the interscapular brown adipose tissue of cold-adapted rats is increased in vivo by insulin by mechanisms involving parallel activation of pyruvate dehydrogenase and acetyl-coenzyme A carboxylase.

Plasma insulin concentrations in cold-adapted rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in interscapular brown adipose tissue were determined from the incorporation of 3H from 3H2O into tissue lipid. Rates of synthesis were greatly elevated after glucose administration and markedly decreased after injection with anti-insulin serum. Parallel changes in the initial activities of both acetyl-CoA carboxylase and pyruvate dehydrogenase were observed under these conditions, but no changes in total activities were evident. The results suggest that this tissue is an important site of fatty acid synthesis in the cold-adapted rat and that this feature of the tissue is sensitive to changes in plasma insulin concentrations.

Acetyl-CoA Carboxylase↗
PubMed1976

Acute effects in vivo of anti-insulin serum on rates of fatty acid synthesis and activities of acetyl-coenzyme A carboxylase and pyruvate dehydrogenase in liver and epididymal adipose tissue of fed rats.

Plasma insulin concentrations in fed rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in adipose tissue and liver were estimated from the incorporation of 3H from 3H2O. In the adipose tissue dehydrogenase and acetyl-CoA carboxylase were evident. In liver, although changes in rates of fatty acid synthesis were found, the initial activity of pyruvate dehydrogenase did not alter, but small parallel changes in acetyl-CoA carboxylase activity were observed.

Acetyl-CoA Carboxylase↗
PubMed1976

The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...

Acetaldehyde↗
PubMed1976

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.

Acetates↗
PubMed1976

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

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.

Adipose Tissue↗
PubMed1976

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

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.

Adipose Tissue↗
PubMed1976

Does hyperinsulinemia in ob/ob mice cause an insulin-stimulated adipose tissue?

After a 1-h preincubation to remove endogenous insulin, adipose tissue of obese mice (C57BL/L4 ob/ob) had a lower rate of glucose metabolism than tissue which was not preincubated. In contrast, preincubation did not change the metabolism of adipose tissue from lean mice (C57B1/6J +/+). The preincubation effect was abolished in obese mice which had had their serum insulin levels lowered toward normal by streptozotocin treatment. Injection of anti-insulin serum to obese mice caused adipose tissue removed 15 min after the injection to display a rate of glucose metabolsim lower than that of tissue removed before the injection. No such effect was seen in lean mice. These data are consistent with the hypothesis that hyperinsulinemia in the obese mice causes a chronic state of insulin stimulation of their adipose tissue, possibly contributing to their high rates of lipogenesis and their obesity. Several lipogenic enzymes were measured in adipose tissue of both lean and obese mice, and no single enzymatic abnormality was detected which might explain the hyperlipogenesis. Pyruvate dehydrogenase and acetyl-CoA carboxylase were both insulin-sensitive enzymes in lean and obese mice.

ATP Citrate (pro-S)-Lyase↗
PubMed1975

Regulation of mammalian pyruvate dehydrogenase.

In mammalian tissues, two types of regulation of the pyruvate dehydrogenase complex have been described: end product inhibition by acetyl CoA and NADH: and the interconversion of an inactive phosphorylated form and an active nonphosphorylated form by an ATP requiring kinase and a specific phosphatase. This article is largely concerned with the latter type of regulation of the complex in adipose tissue by insulin (and other hormones) and in heart muscle by lipid fuels. Effectors of the two interconverting enzymes include pyruvate and ADP which inhibit the kinase, acetoin which activates the kinase and Ca2+ and Mg2+ which both activate the phosphatase and inhibit the kinase. Evidence is presented that all components of the pyruvate dehydrogenase complex including the phosphatase and kinase are located within the inner mitochondrial membrane. Direct measurements of the matrix concentration of substrates and effectors is not possible by techniques presently available. This is the key problem in the identification of the mechansims involved in the alterations in pyruvate dehydrogenase activity observed in adipose tissue and muscle. A number of indirect approaches have been used and these are reviewed. Most hopeful is the recent finding in this laboratory that in both adipose tissue and heart muscle, differences in activity of pyruvate dehydrogenase in the intact tissue persist during preparation and subsequent incubation of mitochondria.

Acetates↗
PubMed1975

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.

Animals↗
PubMed1974

Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by alpha-cyano-4-hydroxycinnamate.

alpha-Cyano-4-hydroxycinnamate greatly inhibits the transport of pyruvate but not that of acetate or butyrate in liver mitochondria and erythrocytes. In the latter, lactate uptake is also inhibited. It is concluded that a specific carrier is involved in membrane transport of pyruvate and that the plasma-membrane carrier may also be involved in lactate transport.

Acetates↗