scholarly journals Modulation of pyruvate dehydrogenase kinase activity in cultured hepatocytes by glucagon and n-octanoate

1986 ◽  
Vol 234 (1) ◽  
pp. 233-236 ◽  
Author(s):  
H R Fatania ◽  
T C Vary ◽  
P J Randle
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Chain Complex ◽  
Rat Hepatocytes ◽  
Liver Mitochondria ◽  
Pyruvate Dehydrogenase Kinase ◽  
Rat Liver Mitochondria ◽  
Potential Contribution ◽  
Complex Activity ◽  
Branched Chain

The activity of pyruvate dehydrogenase kinase in extracts of mitochondria from rat hepatocytes cultured for 21 h in medium 199 was increased 2.5-fold by the presence of 55 nM-glucagon and 1 mM-sodium n-octanoate in the culture medium. The change was comparable with that induced in vivo by 48 h starvation. The potential contribution of branched-chain complex to estimates of PDH-complex activity in rat liver mitochondria has been defined.

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 Studies on the activation of rat liver pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase by adrenaline and glucagon. Role of increases in intramitochondrial Ca2+ concentration

1985 ◽  
Vol 231 (3) ◽  
pp. 597-608 ◽  
Author(s):  
J G McCormack
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Liver Mitochondria ◽  
Active Enzyme ◽  
Female Rats ◽  
Rat Liver Mitochondria ◽  
Na Ions ◽  
Oxoglutarate Dehydrogenase

The administration in vivo of either adrenaline or glucagon alone resulted in increases of about 2-fold in the amounts of active, non-phosphorylated, pyruvate dehydrogenase in the livers of fed male or female rats, whereas when administered together increases of about 4-fold were obtained. Ca2+-dependent increases in the amount of active enzyme of up to about 5-fold could be achieved in isolated rat liver mitochondria by incubating them with increasing extramitochondrial [Ca2+]; from this, two conditions of Ca loading were chosen which caused increases in active enzyme similar to those with the hormone treatments given above. The increases in enzyme activity owing to these Ca loads persisted through the ‘re-isolation’ of mitochondria and their incubation in Na+-free KCl-based media containing EGTA. Differences from values obtained with unloaded controls could be diminished by adding Na+ ions to cause the egress of Ca2+ from the mitochondria, or enough extramitochondrial Ca2+ to saturate the enzyme in its Ca2+-dependent activation; the effects of Na+ could be blocked by diltiazem, an inhibitor of mitochondrial Na+/Ca2+ exchange. The re-isolated, Ca-preloaded, mitochondria also exhibited enhanced activities of 2-oxoglutarate dehydrogenase when assayed at non-saturating [2-oxoglutarate] by two different methods; effects of Na+, Ca2+ or diltiazem on the persistent activations of this enzyme were similar to those for pyruvate dehydrogenase. Na+ caused a marked depletion, which could be blocked by diltiazem, of the 45Ca content of re-isolated mitochondria which had pre-loaded with Ca, containing 45Ca, to the same degrees as above. The activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase in incubated liver mitochondria prepared from rats subjected to the hormone treatments given above were found to behave in a very similar manner to those exhibited in the re-isolated, Ca-preloaded, mitochondria. It is concluded that these hormones each bring about the activations of these rat liver enzymes by causing increases in intramitochondrial [Ca2+], and that their effects, as such, are additive.

scholarly journals Kinase activator protein mediates longer-term effects of starvation on activity of pyruvate dehydrogenase kinase in rat liver mitochondria

1986 ◽  
Vol 239 (2) ◽  
pp. 347-354 ◽  
Author(s):  
G S Denyer ◽  
A L Kerbey ◽  
P J Randle
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Protein Fraction ◽  
Kinase Activity ◽  
Gel Filtration ◽  
Liver Mitochondria ◽  
Pyruvate Dehydrogenase Kinase ◽  
Rat Liver Mitochondria ◽  
Fractional Precipitation ◽  
Activator Protein

Starvation of rats for 48 h increased the activity of PDH (pyruvate dehydrogenase) kinase 2.2-fold in extracts of liver mitochondria, 2.9-fold in PDH complex partially purified therefrom by fractional precipitation, and 5-fold in PDH complex partially purified by gel filtration on Sephacryl S-300. A protein fraction was separated from PDH complex in extracts of rat liver mitochondria by gel filtration or fractional precipitation, which increased the activity of PDH kinase in rat liver and pig heart PDH complexes. The activity of this protein fraction was increased approx. 2.5-fold by 48 h starvation of rats. With highly purified pig heart PDH complex it was shown that the protein fraction increased the Vmax. of the PDH kinase reaction 35-fold (fraction from fed rats) or 82-fold (fraction from starved rats); starvation had no effect on the concentration of protein fraction required to give 0.5 Vmax. Evidence is given that the increase in PDH kinase activity effected in extracts of liver mitochondria by starvation is due to increased activity of kinase activator protein, which is tightly bound by rat liver PDH complex and not removed by a single gel filtration. With pig heart PDH complex, increased PDH kinase activity was retained after gel filtration of an admixture with kinase activator protein from starved rats, but was restored to the control value by a second gel filtration; the alterations in PDH kinase activity were associated with obvious changes in protein bands in SDS gels.

