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R M Denton

Publications and source records attributed to R M Denton.

At least 109 records · Page 6Linked to original sources
PubMed1984

Studies on insulin-stimulated phosphorylation of acetyl-CoA carboxylase, ATP citrate lyase and other proteins in rat epididymal adipose tissue. Evidence for activation of a cyclic AMP-independent protein kinase.

Protein kinase activity in high-speed supernatant fractions prepared from rat epididymal adipose tissue previously incubated in the absence or presence of insulin was investigated by following the incorporation of 32P from [gamma-32P]ATP into phosphoproteins separated by sodium dodecyl sulphate/polyacrylamide-gel electro-phoresis. Incorporation of 32P into several endogenous proteins in the supernatant fractions from insulin-treated tissue was significantly increased. These included acetyl-CoA carboxylase and ATP citrate lyase (which exhibit increased phosphorylation within fat-cells exposed to insulin), together with two unknown proteins of subunit Mr 78000 and 43000. The protein kinase activity increased by insulin was distinct from cyclic AMP-dependent protein kinase, was not dependent on Ca2+ and was not appreciably affected by dialysis or gel filtration. The rate of phosphorylation of added purified fat-cell acetyl-CoA carboxylase and ATP citrate lyase was also increased by 60-90% in high-speed-supernatant fractions prepared from insulin-treated tissue. No evidence for any persistent changes in phosphoprotein phosphatase activity was found. It is concluded that insulin action on acetyl-CoA carboxylase, ATP citrate lyase and other intracellular proteins exhibiting increased phosphorylation involves an increase in cyclic AMP-independent protein kinase activity in the cytoplasm. The possibility that the increase reflects translocation from the plasma membrane, perhaps after phosphorylation by the protein tyrosine kinase associated with insulin receptors, is discussed.

ATP Citrate (pro-S)-Lyase↗
PubMed1984

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes by increasing the intramitochondrial concentration of Ca2+.

Increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase which result from the perfusion of rat hearts with adrenaline were still evident during the preparation of mitochondria in sucrose-based media containing EGTA (at 0 degrees C) and their subsequent incubation at 30 degrees C in Na+-free KCl-based media containing respiratory substrates and EGTA. The differences from control values gradually diminished with time of incubation, but were still present after 8 min. Similar increases resulting from an increase in the concentration of Ca2+ in the perfusing medium also persisted. However, similar increases caused by 5 mM-pyruvate were only maintained during the preparation of mitochondria, not their incubation. Parallel increases, within incubated mitochondria, were found in the activity of the 2-oxoglutarate dehydrogenase complex assayed at a non-saturating concentration of 2-oxoglutarate. The enhancement of the activities of both of these Ca2+-sensitive enzymes within incubated mitochondria as a result of perfusion with adrenaline or a raised concentration of Ca2+ in the medium could be abolished within 1 min by the presence of 10 mM-NaCl. This effect of Na+ was blocked by 300 microM-diltiazem, which has been shown to inhibit Na+-induced egress of Ca2+ from rabbit heart mitochondria [Vághy, Johnson, Matlib, Wang & Schwartz (1982) J. Biol. Chem. 257, 6000-6002]. The enhancements could also be abolished by increasing the extramitochondrial concentration of Ca2+ to a value where it caused maximal activation of the enzymes within control mitochondria. The results are consistent with the hypothesis that adrenaline activates rat heart pyruvate dehydrogenase by increasing the intramitochondrial concentration of Ca2+ and that this increase persists through to incubated mitochondria. Support for this conclusion was obtained by the yielding of a similar set of results from parallel experiments performed on control mitochondria that had firstly been preincubated (under conditions of steady-state Ca2+ cycling across the inner membrane) with sufficient proportions of Ca-EGTA buffers to achieve a similar degree of Ca2+-activation of pyruvate dehydrogenase (as caused by adrenaline) and had then undergone the isolation procedure again.

Animals↗
PubMed1984

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.

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.

Adipose Tissue↗
PubMed1984

Persistence of the effect of insulin on pyruvate dehydrogenase activity in rat white and brown adipose tissue during the preparation and subsequent incubation of mitochondria.

