Mechanism responsible for the hypoglycemic actions of dichloroacetate and 2-chloropropionate

Dichloroacetate, an activator of the pyruvate dehydrogenase complex, is known to lower blood glucose, lactate, pyruvate, and alanine when given to diabetic and 24 h fasted rats. Under certain conditions, especially when pyruvate carboxylase is made rate limiting for want of bicarbonate, dichloroacetate effectively inhibits glucose synthesis from lactate by isolated hepatocytes. 2-Chloropropionate also activates the pyruvate dehydrogenase complex, lowers blood glucose, lactate, and pyruvate in 24 h fasted rats, but stimulates gluconeogenesis from lactate or alanine by isolated hepatocytes. Dichloroacetate is catabolized to glyoxylate and thence to oxalate by liver cells, whereas 2-chloropropionate cannot be catabolized to these products. Glyoxylate and oxalate are potent inhibitors of glucose synthesis from lactate, pyruvate, and alanine, but not from dihydroxyacetone. Inhibition is much more pronounced in a bicarbonate-deficient medium, in which pyruvate carboxylase is probably rate limiting for gluconeogenesis. It seems likely, therefore, that the inhibition of lactate gluconeogenesis by dichloroacetate is actually caused by oxalate, which inhibits pyruvate carboxylation. Nevertheless, the major effect of dichloroacetate, and probably the sole effect of 2-chloropropionate, on blood glucose concentration is to limit substrate availability in the blood for hepatic gluconeogenesis. Since oxalic acid stone formation and renal dysfunction may prove to be side effects of any therapeutic application of dichloroacetate, we suggest that further studies on the treatment of hyperglycemia and lactic acidosis with pyruvate dehydrogenase activators be carried out with 2-chloropropionate rather than dichloroacetate.     

Cloning of yeast glycolysis genes by complementation

In hepatocytes isolated from fed rats, low concentrations of oxalate (50 to 100 μM) greatly enhance ketogenesis and decrease fatty acid synthesis. These metabolic changes are due to pyruvate carboxylase inhibition. Dichloroacetate, which can be catabolized into oxalate enhances ketogenesis only when cells are enriched with lactate and pyruvate and has no obvious effect on lipogenesis. The enhancement of ketogenesis, in both cases, is due to an imbalance between pyruvate dehydrogenase and pyruvate carboxylase but with oxalate, the primary event is oxaloacetate shortage and with dichloroacetate, mitochondrial acetyl CoA excess. This work demonstrates that the studied effects of dichloroacetate are not mediated by oxalate and that low concentrations of oxalate alter the lipid metabolism of hepatocytes.     

Effects of dichloroacetate and 2-chloropropionate on carbohydrate metabolism of isolated hepatocytes. Therapeutic applications

In isolated hepatocytes, dichloroacetate directly activates pyruvate dehydrogenase whereas its biotransformation product, oxalate, inhibits pyruvate carboxylase and pyruvate kinase. Dichloroacetate, which decreases blood lactate very efficiently, has been sucessfully tested in the acute treatment of congenital lactic acidosis, but its transformation into oxalate and potential chronic toxicity prompt to replace it by 2-chloropropionate as a therapeutic agent.     

Studies on the inhibition of gluconeogenesis by oxalate

Oxalate was shown to enter isolated rat hepatocytes and to inhibit gluconeogenesis from lactate, pyruvate, and alanine, but not from glutamine, proline, propionate or dihydroxyacetone. Oxalate apparently acts by inhibiting pyruvate carboxylase (EC 6.4.1.1). It is known to inhibit the isolated enzyme, and inhibition of gluconeogenesis was much greater in a bicarbonate-deficient medium where pyruvate carboxylase activity limits the overall rate of the pathway. A slight inhibition of gluconeogenesis from asparagine was observed, suggesting that oxalate may also inhibit gluconeogenesis at another site. Chelation of extracellular Ca²⁺ does not contribute to the inhibition of gluconeogenesis. Compared to oxalate, other Ca²⁺ chelators have little effect upon gluconeogenesis. Also, oxalate inhibits gluconeogenesis effectively both in low Ca²⁺ medium and in medium containing 2.6 mM Ca²⁺. Chelation of intracellular Ca²⁺ also appears to be of little importance, since oxalate does not block the glycogenolytic effects of epinephrine, vasopressin, and angiotensin which are thought to act via Ca²⁺ as the second messenger. The inhibition of gluconeogenesis could conceivably contribute to the toxic actions of oxalate and to the hypoglycemic action of dichloroacetate, a compound that is metabolized to oxalate. However, oxalate did not cause hypoglycemia in the suckling rat, a model in vivo system very dependent upon gluconeogenesis for maintenance of normal blood glucose levels. Thus, inhibition of gluconeogenesis is probably of little importance in oxalate toxicity and the hypoglycemic effects of dichloroacetate.     

