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.     

Characterization of oxalate as a catabolite of dichloroacetate responsible for the inhibition of gluconeogenesis and pyruvate carboxylation in rat liver cells

In isolated hepatocytes, dichloroacetate decreased glucose synthesis from lactate, pyruvate and alanine, but not from substrates which bypass pyruvate carboxylase (propionate, glycerol). It was also found to inhibit pyruvate carboxylation in isolated mitochondria, but only after a preincubation period, and had no effect on partially purified pyruvate carboxylase. Hepatocytes and liver mitochondria metabolized [¹⁴C] dichloroacetate to oxalate which inhibits pyruvate carboxylase and mimics, without preincubation, the effects of dichloroacetate in mitochondrial pyruvate carboxylation. Thus, oxalate appears to be responsible for the inhibition of gluconeogenesis by dichloroacetate at the level of pyruvate carboxylation.     

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.     

Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids

1. Monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, 2-chloropropionate, 2,2'-dichloropropionate and 3-chloropropionate were inhibitors of pig heart pyruvate dehydrogenase kinase. Dichloroacetate was also shown to inhibit rat heart pyruvate dehydrogenase kinase. The inhibition was mainly non-competitive with respect to ATP. The concentration required for 50% inhibition was approx. 100mum for the three chloroacetates, difluoroacetate and 2-chloropropionate and 2,2'-dichloropropionate. Dichloroacetamide was not inhibitory. 2. Dichloroacetate had no significant effect on the activity of pyruvate dehydrogenase phosphate phosphatase when this was maximally activated by Ca(2+) and Mg(2+). 3. Dichloroacetate did not increase the catalytic activity of purified pig heart pyruvate dehydrogenase. 4. Dichloroacetate, difluoroacetate, 2-chloropropionate and 2,2'-dichloropropionate increased the proportion of the active (dephosphorylated) form of pyruvate dehydrogenase in rat heart mitochondria with 2-oxoglutarate and malate as respiratory substrates. Similar effects of dichloroacetate were shown with kidney and fat-cell mitochondria. Glyoxylate, monochloroacetate and dichloroacetamide were inactive. 5. Dichloroacetate increased the proportion of active pyruvate dehydrogenase in the perfused rat heart, isolated rat diaphragm and rat epididymal fat-pads. Difluoroacetate and dichloroacetamide were also active in the perfused heart, but glyoxylate, monochloroacetate and trichloroacetate were inactive. 6. Injection of dichloroacetate into rats starved overnight led within 60 min to activation of pyruvate dehydrogenase in extracts from heart, psoas muscle, adipose tissue, kidney and liver. The blood concentration of lactate fell within 15 min to reach a minimum after 60 min. The blood concentration of glucose fell after 90 min and reached a minimum after 120 min. There was no significant change in plasma glycerol concentration. 7. In epididymal fatpads dichloroacetate inhibited incorporation of (14)C from [U-(14)C]glucose, [U-(14)C]fructose and from [U-(14)C]lactate into CO(2) and glyceride fatty acid. 8. It is concluded that the inhibition of pyruvate dehydrogenase kinase by dichloroacetate may account for the activation of pyruvate dehydrogenase and pyruvate oxidation which it induces in isolated rat heart and diaphragm muscles, subject to certain assumptions as to the distribution of dichloroacetate across the plasma membrane and the mitochondrial membrane. 9. It is suggested that activation of pyruvate dehydrogenase by dichloroacetate could contribute to its hypoglycaemic effect by interruption of the Cori and alanine cycles. 10. It is suggested that the inhibitory effect of dichloroacetate on fatty acid synthesis in adipose tissue may involve an additional effect or effects of the compound.     

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.     

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

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.     

Metabolic interactions of dichloroacetate and insulin in experimental diabetic ketoacidosis.