scholarly journals Purification and properties of a protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex

1985 ◽  
Vol 225 (2) ◽  
pp. 509-516 ◽  
Author(s):  
J Espinal ◽  
P A Patston ◽  
H R Fatania ◽  
K S Lau ◽  
P J Randle
Keyword(s):  
Rat Liver ◽  
Chain Complex ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Activator Protein ◽  
Protein Activator ◽  
Liver And Kidney ◽  
Branched Chain ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

The protein activator of phosphorylated branched-chain 2-oxo acid dehydrogenase complex was purified greater than 1000-fold from extracts of rat liver mitochondria; the specific activity was greater than 1000 units/mg of protein (1 unit gives half-maximum re-activation of 10 munits of phosphorylated complex). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave two bands (Mr 47700 and 35300) indistinguishable from the alpha- and beta-subunits of the branched-chain dehydrogenase component of the complex. On gel filtration (Sephacryl S-300), apparent Mr was 190000. This and other evidence suggests that activator protein is free branched-chain dehydrogenase; this conclusion is provisional until identical amino acid composition of the subunits has been demonstrated. Activator protein (i.e. free branched-chain dehydrogenase) was inhibited (up to 30%) by NaF, whereas branched-chain complex was not inhibited. There was no convincing evidence for interconvertible active and inactive forms of activator protein in rat liver mitochondria. Activator protein was detected in mitochondria from liver (ox, rabbit and rat) and kidney (ox and rat), but not in rat heart or skeletal-muscle mitochondria. In rat liver mitochondrial extracts, branched-chain complex sedimented with the mitochondrial membranes, whereas activator protein remained in the supernatant. Activator protein re-activated phosphorylated (inactive) particulate complex from rat liver mitochondria, but it did not activate dephosphorylated complex. Liver and kidney, but not muscle, mitochondria apparently contain surplus free branched-chain dehydrogenase, which is bound by the complex with lower affinity than is the branched-chain dehydrogenase intrinsic to the complex. It is suggested that this functions as a buffering mechanism to maintain branched-chain complex activity in liver and kidney mitochondria.

scholarly journals The metabolism of tetradecylthiopropionic acid, a 4-thia stearic acid, in the rat. In vivo and in vitro studies

1992 ◽  
Vol 286 (3) ◽  
pp. 879-887 ◽  
Author(s):  
E Hvattum ◽  
S Skrede ◽  
J Bremer ◽  
M Solbakken
Keyword(s):  
Stearic Acid ◽  
Rat Hepatocytes ◽  
Liver Mitochondria ◽  
Oxidation Products ◽  
Rat Liver Mitochondria ◽  
Beta Oxidation ◽  
Isolated Rat Hepatocytes ◽  
Semialdehyde Dehydrogenase

The metabolism of [1-14C]tetradecylthiopropionic acid (TTP), a 4-thia stearic acid, and its sulphoxide, [1-14C]texadecylsulphoxypropionic acid (TTP-SO), has been studied in intact rats, in isolated rat hepatocytes, and in rat liver mitochondria. Two pathways of oxidation (beta-oxidation and omega-oxidation) have been demonstrated. TTP is incorporated, in vivo, into tissue triacylglycerol and phospholipids, it is oxidized to CO2, and it is excreted in urine, mainly as carboxypropylsulphoxypropionic acid and a little as carboxymethylsulphoxypropionic acid. TTP-SO is metabolized, in vivo, more rapidly to the same two omega-oxidation products. In hepatocytes TTP is incorporated into triacylglycerol and phospholipids even more rapidly than stearic acid. It is recovered mainly in the 1-position of phosphatidylcholine. Some is oxidized to CO2 and acid-soluble products. TTP-SO is mainly omega-oxidized to the same metabolites as are found in urine. A small fraction is incorporated into phospholipids or oxidized to CO2. In isolated mitochondria [1-14C]TTP is converted into 14CO2, radioactive malonic semialdehyde, and addition products of malonic semialdehyde. In the presence of phenylhydrazine, malonic semialdehyde phenylhydrazone is the dominating product. In soluble extracts of mitochondria [1-14C]malonic semialdehyde is oxidized directly to 14CO2 in the presence of CoA and NAD+, probably by the (methyl)malonic acid semialdehyde dehydrogenase (EC 1.2.1.27).

scholarly journals Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex

1998 ◽  
Vol 329 (1) ◽  
pp. 191-196 ◽  
Author(s):  
Melissa M. BOWKER-KINLEY ◽  
I. Wilhelmina DAVIS ◽  
Pengfei WU ◽  
A. Robert HARRIS ◽  
M. Kirill POPOV
Keyword(s):  
Tissue Distribution ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Synthetic Analogue ◽  
Complex Activity ◽  
Acetyl Coa ◽  
Tissue Specific ◽  
Specific Regulation ◽  
Dehydrogenase Complex

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50 nmol/min per mg for PDK2 to 1250 nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.

Sign in / Sign up

Export Citation Format

Share Document