Increases in the amount of the active non-phosphorylated form of pyruvate dehydrogenase in rat epididymal adipose tissue, as a result of incubation with insulin, persist not only during the preparation of mitochondria but also during subsequent incubation of coupled mitochondria in the presence of respiratory substrates. No effect on insulin was found if the hormone was added directly to mitochondria in the presence or absence of added plasma membranes. Concentrations of several possible regulators of pyruvate dehydrogenase kinase (ATP, ADP, NADH, NAD+, acetyl-CoA, CoA and potassium) were measured in rat epididymal-adipose-tissue mitochondria incubated under conditions where differences in pyruvate dehydrogenase activity persist as a result of insulin action. No alterations were found, and it is suggested that inhibition of the kinase is not the principal means by which insulin activates pyruvate dehydrogenase. The intramitochondrial concentration of magnesium was also unaffected. Differences in pyruvate dehydrogenase activity in interscapular brown adipose tissue associated with manipulation of plasma insulin concentrations of cold-adapted rats were also shown to persist during the preparation and subsequent incubation of mitochondria in the presence or absence of GDP. It is pointed out that the persistence of the effect of insulin on pyruvate dehydrogenase in incubated mitochondria will facilitate the recognition of the mechanism of this action of the hormone. Evidence that the short-term action of insulin involves an increase in pyruvate dehydrogenase phosphate phosphatase activity rather than inhibition of that of pyruvate dehydrogenase kinase is discussed.

Adenosine Diphosphate↗
PubMed1983

The role of phosphorylation in the regulation of fatty acid synthesis by insulin and other hormones.

Insulin stimulates fatty acid synthesis in white and brown fat cells as well as in liver and mammary tissue. Hormones that increase cellular cyclic AMP concentrations inhibit fatty acid synthesis, at least in white adipose tissue and liver. These changes in fatty acid synthesis occur within minutes. In white fat cells, they are brought about not only by changes in glucose transport but also changes in the activities of pyruvate kinase, pyruvate dehydrogenase and acetyl-CoA carboxylase. The basis of the alterations in pyruvate kinase activity in fat cells is not understood. Unlike the liver isoenzyme, the isoenzyme present in fat cells does not appear to be phosphorylated either in the absence or presence of hormones. The changes in pyruvate dehydrogenase activity in fat cells are undoubtedly due to changes in phosphorylation of the alpha subunits. Insulin appears to act by causing the parallel dephosphorylation of all three sites. The persistence of the effect of insulin during the preparation and subsequent incubation of mitochondria has allowed the demonstration that insulin acts mainly by stimulating pyruvate dehydrogenase phosphatase rather than inhibiting the kinase. Acetyl-CoA carboxylase within fat cells is phosphorylated on a number of different sites. The exposure of cells to insulin leads to activation of the enzyme and this is associated with increased phosphorylation of a specific site on the enzyme. Exposure to adrenalin, which results in a marked diminution in activity, also causes a small increase in the overall level of phosphorylation, but this increase is due to an enhanced phosphorylation of different sites; probably those phosphorylated by cyclic-AMP-dependent protein kinase. Acetyl-CoA carboxylase is one of a number of proteins in fat cells that exhibit increased phosphorylation with insulin. Others include ATP-citrate lyase, the ribosomal protein S6, the beta subunit of the insulin receptor and a heat and acid stable protein of Mr 22000. Changes in phosphorylation of ATP-citrate lyase do not appear to result in any appreciable changes in catalytic activity. A central aspect of insulin action may be the activation and perhaps release of a membrane-associated protein kinase. Plasma membranes from fat cells have been shown to contain a cyclic-nucleotide-independent kinase able to phosphorylate and activate acetyl-CoA carboxylase. Furthermore, high-speed supernatant fractions from cells previously exposed to insulin contain elevated levels of the same or similar kinase activity capable of phosphorylating both ATP-citrate lyase and acetyl-CoA carboxylase.

ATP Citrate (pro-S)-Lyase↗
PubMed1982

The effects of glucagon, phenylephrine and insulin on the phosphorylation of cytoplasmic, mitochondrial and membrane-bound proteins of intact liver cells from starved rats.

1. The effects of glucagon, insulin and phenylephrine on the phosphorylation of cytoplasmic, mitochondrial and membrane proteins were studied in intact hepatocytes from 24 h-starved rats incubated with [32P]Pi. A rapid cell-fractionation technique was used, followed by radioautography of the proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. Glucagon consistently caused a significant increase in the phosphorylation of four readily separable cytoplasmic phosphoproteins, of Mr 93000, 50000, 46000 and 20000, and a decrease in phosphorylation of a phosphoprotein of Mr 22000. Phosphorylation of the protein of Mr 46000 was also enhanced by both phenylephrine and insulin, and that of Mr 93000 by phenylephrine. 3. The phosphoprotein of Mr 22000 was not precipitated by boiling for 5 min, and had a mobility identical with that of similar protein whose phosphorylation is enhanced in the adipocyte by insulin [Belsham & Denton (1980) Biochem. Soc. Trans. 8, 382-383]. 4. Glucagon, but not phenylephrine or insulin, enhanced the phosphorylation of a mitochondrial protein of Mr 35000 and of four plasma- or microsomal-membrane proteins of Mr 50000, 30000, 23000 and 19000. 5. Mitochondria from glucagon-treated animals or hepatocytes phosphorylated a protein of Mr 30000 when incubated in vitro with [32P]Pi and ADP. Phosphorylation of this protein did not occur with mitochondria from control, phenylephrine- or insulin-treated cells. 6. The significance of these hormonally induced changes in protein phosphorylation is discussed.

