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 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 A comparison of the regulation of pyruvate dehydrogenase in mitochondria from rat brain and liver

1975 ◽  
Vol 150 (3) ◽  
pp. 397-403 ◽  
Author(s):  
R Jope ◽  
J P Blass
Keyword(s):  
Rat Brain ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Active Form ◽  
Energy Charge ◽  
Liver Mitochondria ◽  
Brain Mitochondria ◽  
Ruthenium Red ◽  
Rat Brain And Liver ◽  
Pyruvate Dehydrogenase Phosphatase

The total activity of pyruvate dehydrogenase in mitochondria isolated from rat brain and liver was 53.5 and 14.2nmol/min per mg of protein respectively. Pyruvate dehydrogenase in liver mitochondria incubated for 4 min at 37 degrees C with no additions was 30% in the active form and this activity increased with longer incubations until it was completely in the active form after 20 min. Brain mitochondrial pyruvate dehydrogenase activity was initially high and did not increase with addition of Mg2+ plus Ca2+ or partially purified pyruvate dehydrogenase phosphatase or with longer incubations. The proportion of pyruvate dehydrogenase in the active form in both brain and liver mitochondria changed inversely with changes in mitochondrial energy charge, whereas total pyruvate dehydrogenase did not change. The chelators citrate, isocitrate, EDTA, ethanedioxybis(ethylamine)tetra-acetic acid and Ruthenium Red each lowered pyruvate dehydrogenase activity in brain mitochondria, but only citrate and isocitrate did so in liver mitochondria. These chelators did not affect the energy charge of the mitochondria. Mg2+ plus Ca2+ reversed the pyruvate dehydrogenase inactivation in liver, but not brain, mitochondria. The regulation of the activation-inactivation of pyruvate dehydrogenase in mitochondria from rat brain and liver with respect to energy charge is similar and may be at least partially regulated by this parameter, and the effects of chelators differ in the two types of mitochondria.

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.

Control of Fatty Acid Synthesis in White Adipose Tissue by Insulin: Coordination Between the Mitochondrial Citrate Transporter and Pyruvate Dehydrogenase

1974 ◽  
Vol 52 (10) ◽  
pp. 813-821 ◽  
Author(s):  
Carol M. Schiller ◽  
Wayne M. Taylor ◽  
Mitchell L. Halperin
Keyword(s):  
Fatty Acids ◽  
Adipose Tissue ◽  
Pyruvate Dehydrogenase ◽  
Active Form ◽  
Acid Synthesis ◽  
Citrate Transporter ◽  
Maximal Activation ◽  
Initial Decrease ◽  
Preincubation Medium

The transport of citrate out of adipose tissue mitochondria is inhibited by palmitoyl-CoA. This inhibition varied inversely with the concentration of extramitochondrial exchanging anion.When adipose tissue is incubated in vitro, the rate of citrate output into the medium was increased by the addition of insulin. The tissue citrate content did not change significantly. Norepinephrine caused an initial decrease in the rate of citrate output (2.5 min). The tissue citrate content was approximately twofold higher at this time.When rats were fasted for 36 h, less than 40% of adipose tissue pyruvate dehydrogenase was in the active form. Optimal interconversion to the active form was achieved by preincubation with 4 mM Mg2+ in the absence of added Ca2+ (endogenous Ca2+ was approximately 25 μM). Citrate addition to the preincubation medium decreased this activation of pyruvate dehydrogenase. The inhibition induced by citrate correlated best with the concentration of 'free' citrate when the 'free' Mg2+ concentration was sufficient to cause near-maximal activation of pyruvate dehydrogenase.A hypothesis regarding the coordination of regulation of pyruvate conversion to fatty acids is formulated based on these findings.

scholarly journals Effect of nutrition on subcellular localization of rat fat-cell lipoprotein lipase

