scholarly journals Role of protein synthesis and of fatty acid metabolism in the longer-term regulation of pyruvate dehydrogenase kinase

1994 ◽  
Vol 300 (3) ◽  
pp. 659-664 ◽  
Author(s):  
D A Priestman ◽  
S C Mistry ◽  
A Halsall ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Kinase Activity ◽  
Specific Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Rat Liver Mitochondria ◽  
Dibutyryl Cyclic Amp ◽  
Alpha Chain

Antibodies were raised in rabbits to free rat liver pyruvate dehydrogenase (PDH) kinase alpha-chain and shown to react with PDH kinase alpha-chain in rat heart and liver PDH complexes, in purified pig heart PDH complex and in bovine kidney dihydrolipoamide acetyltransferase-protein X-PDH kinase subcomplex. E.l.i.s.a for PDHE1 (pyruvate dehydrogenase) and PDH kinase have been developed and applied to assays of these proteins in extracts of rat liver and rat heart mitochondria; the measured immunoreactivities for PDHE1 (heart > liver) and for PDH kinase alpha-chain (liver > heart) paralleled known differences in PDH complex and PDH kinase activities respectively. The results of e.l.i.s.a of PDH kinase alpha-chain in extracts of rat liver mitochondria showed that the effects of starvation to increase PDH kinase activity in vivo, and the effects of dibutyryl cyclic AMP or palmitate to increase PDH kinase activity in hepatocytes cultured in vitro, are due largely (> 90%) to an increase in the specific activity of PDH kinase. The effect, in cultured hepatocytes, of dibutyryl cyclic AMP to increase PDH kinase activity was blocked by cycloheximide; the effect of palmitate was blocked by an inhibitor of carnitine palmitoyltransferase I (Etomoxir), but not by cycloheximide.

scholarly journals Longer-term regulation of pyruvate dehydrogenase kinase in cultured rat cardiac myocytes

1990 ◽  
Vol 267 (1) ◽  
pp. 245-247 ◽  
Author(s):  
D R Marchington ◽  
A L Kerbey ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Cardiac Myocytes ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Dibutyryl Cyclic Amp ◽  
Rat Cardiac Myocytes ◽  
Increased Activity

The increased activity of pyruvate dehydrogenase (PDH) kinase induced in hearts of rats by starvation for 48 h was maintained following preparation of cardiac myocytes, and it was also maintained, though at a decreased level, after 25 h of culture in medium 199. This loss of PDH kinase activity was not prevented by n-octanoate, dibutyryl cyclic AMP or glucagon. The PDH kinase activity of myocytes from fed rats was increased to that of starved rats after 25 h of culture with n-octanoate, dibutyryl cyclic AMP or both agents together.

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 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 Pyruvate dehydrogenase kinase/activator in rat heart mitochondria, Assay, effect of starvation, and effect of protein-synthesis inhibitors of starvation

1982 ◽  
Vol 206 (1) ◽  
pp. 103-111 ◽  
Author(s):  
A L Kerbey ◽  
P J Randle
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
High Speed ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Active Form ◽  
Pyruvate Dehydrogenase Kinase ◽  
Heart Mitochondria ◽  
Supernatant Fraction ◽  
Kidney Mitochondria

Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60 munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by its ability to accelerate NgATP-induced inactivation in dilute solutions of unprocessed complex (50 munits/ml). With this assay it has been shown that the activity of kinase/activator in heart mitochondria is increased 3-6 fold by starvation of rats for 48 h. This increase was prevented completely by cycloheximide treatment and prevented partially by puromycin treatment of rats during starvation. The concentration of kinase/activator in heart mitochondria fell during 20 h of re-feeding of 48 h-starved rats; this fall was correlated with an increase in the proportion of complex in the active form. Kinase/activator was also extracted from ox kidney mitochondria, and on gel filtration (Sephadex G-100, superfine grade) was eluted close to the void volume. Kinase/activator (ox kidney or rat heart) was thermolabile, non-diffusable on dialysis, and inactivated by trypsin. The results of this study appear to show increased cytoplasmic synthesis in starvation of pyruvate dehydrogenase kinase and/or of an activator of the kinase.