Animals↗
PubMed1982

Parallel increases in rates of fatty acid synthesis and in pyruvate dehydrogenase activity in isolated rat hepatocytes incubated with insulin.

The effect of insulin on the activity of pyruvate dehydrogenase is studied in isolated hepatocytes from fed rats. Insulin increases the 'initial' activity of pyruvate dehydrogenase by 30% without modifying the total activity of the enzyme. The maximal increase is reached 3 min after addition of the hormone and is dose-dependent. Insulin also increases the rate of fatty acid synthesis.

Aminoisobutyric Acids↗
PubMed1982

Properties of N-maleoylmethionine sulphone, a novel impermeant maleimide, and its use in the selective labelling of the erythrocyte glucose-transport system.

1. The synthesis of N-maleoylmethionine sulphone (MMS), a membrane-impermeant protein-labelling reagent, is described. Radioactively labelled MMS can be readily prepared at high specific radioactivity from [35S]methionine. 2. The permeability of the erythrocyte membrane to the reagent was assessed by determining the extent of inactivation of glyceraldehyde 3-phosphate dehydrogenase after treatment of erythrocytes with MMS. Some inactivation of this enzyme was found when high concentrations (20mM) of the compound were used, but this could be prevented by pretreatment of the erythrocytes with 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid, suggesting that MMS slowly enters the cells via the anion-transport system. 3. Treatment of erythrocytes with [35S]MMS resulted in the labelling of six major components. Labelling of erythrocyte membranes resulted in the intense labelling of many additional components. 4. MMS inhibited erythrocyte glucose transport. Cytochalasin b protected glucose transport against inactivation by MMS. Labelling experiments in erythrocytes in the presence and in the absence of cytochalasin b showed that the cytochalasin b-protected material was a broad band in the band-4.5 region.

Biological Transport↗
PubMed1982

Reversibility of the insulin-stimulated phosphorylation of ATP citrate lyase and a cytoplasmic protein of subunit Mr 22000 in adipose tissue.

Exposure of rat epididymal adipose tissue to insulin leads to a 2-fold increase in incorporation of [32P]phosphate into ATP citrate lyase and a cytoplasmic protein of Mr 22000. A smaller increase and decrease in incorporation into cytoplasmic proteins of Mr 63000 and 20000 respectively were also observed. Subsequent addition of anti-insulin serum largely reversed these changes within 15 min, indicating that the alterations in 32P incorporation represented changes in net phosphate content of the proteins.

ATP Citrate (pro-S)-Lyase↗
PubMed1982

Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes.

1. Previous studies showed that the activation of pyruvate dehydrogenase within intact rat heart mitochondria of pyruvate is much diminished in mitochondria from starved or diabetic animals [see Kerbey, Randle, Cooper, Whitehouse, Pask & Denton (1976) Biochem. J. 154, 327-348]. In the present study, diminished responses to added Ca2+ and ADP were also found in these mitochondria. 2. Starvation or diabetes did not affect the mitochondrial respiratory control ratio of the ATP content. Moreover, starvation and diabetes did not alter the response of the intramitochondrial Ca2+-sensitive enzyme, 2-oxoglutarate dehydrogenase, to changes in the extramitochondrial concentration of Ca2+ and 2-oxoglutarate, thus indicating that there were no appreciable changes in the distribution of Ca2+ and H+ across the mitochondrial inner membrane. 3. Pyruvate, Ca2+ and ADP were found to have synergistic effects on pyruvate dehydrogenase activity, particularly in mitochondria from starved and diabetic rats. 4. The results suggest that the effects of diabetes and starvation on pyruvate dehydrogenase are not brought about by changes in the distribution of these effectors across the mitochondrial inner membrane or by changes in the intrinsic sensitivity of the kinase or phosphatase of the pyruvate dehydrogenase system to pyruvate, Ca2+ or ADP; rather it is probably that there is an increase in the maximum activity of kinase relative to that of the phosphatase. 6. The results also lend further support to the hypothesis that adrenaline may bring about the activation of pyruvate dehydrogenase in the rat heart by an increase in the intramitochondrial concentration of Ca2+.