1978 ◽  
Vol 172 (2) ◽  
pp. 239-245 ◽  
Author(s):  
A Vanhove ◽  
C Wolf ◽  
M Breton ◽  
M C Glangeaud
Keyword(s):  
Adipose Tissue ◽  
Lipoprotein Lipase ◽  
Plasma Membranes ◽  
Total Activity ◽  
Normal Diet ◽  
Epididymal Adipose Tissue ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Fat Cell ◽  
Intact Tissue

This study supports the possibility for multiple subcellular forms of lipoprotein lipase. 1. The total activity of lipoprotein lipase per g of intact epididymal adipose tissue from fed rats is much higher than that from starved rats. 2. The isolated fat-cells of fed and of starved rats have lipoprotein lipase of almost the same activity per g of fat-pads. The isolated fat-cells of starved rats have a much higher proportion of total activity per g of the intact tissue than do those of fed rats. 3. Under the conditions of homogenization used, only a small proportion of the total activity per g of intact tissue from fed rats was associated with the fat layer which floated to the top of the homogenate during low-speed centrifugation. The different proportions of the specific enzyme activity found in each subcellular fraction are described. 4. Lipoprotein lipase from plasma membranes and microsomal fractions from starved and fed rats was purified by affinity chromatography. 5. The total activity of microsomal lipoprotein lipase per g of intact adipose tissue is enhanced by a normal diet. 6. In intact epididymal adipose tissue from fed rats, the activity per g of tissue of lipoprotein lipase of plasma membranes is much higher than that in the same fraction from starved rats. By contrast, the activities per g of tissue in plasma membranes obtained from starved or from fed rats by collagenase treatment were similar.

scholarly journals Relation between cytosolic free Ca2+ concentration and the control of pyruvate dehydrogenase in isolated cardiac myocytes

1987 ◽  
Vol 241 (1) ◽  
pp. 145-151 ◽  
Author(s):  
R G Hansford
Keyword(s):  
Cardiac Myocytes ◽  
Pyruvate Dehydrogenase ◽  
Energy Supply ◽  
Supply And Demand ◽  
Active Form ◽  
Chelating Agent ◽  
Work Load ◽  
Ruthenium Red ◽  
Isolated Cardiac Myocytes ◽  
Physiological Values

The proportion of pyruvate dehydrogenase existing in the active form (PDHA) in suspensions of unstimulated cardiac myocytes oxidizing glucose is approx. 30%. Depolarization of the cells with concentrations of K+ above physiological values leads to an increase in the content of PDHA. Overloading of the cells with Na+ by treatment with veratridine and ouabain gives the same result. Each of these interventions is shown in experiments with Quin 2-loaded myocytes to lead to an increase in cytosolic free Ca2+ concentration ([Ca2+]c). Treatment of the cells with Ruthenium Red, an inhibitor of Ca2+ transport into mitochondria, largely prevents an increase in PDHA in response to addition of KCl or of veratridine plus ouabain. Ruthenium Red does not attenuate the increase in [Ca2+]c that occurs under these conditions. By contrast, treatment of the cells with ryanodine, an inhibitor of sarcoplasmic-reticulum Ca2+ transport and therefore of contraction, does not diminish the response of PDHA content to agents which raise [Ca2+]c; nor does loading of the cells with the Ca2+-chelating agent Quin 2, which also prevents contraction, at appropriate concentrations. It is concluded that an increase in [Ca2+]c causes an increase in PDHA content of cardiac myocytes independently of an increase in mechanical work. In the normal physiological situation the activation of dehydrogenases by Ca2+ is thought to help to maintain the balance of energy supply and demand during periods of increased work-load, which are associated with an increased myoplasmic [Ca2+]c.

scholarly journals Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria

1985 ◽  
Vol 231 (3) ◽  
pp. 581-595 ◽  
Author(s):  
J G McCormack
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Pyruvate Dehydrogenase Kinase ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Mammalian Tissues ◽  
14Co2 Production

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent ‘State 3.5’ respiration condition. Ca2+ had no effect on NAD(P)H formation induced by β-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.

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.

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