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 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 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.

scholarly journals Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes

1983 ◽  
Vol 216 (2) ◽  
pp. 359-367 ◽  
Author(s):  
E Baumgarten ◽  
M D Brand ◽  
T Pozzan
Keyword(s):  
Dehydrogenase Activity ◽  
Concanavalin A ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Kinase ◽  
Ionophore A23187 ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Maximal Activation ◽  
Pyruvate Dehydrogenase Phosphatase

The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.

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 Increased pyruvate dehydrogenase activity in skeletal muscle of growth-restricted ovine fetuses

2019 ◽  
Vol 317 (4) ◽  
pp. R513-R520 ◽  
Author(s):  
Alexander L. Pendleton ◽  
Laurel R. Humphreys ◽  
Melissa A. Davis ◽  
Leticia E. Camacho ◽  
Miranda J. Anderson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Pyruvate Dehydrogenase ◽  
Intrauterine Growth Restriction ◽  
Citrate Synthase ◽  
Pyruvate Dehydrogenase Kinase ◽  
Semitendinosus Muscle ◽  
Fetal Sheep ◽  
Pyruvate Dehydrogenase Phosphatase ◽  
And Control ◽  
Fetal Muscle

Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower fractional rates of glucose oxidation and greater gluconeogenesis, indicating lactate shuttling between skeletal muscle and liver. Suppression of pyruvate dehydrogenase ( PDH) activity was proposed because of greater pyruvate dehydrogenase kinase (PDK) 4 and PDK1 mRNA concentrations in IUGR muscle. Although PDK1 and PDK4 inhibit PDH activity to reduce pyruvate metabolism, PDH protein concentrations and activity have not been examined in skeletal muscle from IUGR fetuses. Therefore, we evaluated the protein concentrations and activity of PDH and the kinases and phosphatases that regulate PDH phosphorylation status in the semitendinosus muscle from placenta insufficiency-induced IUGR sheep fetuses and control fetuses. Immunoblots were performed for PDH, phosphorylated PDH (E1α), PDK1, PDK4, and pyruvate dehydrogenase phosphatase 1 and 2 (PDP1 and PDP2, respectively). Additionally, the PDH, lactate dehydrogenase (LDH), and citrate synthase (CS) enzymatic activities were measured. Phosphorylated PDH concentrations were 28% lower (P < 0.01) and PDH activity was 67% greater (P < 0.01) in IUGR fetal muscle compared with control. PDK1, PDK4, PDP1, PDP2, and PDH concentrations were not different between groups. CS and LDH activities were also unaffected. Contrary to the previous speculation, PDH activity was greater in skeletal muscle from IUGR fetuses, which parallels lower phosphorylated PDH. Therefore, greater expression of PDK1 and PDK4 mRNA did not translate to greater PDK1 or PDK4 protein concentrations or inhibition of PDH as proposed. Instead, these findings show greater PDH activity in IUGR fetal muscle, which indicates that alternative regulatory mechanisms are responsible for lower pyruvate catabolism.

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
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