Intracellular localization of pyruvate carboxylase in mammalian liver

We propose that an adequate amount of extramitochondrial (soluble) pyruvate carboxylase exists in mammalian liver. It has been previously accepted that pyruvate carboxylase is localized in the mitochondria-containing glutamate dehydrogenase. The overall activity and distribution of pyruvate carboxylase and of phosphoenol-pyruvate carboxykinase in mammalian liver has been studied using an improved technique for the fractional extraction of isolated mitochondria. We found about 40% of the total pyruvate carboxylase and about 60 % of the total PEP-carboxykinase in the soluble fraction. Glutamate dehydrogenase was considered to be the ‘marker enzyme’ for mitochondria. Our results strongly support the view that in murine, porcine, bovine and chicken liver, the pyruvate involved in gluconeogenesis is not required to enter the mitochondria prior to its carboxylation to oxalacetate, because extramitochondrial carboxylation of pyruvate through the ‘soluble pyruvate carboxylase’ is possible.     

Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria

1. The fixation of CO(2) by pyruvate carboxylase in isolated rat brain mitochondria was investigated. 2. In the presence of pyruvate, ATP, inorganic phosphate and magnesium, rat brain mitochondria fixed H(14)CO(3) (-) into tricarboxylic acid-cycle intermediates at a rate of about 250nmol/30min per mg of protein. 3. Citrate and malate were the main radioactive products with citrate containing most of the radioactivity fixed. The observed rates of H(14)CO(3) (-) fixation and citrate formation correlated with the measured activities of pyruvate carboxylase and citrate synthase in the mitochondria. 4. The carboxylation of pyruvate by the mitochondria had an apparent K(m) for pyruvate of about 0.5mm. 5. Pyruvate carboxylation was inhibited by ADP and dinitrophenol. 6. Malate, succinate, fumarate and oxaloacetate inhibited the carboxylation of pyruvate whereas glutamate stimulated it. 7. The results suggest that the metabolism of pyruvate via pyruvate carboxylase in brain mitochondria is regulated, in part, by the intramitochondrial concentrations of pyruvate, oxaloacetate and the ATP:ADP ratio.     

Properties of reconstituted cholesterol 7 alpha-hydroxylase system from rat and rabbit liver microsomes.

1. Pyruvate carboxylase is present in brown adipose tissue mitochondria. 2. In isolated mitochondria, pyruvate, bicarbonate and ATP, the substrates for pyruvate carboxylase, are able to replace added malate in supplying a condensing partner for acetyl-CoA formed from beta-oxidation of fatty acids. 3. In brown adipocytes, pyruvate and CO2 increase the rate of norepinephrine-stimulated respiration synergistically. 4. The norepinephrine-stimulated respiration in brown adipocytes is diminished when pyruvate transport into the mitochondria is inhibited. 5. Pyruvate carboxylation increases the intramitochondrial level of citric acid cycle intermediates, as shown by titrations of malonate inhibition of respiration. 6. Pyruvate carboxylation can continuously supply the mitochondria with citric acid cycle intermediates, as evidenced by its ability to maintain respiration when oxoglutarate conversion to glutamate is stimulated. 7. Pyruvate carboxylation is necessary for maximal oxygen consumption even when drainage of the citric acid cycle for amino acid synthesis is eliminated. 8. Pyruvate carboxylation explains observed effects of CO2 on respiration in brown adipocytes, and may also explain the increased glucose uptake by brown adipose tissue during thermogenesis in vivo.     

Regulation of mitochondrial pyruvate carboxylation in isolated hepatocytes by acute insulin treatment.

The effect of acute insulin treatment of hepatocytes on pyruvate carboxylation in both isolated mitochondria and cells rendered permeable by filipin was examined. Challenging the cells with insulin alone had no effect on either the basal rate of pyruvate carboxylation or gluconeogenesis, although it did suppress the responses to both glucagon and catecholamines. Insulin treatment was unable to antagonize the enhanced rate of pyruvate carboxylation caused by stimulation of the cells with either angiotensin or vasopressin. Neither insulin nor the gluconeogenic hormones altered the total extractable pyruvate carboxylase activity in the isolated mitochondria, suggesting that the effect of hormones at the level of the isolated intact organelle was mediated via alterations in the intramitochondrial concentrations of effector molecules, notably ATP and the [ATP]/[ADP] ratio and substrate availability. The alterations in pyruvate carboxylation correlate well with glucose synthesis in terms of sensitivity to effector molecules, putative second messengers and time of onset of the response, indicating that alterations in the flux through this enzyme are compatible with it being an important site in the control of gluconeogenesis from C3 precursors.     

