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

1986 ◽  
Vol 238 (1) ◽  
pp. 83-91 ◽  
Author(s):  
A P Thomas ◽  
T A Diggle ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Major Effect ◽  
Maximum Activity ◽  
Close Similarity ◽  
Epididymal Adipose Tissue ◽  
Kinetic Properties ◽  
Ionophore A23187 ◽  
Magnesium Ions ◽  
Dehydrogenase System

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.

scholarly journals Use of toluene-permeabilized mitochondria to study the regulation of adipose tissue pyruvate dehydrogenase in situ. Further evidence that insulin acts through stimulation of pyruvate dehydrogenase phosphate phosphatase

1986 ◽  
Vol 238 (1) ◽  
pp. 93-101 ◽  
Author(s):  
A P Thomas ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Phosphatase Activity ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Kinase ◽  
Epididymal Adipose Tissue ◽  
Ionophore A23187 ◽  
Dilute Suspensions ◽  
Dehydrogenase System ◽  
Pyruvate Dehydrogenase Phosphatase

Rat epididymal-adipose-tissue mitochondria were made selectively permeable to small molecules without the loss of matrix enzymes by treating the mitochondria with toluene under controlled conditions. With this preparation the entire pyruvate dehydrogenase system was shown to be retained within the mitochondrial matrix and to retain its normal catalytic activity. By using dilute suspensions of these permeabilized mitochondria maintained in the cuvette of a spectrophotometer, it was possible to monitor changes of pyruvate dehydrogenase activity continuously while the activities of the interconverting kinase and phosphatase could be independently manipulated. Permeabilized mitochondria were prepared from control and insulin-treated adipose tissue, and the properties of both the pyruvate dehydrogenase kinase and the phosphatase were compared in situ. No difference in kinase activity was detected, but increases in phosphatase activity were observed in permeabilized mitochondria from insulin-treated tissue. Further studies showed that the main effect of insulin treatment was a decrease in the apparent Ka of the phosphatase for Mg2+, in agreement with earlier studies with mitochondria made permeable to Mg2+ by using the ionophore A23187 [Thomas, Diggle & Denton (1986) Biochem. J. 238, 83-91]. No effects of spermine were detected, although spermine diminishes the Ka of purified phosphatase preparations for Mg2+. Since effects of insulin on pyruvate dehydrogenase phosphatase activity are not evident in mitochondrial extracts, it is concluded that insulin may act by altering some high-Mr component which interacts with the pyruvate dehydrogenase system within intact or permeabilized mitochondria, but not when the mitochondrial membranes are disrupted.

scholarly journals Role of calcium ions in the regulation of intramitochondrial metabolism. Properties of the Ca2+-sensitive dehydrogenases within intact uncoupled mitochondria from the white and brown adipose tissue of the rat

1980 ◽  
Vol 190 (1) ◽  
pp. 95-105 ◽  
Author(s):  
J G McCormack ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Epididymal Adipose Tissue ◽  
Ionophore A23187 ◽  
Mitochondrial Inner Membrane ◽  
Brown Adipose ◽  
Oxoglutarate Dehydrogenase

1. Increasing concentrations of both Ca2+ and Sr2+ (generated by using EGTA buffers) resulted in 4-fold increases in the initial activity of pyruvate dehydrogenase within intact uncoupled mitochondria from rat epididymal adipose tissue incubated in the presence of the ionophore A23187, ATP, Mg2+ and oligomycin. The k0.5 values (concentrations required for half-maximal effects) for Ca2+ and Sr2+ were 0.54 and 7.1 microM respectively. In extracts of the mitochondria, pyruvate dehydrogenase phosphate phosphatase activity was stimulated about 4-fold by Ca2+ and Sr2+, with k0.5 values of 1.08 and 6.4 microM respectively. 2. NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase appeared to be rate-limiting in the oxidation of threo-Ds-isocitrate and oxoglutarate by uncoupled mitochondria from brown adipose tissue of cold-adapted rats. Ca2+ (and Sr2+) diminished the Km for the oxidation of both threo-Ds-isocitrate and oxoglutarate. The kinetic constants for these oxidations were very similar to those obtained for the activities of NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of the mitochondria. In particular, the k0.5 values for Ca2+ were all in the range 0.2–1.6 microM and Sr2+ was found to mimic Ca2+, but with k0.5 values about 10 times greater. 3. Overall, the results of this study demonstrate that the activities of pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase may all be increased by Ca2+ and Sr2+ within intact mitochondria. In all cases the k0.5 values are close to 1 and 10 microM respectively, as found for the separated enzymes. Experiments on brown-adipose-tissue mitochondria incubated in the presence of albumin suggest that it may be possible to use the sensitivity of the dehydrogenases to Ca2+ as a means of assessing the distribution of Ca2+ across the mitochondrial inner membrane.