scholarly journals Longer-term regulation of pyruvate dehydrogenase kinase in cultured rat hepatocytes

1989 ◽  
Vol 257 (2) ◽  
pp. 487-491 ◽  
Author(s):  
D R Marchington ◽  
A L Kerbey ◽  
M G Giardina ◽  
A E Jones ◽  
P J Randle
Keyword(s):  
Fatty Acids ◽  
Cyclic Amp ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Rat Hepatocytes ◽  
Pyruvate Dehydrogenase Kinase ◽  
Activator Protein ◽  
Cultured Rat Hepatocytes ◽  
Culture Of Hepatocytes

The activities of pyruvate dehydrogenase (PDH) kinase and of PDH kinase activator protein (KAP) were increased 2-2.4-fold during 25 h of culture of hepatocytes from fed rats with glucagon plus n-octanoate. PDH kinase activity in hepatocytes from starved rats (initially 2.2 x fed control) fell during 25 h of culture in medium 199 (to 1.5 x fed control), but was maintained by glucagon plus octanoate. Dibutyryl or 8-bromo cyclic AMP increased PDH kinase activity 2-2.2-fold in hepatocytes from fed rats, but phenylephrine and isoproterenol (isoprenaline) were without effect. Insulin blocked the action of glucagon to increase PDH kinase activity and decreased the effect of octanoate and octanoate plus glucagon. It is suggested that the effects of starvation to increase activities of PDH kinase and of KAP in liver are mediated by alterations in circulating concentrations of glucagon, fatty acids and insulin and in hepatic cyclic AMP.

scholarly journals The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by α-Cyano-4-hydroxycinnamate and related compounds

1975 ◽  
Vol 148 (1) ◽  
pp. 97-106 ◽  
Author(s):  
A P Halestrap ◽  
R M Denton
Keyword(s):  
Fatty Acid ◽  
Cell Membrane ◽  
Fatty Acid Synthesis ◽  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Pyruvate Carboxylase ◽  
Acid Synthesis ◽  
Pyruvate Dehydrogenase Kinase ◽  
Kidney Cortex ◽  
Rat Liver Mitochondria

1. Effects of α-cyano-4-hydroxycinnamate and α-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. α-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that α-cyano-4-hydroxycinnamate or α-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of α-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. α-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.

scholarly journals Evidence that rat liver pyruvate dehydrogenase kinase activator protein is a pyruvate dehydrogenase kinase

1991 ◽  
Vol 275 (3) ◽  
pp. 775-779 ◽  
Author(s):  
S C Mistry ◽  
D A Priestman ◽  
A L Kerbey ◽  
P J Randle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activator Protein ◽  
Dependent Inactivation

It is shown here that rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP) catalyses ATP-dependent inactivation and [32P]phosphorylation of pig heart PDHE1 and of yeast (Saccharomyces cerevisiae) PDH complex devoid of PDH kinase activity, that fluorosulphonylbenzoyladenosine inactivates rat liver KAP and the intrinsic PDH kinase of rat liver PDH complex, and that KAP, like PDH kinase, is inactivated by thiol-reactive reagents. It is concluded that KAP is a free PDH kinase.

scholarly journals Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes

1990 ◽  
Vol 267 (1) ◽  
pp. 85-90 ◽  
Author(s):  
M P Kolodziej ◽  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Mitochondria ◽  
Subsequent Treatment ◽  
Carnitine Palmitoyltransferase ◽  
Rat Liver Mitochondria ◽  
Outer Membranes ◽  
High Affinity ◽  
Malonyl Coa ◽  
Order Of Magnitude

1. The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. 2. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48 h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. 6. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.

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