THE RELATIVE SIGNIFICANCE OF CO2-FIXING ENZYMES IN THE METABOLISM OF RAT BRAIN

To evaluate the relative significance of CO₂-fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase, as well as the fixation of H¹⁴CO₃^? by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol-pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H¹⁴CO₃^? at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H¹⁴CO₃^? in the presence of α-ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H¹⁴CO₃^? at a rate of about 7 nmol/g of tissue/min and it was increased by two-fold when pyruvate was added to the incubation medium. The carboxylation of α-ketoglutarate and pyruvate by the reversal of the cytosolic NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO₂-fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α-ketoglutarate by a sequential action of aconitate hydratase and NADP-isocitrate dehydrogenase. The operation of the citrate-cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO₂-fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO₂-fixation in the brain.     

Metabolism of pyruvate by isolated rat mesenteric lymphocytes, lymphocyte mitochondria and isolated mouse macrophages.

1. The activities of pyruvate dehydrogenase in rat lymphocytes and mouse macrophages are much lower than those of the key enzymes of glycolysis and glutaminolysis. However, the rates of utilization of pyruvate (at 2 mM), from the incubation medium, are not markedly lower than the rate of utilization of glucose by incubated lymphocytes or that of glutamine by incubated macrophages. This suggests that the low rate of oxidation of pyruvate produced from either glucose or glutamine in these cells is due to the high capacity of lactate dehydrogenase, which competes with pyruvate dehydrogenase for pyruvate. 2. Incubation of either macrophages or lymphocytes with dichloroacetate had no effect on the activity of subsequently isolated pyruvate dehydrogenase; incubation of mitochondria isolated from lymphocytes with dichloroacetate had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, and the double-reciprocal plot of [1-14C]pyruvate concentration against rate of 14CO2 production was linear. In contrast, ADP or an uncoupling agent increased the rate of 14CO2 production from [1-14C]pyruvate by isolated lymphocyte mitochondria. These data suggest either that pyruvate dehydrogenase is primarily in the a form or that pyruvate dehydrogenase in these cells is not controlled by an interconversion cycle, but by end-product inhibition by NADH and/or acetyl-CoA. 3. The rate of conversion of [3-14C]pyruvate into CO2 was about 15% of that from [1-14C]pyruvate in isolated lymphocytes, but was only 1% in isolated lymphocyte mitochondria. The inhibitor of mitochondrial pyruvate transport, alpha-cyano-4-hydroxycinnamate, inhibited both [1-14C]- and [3-14C]-pyruvate conversion into 14CO2 to the same extent, and by more than 80%. 4. Incubations of rat lymphocytes with concanavalin A had no effect on the rate of conversion of [1-14C]pyruvate into 14CO2, but increased the rate of conversion of [3-14C]pyruvate into 14CO2 by about 50%. This suggests that this mitogen causes a stimulation of the activity of pyruvate carboxylase.     

Influence of 1,2,3-benzene-tricarboxylate on pyruvate metabolism in rat-liver mitochondria.

1,2,3-Benzene-tricarboxylate, a known inhibitor of the mitochondrial tricarboxylate carrier, was found to inhibit pyruvate carboxylation as well as the transport of citrate out of the matrix in rat liver mitochondria incubated with pyruvate. The inhibition of pyruvate carboxylation was observed with both intact mitochondria and with the solubilized pyruvate carboxylase. The inhibition of the pyruvate carboxylase by 1,2,3-benzene-tricarboxylase was not mediated via one of the parameters known to regulate the activity of the enzyme and therefore a direct inhibition of the enzyme by the tricarboxylate was assumed. Since the pyruvate carboxylase is exclusively localized in the mitochondrial matrix space it was concluded that 1,2,3-benzene-tricarboxylate penetrates into this compartment.     

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.     

Inhibition of lactate glucogneogenesis in rat kidney by dichloroacetate.