1. The infusion of sodium dichloroacetate into rats with severe diabetic ketoacidosis over 4h caused a 2mM decrease in blood glucose, and small falls in blood lactate and pyruvate concentrations. Similar findings had been reported in normal rats (Blackshear et al., 1974). In contrast there was a marked decrease in blood ketone-body concentration in the diabetic ketoacidotic rats after dichloroacetate treatment. 2. The infusion of insulin alone rapidly decreased blood glucose and ketone bodies, but caused an increase in blood lactate and pyruvate. 3. Dichloroacetate did not affect the response to insulin of blood glucose and ketone bodies, but abolished the increase of lactate and pyruvate seen after insulin infusion. 4. Neither insulin nor dichloroacetate stimulated glucose disappearance after functional hepatectomy, but both agents decreased the accumulation in blood of lactate, pyruvate and alanine. 5. Dichloroacetate inhibited 3-hydroxybutyrate uptake by the extra-splachnic tissues; insulin reversed this effect. Ketone-body production must have decreased, as hepatic ketone-body content was unchanged by dicholoracetate yet blood concentrations decreased. 6. It was concluded that: (a) dichloroacetate had qualitatively similar effects on glucose metabolism in severely ketotic rats to those observed in non-diabetic starved animals; (b) insulin and dichloroacetate both separately and together, decreased the net release of lactate, pyruvate and alanine from the extra-splachnic tissues, possibly through a similar mechanism; (c) insulin reversed the inhibition of 3-hydroxybutyrate uptake caused by dichloroacetate; (d) dichloroacetate inhibited ketone-body production in severe ketoacidosis.     

Stimulation of flux through hepatic pyruvate dehydrogenase by 3-mercaptopicolinate

Isolated hepatocytes from 24-h-starved rats were used to assess the possible effect of Ahe hypoglycaemic agent 3-mercaptopicolinate on flux through the hepatic pyruvate dehydrogenase complex. Increasing the extraceIIular pyruvate concentration from 1 mM to 2 mM or 5 mM resulted in an increase in flux through pyruvate dehydrogenase and the tricarboxylic acid cycle as measured by¹⁴CO₂ evolution from [1-¹⁴C]pyruvate and [3-¹⁴C]pyruvate. Gluconeogenesis was inhibited by 3-mercaptopicolinate from both 1 mM and 2 mM pyruvate, but significant increases in malate and citrate concentrations only occurred in cells incubated with 1 mM pyruvate. Flux through pyruvate dehydrogenase was stimulated by 3-mercaptopicolinate with 1 mM pyruvate but was unaltered with 2 mM pyruvate. Dichloroacetate stimulated flux through pyruvate dehydrogenase with no effect on gluconeogenesis in the presence of I mM pyruvate. There was no effect of 3-mercaptopicolinate, administered in vivo, to 24-h-starved rats on the activity of pyruvate dehydrogenase in freeze-clamped heart or liver tissue, although the drug did decrease blood glucose concentration and increase the blood concentrations of lactate and alanine. Dichloroacetate, administered in vivo to 24-h-starved rats, increased the activity of pyruvate dehydrogenase in freeze-clamped heart and liver, and caused decreases in the blood concentrations of glucose, lactate , and alanine. The results suggest that 3-mercaptopicolinate increases flux through hepatocyte pyruvate dehydrogenase by an indirect mechanism.     

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.     

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.     

The metabolic effects of sodium dichloroacetate in the starved rat

1. Sodium dichloroacetate (300mg/kg body wt. per h) was infused in 24h-starved rats for 4h. 2. Blood glucose decreased significantly, an effect that had previously only been noted in diabetic animals 3. Plasma insulin concentration decreased by 63%; blood lactate and pyruvate concentrations decreased by 50 and 33%, whereas concentrations of 3-hydroxybutyrate and acetoacetate increased by 81 and 73% respectively. 4. Livers were freeze-clamped at the end of the 4h infusion. There were significant decreases in hepatic [glucose], [glucose 6-phosphate], [2-phosphoglycerate], the [lactate]/[pyruvate] ratio, [citrate] and [malate], and also [alanine], [glutamate] and [glutamine], suggesting a diminished supply of gluconeogenic substrates. 5. Animals subjected to a functional hepatectomy at the end of 2h infusions showed no difference in blood-glucose disappearance but a highly significant decrease in the rate of accumulation of lactate, pyruvate, glycerol and alanine, compared with control animals. Dichloroacetate decreased ketone-body clearance. 6. After functional hepatectomy an increase in glutamine accumulation appeared to compensate for the decrease in alanine accumulation. 7. It is concluded that dichloroacetate causes hypoglycaemia by decreasing the net release of gluconeogenic precursors from extrahepatic tissues while inhibiting peripheral ketone-body uptake. 8. These findings are consistent with the activation of pyruvate dehydrogenase (EC 1.2.4.1) in rat muscle by dichloroacetate previously described by Whitehouse & Randle (1973).     

