The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.

/pdf/regulation-of-pyruvate-dehydrogenase-in-rat-heart-mechanism-kb7ovdodld.pdf

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The mammalian pyruvate dehydrogenase complex: structure and regulation.

This article focuses on the functional significance of liver parenchymal cell heterogeneity in nitrogen metabolism

/pdf/nitrogen-metabolism-in-liver-structural-and-functional-1xfd6i9myy.pdf

Nitrogen metabolism in liver: structural and functional organization and physiological relevance.

1. The metabolism of glutamine and ammonia was studied in isolated perfused rat liver in relation to its dependence on the direction of perfusion by comparing the physiological antegrade (portal to caval vein) to the retrograde direction (caval to portal vein). 2. Added ammonium ions are mainly converted to urea in antegrade and to glutamine in retrograde perfusions. In the absence of added ammonia, endogenously arising ammonium ions are converted to glutamine in antegrade, but are washed out in retrograde perfusions. When glutamine synthetase is inhibited by methionine sulfoximine, direction of perfusion has no effect on urea synthesis from added or endogenous ammonia. 3. 14CO2 production from [1-14C]glutamine is higher in antegrade than in retrograde perfusions as a consequence of label dilution during retrograde perfusions. 4. The results are explained by substrate and enzyme activity gradients along the liver lobule under conditions of limiting ammonia supply for glutamine and urea synthesis, and they are consistent with a perivenous localization of glutamine synthetase and a predominantly periportal localization of glutaminase and urea synthesis. Further, the data indicate a predominantly periportal localization of endogenous ammonia production. The results provide a basis for an intercellular (as opposed to intracellular) glutamine cycling and its role under different metabolic conditions.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07458.x

Hepatocyte heterogeneity in glutamine and ammonia metabolism and the role of an intercellular glutamine cycle during ureogenesis in perfused rat liver.

1
Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions.

2
Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3–4 μmol × min−1× gram liver−1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per μmol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity.

3
GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 μmol x min−1× gram liver−1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole.

4
A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed.

5
Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation.

6
Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts.

7
A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1975.tb02325.x

Hydroperoxide-Metabolizing Systems in Rat Liver

1
Ammonium chloride addition to perfused rat liver results in a shift of the steady state of the pyruvate dehydrogenase system towards the active (dephospho) form This is evidenced by direct measurement of enzyme activity in freeze-stopped liver tissue, by net uptake of pyruvate from the perfusate, and also by an increased release of labeled CO2 from [1-14C]pyruvate Half-maximal concentration of NH4Cl for extra pyruvate uptake is 05–07 mM The effect is readily reversible

2
Extra urea formation upon ammonia addition is matched by an extra O2 uptake as that required theoretically for the two ATP-consuming steps of urea synthesis At 15 mM NH4Cl, there is no significant difference of ATP levels compared to the controls

3
When extra urea formation and the concomitant O2 uptake are suppressed due to limitation at carbamoylphosphate synthesis in carbon-dioxide-free media, the activation of pyruvate dehydrogenase by NH4Cl is still observed

4
When ammonia is generated intracellularly from glutamine, an extra uptake of pyruvate is not observed

5
The NH4Cl-induced activation of pyruvate dehydrogenase is increased in presence of acetoacetate, and decreased in presence of 3-hydroxybutyrate

6
Possible mechanisms are discussed While some of the observations could also be explained by a direct interaction of ammonium ions with the pyruvate dehydrogenase interconversion system, the effect of ammonium ions on pyruvate dehydrogenase is interpreted to be mediated in an indirect manner by changes of the mitochondrial concentration of 2-oxoglutarate As 2-oxoglutarate decreases during reductive amination to glutamate, a pool of high-energy compounds (eg GTP or related substances) dependent upon 2-oxoglutarate oxidation is depleted, resulting in a de-inhibition of pyruvate dehydrogenase by the interconversion system The detailed mechanism of the linkage between 2-oxoglutarate oxidation and pyruvate oxidation remains to be elucidated

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1975.tb04010.x

Ludwig Weiss

Papers

Activation of Pyruvate Dehydrogenase during Metabolism of Ammonium Ions in Hemoglobin‐Free Perfused Rat Liver