1. Sodium dichloroacetate (1mM) inhibited glucose production from L-lactate in kidney-cortex slices from fed, starved or alloxan-diabetic rates. In general gluconeogenesis from other substrates was no inhibited. 2. Sodium dichloracetate inhibited glucose production from L-lactate but no from pyruvate in perfused isolated kidneys from normal or alloxan-diabetic rats. 3. Sodium dichloroacetate is an inhibitor of the pyruvate dehydrogenase kinase reaction and it effected conversion of pyruvate dehydrogenase into its its active (dephosphorylated) form in kidney in vivo. In general, pyruvate dehydrogenase was mainly in the active form in kidneys perfused or incubated with L-lactate and the inhibitory effect of dichloroacetate on glucose production was not dependent on activation of pyruvate dehydrogenase. 4. Balance data from kidney slices showed that dichloroacetate inhibits lactate uptake, glucose and pyruvate production from lactate, but no oxidation of lactate. 5. The mechanism of this effect of dichloroactetate on glucose production from lactate has not been fully defined, but evidence suggests that it may involve a fall in tissue pyruvate concentration and inhibition of pyruvate carboxylation.     

Glutathione biosynthesis in murine L5178Y lymphoma cells

The pyruvate dehydrogenase complex from pea leaf mitochondria was rapidly deactivated in the presence of 50 to 200 μm ATP. The deactivation of the complex requires Mg²⁺ as shown by EDTA inhibition of deactivation. Deactivation was inhibited by 0.1 to 1 mm pyruvate or dichloroacetate. Activation required 10 mM Mg²⁺ or Mn²⁺ but Ca²⁺ and K⁺ had no effect. Activation was inhibited by the phosphatase inhibitor, F^?. Autoradiograms of nondissociating electrophoresis gel, crossed immunoelectrophoresis gels, and dissociating sodium dodecyl sulfate electrophoresis gels of the complex showed that one protein is labeled. Labeling of this protein is prevented by Mg²⁺, pyruvate, and dichloroacetate. The pyruvate dehydrogenase complex was isolated in a partially deactivated state and reactivation required exogenous Mg²⁺ and was inhibited by F^?. These results are taken as conclusive evidence that the pyruvate dehydrogenase complex in pea leaf mitochondria undergoes interconversion between deactivated and activated states by covalent modification (phosphorylation-dephosphorylation) catalyzed by a kinase and phosphatase. Isolation of the complex in a partially deactivated (phosphorylated) state suggests a physiologically significant role for this regulatory mechanism.     

Hyperthermia impairs liver mitochondrial function in vitro

The effects of temperature on the relationships among the rates of pyruvate carboxylation, O(2) uptake (J(o)), oxidative phosphorylation (J(p)), and the free energy of ATP hydrolysis (G(p)) were studied in liver mitochondria isolated from 250-g female rats. Pyruvate carboxylation was evaluated at 37, 40, and 43 degrees C. In disrupted mitochondria, pyruvate carboxylase maximal reaction velocity increased from 37 to 43 degrees C with an apparent Q(10) of 2.25. A reduction in ATP/ADP ratio decreased enzyme activity at all three temperatures. In contrast, in intact mitochondria, increasing temperature failed to increase pyruvate carboxylation (malate + citrate accumulation) but did result in increased J(o) and decreased extramitochondrial G(p). J(p) was studied in respiring mitochondria at 37 and 43 degrees C at various fractions of state 3 respiration, elicited with a glucose + hexokinase ADP-regenerating system. The relationship between J(o) and G(p) was similar at both temperatures. However, hyperthermia (43 degrees C) reduced the J(p)/J(o) ratio, resulting in lower G(p) for a given J(p). Fluorescent measurements of membrane phospholipid polarization revealed a transition in membrane order between 40 and 43 degrees C, a finding consistent with increased membrane proton conductance. It is concluded that hyperthermia augments nonspecific proton leaking across the inner mitochondrial membrane, and the resultant degraded energy state offsets temperature stimulation of pyruvate carboxylase. As a consequence, at high temperatures approaching 43 degrees C, the pyruvate carboxylation rate of intact liver mitochondria may fail to exhibit a Q(10) effect.     

Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.     

The effects of pyruvate concentration, dichloroacetate and alpha-cyano-4-hydroxycinnamate on gluconeogenesis, ketogenesis and [3-hydroxybutyrate]/[3-oxobutyrate] ratios in isolated rat hepatocytes.