Effect of dichloroacetate on lactate concentration in exercising humans

The precise mechanism responsible for the increase in plasma lactate concentration during exercise in humans is not known. We have used dichloroacetate to test the hypothesis that a limitation in pyruvate dehydrogenase activity is responsible for the rise in plasma lactate. Dichloroacetate stimulates the activity of pyruvate dehydrogenase, which is normally the regulatory enzyme in the oxidation of glucose when tissue oxygenation is adequate. Six subjects were studied twice according to a randomized, crossover protocol, involving one test with saline infusion and another with dichloroacetate infusion. Exercise load on a bicycle ergometer was increased progressively until exhaustion. Blood samples were drawn each minute throughout exercise and periodically throughout 120 min of recovery. Dichloroacetate significantly lowered the lactate concentration during exercise performed at less than 80% of the average maximal O2 consumption. The peak concentration of lactate at exhaustion was not affected by dichloroacetate treatment, but dichloroacetate did lower lactate concentration throughout recovery. These results suggest that a limitation in pyruvate dehydrogenase activity contributes to the increase in plasma lactate during submaximal exercise and recovery.     

Branched-chain amino acid metabolism and alanine formation in rat diaphragm muscle in vitro. Effects of dichloroacetate.

Dichloroacetate (which activates pyruvate dehydrogenase) decreases the release of alanine, pyruvate and lactate in hemidiaphragm incubations with valine. Dichloroacetate interferes with alanine formation by diverting pyruvate into oxidative pathways, which not only limits pyruvate availability for direct transamination to form alanine but also indirectly affects branched-chain amino acid transamination by limiting 2-oxoglutarate regeneration from glutamate.     

Inhibition of hepatic gluconeogenesis by dichloroacetate

Gluconeogenesis from lactate by isolated hepatocytes suspended in a low bicarbonate medium is effectively inhibited by the hypoglycemic agent dichloroacetate. With this medium dichloroacetate suppresses the accumulation of the components of the malateaspartate shuttle, limits mitochondrial utilization of cytoplasmic reducing equivalents, and makes the availability of pyruvate and/or oxaloacetate limiting for gluconeogenesis. Much less inhibition is observed with hepatocytes suspended in a medium (Krebs?Henseleit saline) containing physiological concentrations of bicarbonate. No inhibition is observed with Krebs-Henseleit saline supplemented with lysine as a source of amino groups for the malate-aspartate shuttle. Thus, dichloroacetate inhibition of gluconeogenesis is observed only when hepatocytes are incubated in a medium deficient in bicarbonate and amino acids. This means that the action of dichloroacetate as a hypoglycemi agent is best explained by stimulation of peripheral tissue utilization of glucose and potential precursors for hepati gluconeogenesis rather than by direct inhibition of hepatic gluconeogenesis.     

3-Aminopicolinate inhibits phosphoenolpyruvate carboxykinase in hepatocytes and increases release of gluconeogenic precursors from peripheral tissues

3- Aminopicolinate , a hyperglycemic agent that activates purified phosphoenolpyruvate carboxykinase in the presence of Fe2+, inhibits glucose synthesis from lactate, pyruvate, asparagine, monomethyl succinate, or glutamine but does not affect that from fructose, dihydroxyacetone, sorbitol, or glycerol in hepatocytes isolated from rats fasted for 24 h. Lactate production from monomethyl succinate by hepatocytes is also inhibited by 3- aminopicolinate . This compound elevates the concentrations of pyruvate, malate, and aspartate but decreases that of phosphoenolpyruvate in hepatocytes incubated with lactate plus pyruvate. In rats, the ability of 3- aminopicolinate to elevate blood glucose concentration is unimpaired by renalectomy . The drug does not significantly affect glycemia in functionally hepatectomized rats but accelerates blood lactate and pyruvate accumulation to higher maximum concentrations even when kidney function is also ablated. It is concluded that 3- aminopicolinate inhibits phosphoenolpyruvate carboxykinase in hepatocytes, that the reported stimulation of renal glutaminase and glutamine gluconeogenesis by this compound does not contribute significantly to its hyperglycemic property, and that the drug increases gluconeogenic substrate supply from peripheral tissues.     