Insulin activates Pyruvate Dehydrogenase in Rat Epididymal Adipose Tissue

1971 ◽  
Vol 231 (21) ◽  
pp. 115-116 ◽  
Author(s):  
R. M. DENTON ◽  
H. G. COORE ◽  
B. R. MARTIN ◽  
P. J. RANDLE
Keyword(s):  
Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Epididymal Adipose Tissue

scholarly journals Effects of Ca2+ and Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within toluene-permeabilized mitochondria

1987 ◽  
Vol 241 (2) ◽  
pp. 371-377 ◽  
Author(s):  
P J Midgley ◽  
G A Rutter ◽  
A P Thomas ◽  
R M Denton
Keyword(s):  
Adipose Tissue ◽  
Small Molecules ◽  
White Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Maximum Rate ◽  
Major Effect ◽  
Lag Phase ◽  
Inhibitory Effects ◽  
Highly Sensitive ◽  
Epididymal White Adipose Tissue

Mitochondria from rat epididymal white adipose tissue were made permeable to small molecules by toluene treatment and were used to investigate the effects of Mg2+ and Ca2+ on the re-activation of pyruvate dehydrogenase phosphate by endogenous phosphatase. Re-activation of fully phosphorylated enzyme after addition of 0.18 mM-Mg2+ showed a marked lag of 5-10 min before a maximum rate of reactivation was achieved. Increasing the Mg2+ concentration to 1.8 mM (near saturating) or the addition of 100 microM-Ca2+ resulted in loss of the lag phase, which was also greatly diminished if pyruvate dehydrogenase was not fully phosphorylated. It is concluded that, within intact mitochondria, phosphatase activity is highly sensitive to the degree of phosphorylation of pyruvate dehydrogenase and that the major effect of Ca2+ may be to overcome the inhibitory effects of sites 2 and 3 on the dephosphorylation of site 1. Apparent K0.5 values for Mg2+ and Ca2+ were determined from the increases in pyruvate dehydrogenase activity observed after 5 min. The K0.5 for Mg2+ was diminished from 0.60 mM at less than 1 nM-Ca2+ to 0.32 mM at 100 microM-Ca2+; at 0.18 mM-Mg2+, the K0.5 for Ca2+ was 0.40 microM. Ca2+ had little or no effect at saturating Mg2+ concentrations. Since effects of Ca2+ are readily observed in intact coupled mitochondria, it follows that Mg2+ concentrations within mitochondria are sub-saturating for pyruvate dehydrogenase phosphate phosphatase and hence less than 0.5 mM.

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

1982 ◽  
Vol 202 (2) ◽  
pp. 419-427 ◽  
Author(s):  
J G McCormack ◽  
N J Edgell ◽  
R M Denton
Keyword(s):  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Synergistic Effects ◽  
Respiratory Control ◽  
Diabetic Rats ◽  
Maximum Activity ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Dehydrogenase System

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+.

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

1975 ◽  
Vol 148 (2) ◽  
pp. 229-235 ◽  
Author(s):  
C Mukherjee ◽  
R L Jungas
Keyword(s):  
Creatine Kinase ◽  
Adipose Tissue ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Insulin Treatment ◽  
Creatine Phosphate ◽  
Epididymal Adipose Tissue ◽  
Crude Extracts ◽  
Rate Of Increase

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.

scholarly journals 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

1984 ◽  
Vol 217 (2) ◽  
pp. 441-452 ◽  
Author(s):  
R M Denton ◽  
J G McCormack ◽  
S E Marshall
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Plasma Membranes ◽  
Pyruvate Dehydrogenase Kinase ◽  
Epididymal Adipose Tissue ◽  
Short Term ◽  
Interscapular Brown Adipose Tissue ◽  
Brown Adipose

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.

scholarly journals Effect of insulin on pyruvate metabolism in epididymal adipose tissue of the rat. Correlation of intracellular pyruvate contents and pyruvate dehydrogenase activity

1973 ◽  
Vol 134 (4) ◽  
pp. 885-889 ◽  
Author(s):  
Bruce G. Berman ◽  
Mitchell L. Halperin
Keyword(s):  
Adipose Tissue ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Epididymal Adipose Tissue ◽  
Pyruvate Metabolism ◽  
Time Period ◽  
Intracellular Content

A method is described to measure the intracellular content of pyruvate and lactate in epididymal adipose tissue of the rat. The intracellular pyruvate concentration was approx. 330μm. Intracellular pyruvate contents and the rates of pyruvate output were increased when NNN′N′-tetramethyl-p-phenylenediamine was added, and decreased in the presence of alanine. Insulin addition caused an increase in intracellular pyruvate contents only at the earlier time-period studied (1.5min as against 20min). Pyruvate dehydrogenase activity was increased in adipose tissue incubated in vitro with insulin. This increase occurred subsequent to the rise in the intracellular pyruvate content induced by insulin addition. The possible physiological implications are discussed.

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