1. In isolated rat hepatocytes incubated with pyruvate, ketogenesis increased with increasing pyruvate concentrations and decreased under the influence of 1 mM-alpha-cyano-4-hydroxycinnamate, a known inhibitor of pyruvate transport. Ketogenesis from pyruvate was higher by 30% in hepatocytes prepared from starved than from fed rats. 2. With pyruvate as substrate, 2 mM-dichloroacetate had no effect on ketogenesis of starved-rat hepatocytes, but increased ketogenesis of fed-rat hepatocytes to the 'starved' value. Gluconeogenesis from pyruvate, lactate and alanine, but not from glycerol, was inhibited by dichloroacetate. Both increased ketogenesis and decreased gluconeogenesis may result from an inhibition of pyruvate carboxylase by dichloroacetate. 3. Mitochondria were rapidly isolated from incubated hepatocytes, and [3-hydroxybutyrate]/[3-oxobutyrate] ratios were measured in the mitochondrial pellet ('mitochondrial' ratios) and in whole-cell suspensions ('total' ratios). Increasing pyruvate concentrations increased mitochondrial and decreased total ratios. In the presence of pyruvate (2 to 10 mM), dichloroacetate decreased mitochondrial and increased total ratios.     

Mitochondrial pyruvate metabolism in liver and kidney during acidosis

Pyruvate transport and carboxylation have been determined in mitochondria from liver and kidney cortex isolated from Wistar rats with acidosis produced by three different treatments: fasting, exercise and ingestion of ammonium chloride. Fasting for 48 h or swimming for 2 h resulted in an increased rate of CO₂ fixation by mitochondria from both organs incubated with pyruvate. This increase was accompanied by a rise in the rate of pyruvate transport in all cases except in mitochondria derived from the kidney of the fasted animals. Acute acidosis produced by the ingestion of ammonium chloride resulted in increases in pyruvate transport and carboxylation in kidney mitochondria, but a drop in pyruvate carboxylation was observed in mitochondria from the liver. The results are discussed in terms of the differential regulation of the mitochondria steps for gluconeogenesis from three carbon precursors in liver and kidney, taking into consideration the hormonal status of the animals and the prevailing available substrates in each condition.     

Control of pyruvate carboxylase activity by the pyridine-nucleotide redox state in mitochondria from rat liver

Pyruvate carboxylation by isolated mitochondria from rat liver is inhibited by t-butylhydroperoxide in a fully reversible manner. The rate of malate formation at 10 mM pyruvate was decreased by some 80% by 30 microM t-butylhydroperoxide. The effective peroxide concentration was dependent on the mitochondrial hydrogen supply, being increased to about 120 microM in the presence of 50 microM palmitoylcarnitine. Regarding the mechanism(s) of the t-butylhydroperoxide action, pyruvate transport and intramitochondrial energy or activator supply are unlikely involved, because the effect also took place with alanine as the substrate and was not accompanied by a change in the intramitochondrial levels of adenine nucleotides and acetyl-CoA respectively. However, t-butylhydroperoxide caused a rapid fall in the 3-hydroxybutyrate/acetoacetate ratio and a marked increase in the oxidized glutathione content. Therefore, experiments were designed to disclose the participation of the respective redox couples in the expression of pyruvate carboxylase activity. From measurements of NADPH, NADH, oxidized and reduced glutathione contents of mitochondria incubated under a variety of conditions, evidence has been obtained indicating that the mitochondrial NADH supply represents an important factor in the regulation of pyruvate carboxylase activity. The results presented seemingly provide a new basis for the understanding of the functional relationship between beta-oxidation and pyruvate carboxylation.     

Likely individuality of the enzymes catalyzing the phosphorylation of choline and ethanolamine.

Dichloroacetate has effects upon hepatic metabolism which are profoundly different from its effects on heart, skeletal muscle, and adipose tissue metabolism. With hepatocytes prepared from meal-fed rats, dichloroacetate was found to activate pyruvate dehydrogenase, to increase the utilization of lactate and pyruvate without effecting an increase in the net utilization of glucose, to increase the rate of fatty acid synthesis, and to decrease slightly [1-¹⁴C]oleate oxidation to ¹⁴CO₂ without decreasing ketone body formation. With hepatocytes isolated from 48-h-starved rats, dichloroacetate was found to activate pyruvate dehydrogenase, to have no influence on net glucose utilization, to inhibit gluconeogenesis slightly with lactate as substrate, and to stimulate gluconeogenesis significantly with alanine as substrate. The stimulation of fatty acid synthesis by dichloroacetate suggests that the activity of pyruvate dehydrogenase can be rate determining for fatty acid synthesis in isolated liver cells. The minor effects of dichloroacetate on gluconeogenesis suggest that the regulation of pyruvate dehydrogenase is only of marginal importance in the control of gluconeogenesis.