The effect of oxalate on gluconeogenesis by isolated chicken hepatocytes. Increased sensitivity to inhibition as a result of biotin deficiency

Addition of varying concentrations of oxalate to isolated chicken hepatocytes reduced gluconeogenesis from lactate in a manner indicating that pyruvate carboxylase was not the rate-limiting step. With hepatocytes from biotin-deficient chicks, sensitivity to inhibition was increased, and was consistent with pyruvate carboxylase being rate-limiting. Administration of biotin to deficient chicks overnight restores sensitivity to oxalate to normal.     

The effect of oxalate on gluconeogenesis by isolated chicken hepatocytes: Increased sensitivity to inhibition as a result of biotin deficiency

Addition of varying concentrations of oxalate to isolated chicken hepatocytes reduced gluconeogenesis from lactate in a manner indicating that pyruvate carboxylase was not the rate-limiting step. With hepatocytes from biotin-deficient chicks, sensitivity to inhibition was increased, and was consistent with pyruvate carboxylase being rate-limiting. Administration of biotin to deficient chicks overnight restores sensitivity to oxalate to normal.     

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO2 from [1-14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2-14C]pyruvate to 14CO2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H2O into fatty acid, and an increased (134%) accumulation of ketone bodies (acetoacetate + D-3-hydroxybutyrate). Dichloroacetate did not affect ketone body formation from [14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.     

Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-hydroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo

1. The extractions of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo were calculated from measurements of their arterial and coronary sinus blood concentration. Elevation of plasma free fatty acid concentrations by infusion of intralipid and heparin resulted in increased extraction of free fatty acids and diminished extractions of glucose, lactate and pyruvate by the heart. It is suggested that metabolism of free fatty acids by the heart in vivo, as in vitro, may impair utilization of these substrates. These effects of elevated plasma free fatty acid concentrations on extractions by the heart in vivo were reversed by injection of dichloroacetate, which also improved extraction of lactate and pyruvate by the heart in vivo in alloxan diabetes. 2. Sodium dichloroacetate increased glucose oxidation and pyruvate oxidation in hearts from fed normal or alloxan-diabetic rats perfused with glucose and insulin. Dichloroacetate inhibited oxidation of acetate and 3-hydroxybutyrate and partially reversed inhibitory effects of these substrates on the oxidation of glucose. In rat diaphragm muscle dichloroacetate inhibited oxidation of acetate, 3-hydroxybutyrate and palmitate and increased glucose oxidation and pyruvate oxidation in diaphragms from alloxan-diabetic rats. Dichloroacetate increased the rate of glycolysis in hearts perfused with glucose, insulin and acetate and evidence is given that this results from a lowering of the citrate concentration within the cell, with a consequent activation of phosphofructokinase. 3. In hearts from normal rats perfused with glucose and insulin, dichloroacetate increased cell concentrations of acetyl-CoA, acetylcarnitine and glutamate and lowered those of aspartate and malate. In perfusions with glucose, insulin and acetate, dichloroacetate lowered the cell citrate concentration without lowering the acetyl-CoA or acetylcarnitine concentrations. Measurements of specific radioactivities of acetyl-CoA, acetylcarnitine and citrate in perfusions with [1-(14)C]acetate indicated that dichloroacetate lowered the specific radio-activity of these substrates in the perfused heart. Evidence is given that dichloroacetate may not be metabolized by the heart to dichloroacetyl-CoA or dichloroacetylcarnitine or citrate or CO(2). 4. We suggest that dichloroacetate may activate pyruvate dehydrogenase, thus increasing the oxidation of pyruvate to acetyl-CoA and acetylcarnitine and the conversion of acetyl-CoA into glutamate, with consumption of aspartate and malate. Possible mechanisms for the changes in cell citrate concentration and for inhibitory effects of dichloroacetate on the oxidation of acetate, 3-hydroxybutyrate and palmitate are discussed.