Adenosine Diphosphate↗
PubMed1982

Evidence that insulin activates fat-cell acetyl-CoA carboxylase by increased phosphorylation at a specific site.

1. A new rapid method for the purification of fat-cell acetyl-CoA carboxylase is described; the key step is sedimentation after specific polymerization by citrate. 2. Incubation of epididymal fat-pads or isolated fat-cells with insulin or adrenaline leads to a rapid increase or decrease respectively in the activity of acetyl-CoA carboxylase measured in fresh tissue extracts. The persistence of the effect of insulin through high dilution of tissue extracts and through purification involving precipitation with (NH4)2SO4 suggests that the enzyme undergoes a covalent modification after exposure of intact tissue to the hormone. The opposed effects of insulin and adrenaline are not adequately explained through modification of a common site on acetyl-CoA carboxylase, since these hormones bring about qualitatively different alterations in the kinetic properties of the enzyme measured in tissue extracts. 3. The state of phosphorylation of acetyl-CoA carboxylase within intact fat-cells exposed to insulin was determined, and results indicate a small but consistent rise in overall phosphorylation of the Mr-230000 subunit after insulin treatment. 4. Acetyl-CoA carboxylase from fat-cells previously incubated in medium containing [32P]phosphate was purified by immunoprecipitation and then digested with performic acid and trypsin before separation of the released phosphopeptides by two-dimensional analysis. Results obtained show that the exposure of fat-cells to insulin leads to a 5-fold increase in incorporation of 32P into a peptide which is different from those most markedly affected after exposure of fat-cells to adrenaline. 5. These studies indicate that the activation of acetyl-CoA carboxylase in cells incubated with insulin is brought about by the increased phosphorylation of a specific site on the enzyme, possibly catalysed by the membrane-associated cyclic AMP-independent protein kinase described by Brownsey, Belsham & Denton [(1981) FEBS Lett. 124, 145-150].

Acetyl-CoA Carboxylase↗
PubMed1981

Stimulation of hepatic lipogenesis and acetyl-coenzyme A carboxylase by vasopressin.

The effect of vasopressin on the short-term regulation of fatty acid synthesis was studied in isolated hepatocytes from rats fed ad libitum. Vasopressin stimulates fatty acid synthesis by 30-110%. This increase is comparable with that obtained with insulin. Angiotensin also stimulates fatty acid synthesis, whereas phenylephrine does not. The dose-response curve for vasopressin-stimulated lipogenesis is similar to the dose-response curve for glycogenolysis and release of lactate plus pyruvate. Vasopression also stimulates acetyl-CoA carboxylase activity in a dose-dependent manner. Vasopressin does not relieve glucagon-inhibited lipogenesis, whereas insulin does. The action of vasopressin on hepatic lipogenesis is decreased, but not suppressed, in Ca2+-depleted hepatocytes. The results suggest that vasopressin acts on lipogenesis by increasing availability of lipogenic substrate (lactate + pyruvate) and by activating acetyl-CoA carboxylase.

Acetyl-CoA Carboxylase↗
PubMed1981

A comparative study of the regulation of Ca2+ of the activities of the 2-oxoglutarate dehydrogenase complex and NAD+-isocitrate dehydrogenase from a variety of sources.

Ca2+ was shown to activate oxoglutarate dehydrogenase and NAD+-isocitrate dehydrogenase from heart and other rat tissues by markedly decreasing the Km values of the enzymes for their respective substrates [see Denton & McCormack (1980) FEBS Lett. 119, 1-8]. Similar effects of Ca2+ were observed in the present study with both enzymes from other vertebrate sources (pigeon, trout, frog and human heart), but not with the enzymes from blowfly or locust flight muscle, or potato or Escherichia coli. In contrast, the Km values of the oxoglutarate dehydrogenases were affected by ADP, ATP and H+ to a similar extent in every case, except for the enzyme from E. coli, which was not sensitive to regulation by these agents.

Adenine Nucleotides↗
PubMed1981

The activation of pyruvate dehydrogenase in the perfused rat heart by adrenaline and other inotropic agents.

Adrenaline resulted in a reversible 4-fold increase in the amount of pyruvate dehydrogenase in its active non-phosphorylated form in the perfused rat heart within 1 min. The increase was less in extent in hearts from starved or diabetic rats or in hearts from control rats oxidizing acetate, unless pyruvate was added to the perfusion medium. Increases could also be induced by other inotropic agents, supporting the hypothesis that increases in cytoplasmic Ca2+ can be relayed into mitochondria and influence oxidative metabolism.

Animals↗