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.     

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.     

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.     

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.     

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.     

Effect of lactation on gluconeogenesis and ketogenesis in ovine hepatocytes

1. Rates of glucose synthesis from radioactive precursors and ketogenesis were determined in hepatocytes from control and lactating sheep. 2. Gluconeogenesis from propionate was the same in both groups. Gluconeogenesis from lactate + pyruvate was three-fold higher in hepatocytes from lactating sheep. Palmitate stimulated gluconeogenesis from lactate + pyruvate in both groups. 3. Rates of ketogenesis from palmitate but not butyrate were slightly higher in hepatocytes from lactating sheep. No other differences in the metabolism of palmitate or butyrate were seen in the two groups. Exogenous carnitine stimulated ketogenesis from palmitate. Propionate inhibited ketogenesis from palmitate and butyrate. Lactate + pyruvate also inhibited ketogenesis slightly but stimulated oxidation and esterification. 4. It is concluded that the major changes in glucose and ketone production seen in the lactating ruminant are not the result of long-term changes within the hepatocyte but occur because of the changes in substrate supply to the liver and changes in intracellular concentrations of metabolites.     

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.     

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.     

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 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.     

Conversion of pyruvate into ketone bodies in rat hepatocyte suspensions.

The contribution of pyruvate to ketogenesis was determined in rat hepatocyte suspensions by using [14C]pyruvate. The rates of conversion of pyruvate into ketone bodies in hepatocytes from fed and 24 h-starved rats were 10 and 17 mumol/h per g wet wt. respectively, and accounted for 50 and 29% of the total ketone bodies formed. In hepatocytes from fed rats, the addition of palmitate (0.25-1 mM) increased the rate of conversion of pyruvate into ketone bodies (80-140%), but decreased the relative contribution of pyruvate to total ketogenesis. In hepatocytes from starved rats, palmitate did not increase pyruvate conversion into ketone bodies.     

Influence of oxalate on the rate of the tricarboxylic acid cycle in rat hepatocytes

In hepatocytes isolated from fed rats, physiological concentrations of oxalate lower the flux through the tricarboxylic acid cycle (-48%) and reduce the steady-state levels of oxaloacetate and other Krebs cycle intermediates. All the metabolic modifications observed are explained by pyruvate carboxylase inhibition, since oxalate hardly modifies the flux through pyruvate dehydrogenase.     

Regulation of ketogenesis during the suckling-weanling transition in the rat. Studies with isolated hepatocytes.

The rates of ketogenesis from endogenous substrates, butyrate or oleate, have been measured in isolated hepatocytes from suckling and weanling rats. Ketogenesis from endogenous substrate and from oleate decreased on weaning, whereas the rate from butyrate remained unchanged. It is concluded that the major site of regulation of ketogenesis during this period of development involves the disposal of long-chain fatty acyl-CoA between the esterification and beta-oxidation pathways. Modulators of lipogenesis [dihydroxyacetone and 5-(tetradecyloxy)-2-furoic acid] did not alter the rate of ketogenesis in hepatocytes from suckling rats, and it is suggested that this is due to the low rate of lipogenesis in these cells. Hepatocytes from fed weanling rats have a high rate of lipogenesis and evidence is presented for a reciprocal relationship between ketogenesis and lipogenesis, and ketogenesis, and esterification in these cells. Dibutyryl cyclic AMP stimulated ketogenesis from oleate in hepatocytes from fed weanling rats, even in the presence of an inhibitor of lipogenesis [5-(tetradecyloxy)-2-furoic acid], but not in cells from suckling rats. It is suggested that cyclic AMP may act via inhibition of esterification and that in hepatocytes from suckling rats ketogenesis is already maximally stimulated by the high basal concentrations of cyclic AMP [Beaudry, Chiasson & Exton (1977) Am. J. Physiol. 233, E175--E180].     

The effect of acetate on the regulation of the branched chain α-keto acid and the pyruvate dehydrogenase complexes in the perfused rat liver

The regulatory consequences of acetate infusion on the pyruvate and the branched chain α-keto acid dehydrogenase reactions in the isolated, perfused rat liver were investigated. Metabolic flux through these two decarboxylation reactions was monitored by measuring the rate of ¹⁴CO₂ production from infused 1-¹⁴C-labeled substrates. When acetate was presented to the liver as the sole substrate the rate of ketogenesis which resulted was maximal at concentrations of acetate in excess of 10 mm. The increase in hepatic ketogenesis during acetate infusion was not accompanied by an alteration of the mitochondrial oxidation-reduction state as measured by the ratio of β-hydroxybutyrate/ acetoacetate in the effluent perfusate. While acetate infusion did not affect the rate of α-keto[1-¹⁴C]isocaproate decarboxylation, the rate of α-keto[1-¹⁴C]isovalerate decarboxylation was stimulated appreciably upon acetate addition. No change was observed in the amount of extractable branched chain α-keto acid dehydrogenase during acetate infusion. The rate of [1-¹⁴C]pyruvate decarboxylation was stimulated in the presence of acetate at low (<1 mm) but not at high (>1 mm) perfusate pyruvate concentrations. The stimulation of the metabolic flux through the pyruvate dehydrogenase reaction upon acetate infusion was accompanied by an increase in the activation state of the pyruvate dehydrogenase complex from 25.7 to 35.6% in the active form. In a liver perfused in the presence of the pyruvate dehydrogenase kinase inhibitor, dichloroacetate, at a low concentration of pyruvate (0.05 mm) the infusion of acetate did not affect the rate of pyruvate decarboxylation. As the rate of mitochondrial acetoacetate efflux is increased during acetate infusion the stimulation of pyruvate and α-ketoisovalerate decarboxylation is attributed to an accelerated rate of exchange of mitochondrial acetoacetate for cytosolic pyruvate or α-ketoisovalerate on the monocarboxylate transporter.     

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.     

Studies on the regulation of leucine catabolism in the liver. Stimulation by pyruvate and dichloroacetate

The rate of oxidation of L-[1-14C]leucine to 14CO2 by isolated rat hepatocytes is increased by pyruvate and dichloroacetate. This effect is specific for L-leucine, not being observed for L-valine, L-isoleucine, or D-leucine. Transamination, the rate-limiting step of L-leucine catabolism in the liver, is the site of stimulation, because uptake of L-leucine by the cells and the oxidation of its transamination product, alpha-ketoisocaproate, are not increased. Measurement of steady state levels of alpha-ketoisocaproate indicate that both pyruvate and dichloroacetate promote the transamination of L-leucine, thereby increasing the availability of substrate for decarboxylation by the alpha-ketoisocaproate dehydrogenase complex (EC 1.2.4.3). Pyruvate stimulation of transamination is secondary to the provision of keto acid acceptors for the amino group of L-leucine. The mechanism of the effect of dichloroacetate remains unknown.     

Action of the antiepileptic drug, valproic acid, on fatty acid oxidation in isolated rat hepatocytes

Valproate at 0.1 to 5 mM strongly inhibited oxidation of 1-(14C)-palmitate in isolated rat hepatocytes. Valproate at the same concentrations markedly decreased ketogenesis from 1 mM oleate. Valproate in a dose up to 5 mM did not significantly affect cellular concentration of ATP but lowered beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios which paralleled its effect on ketogenesis. Moreover concomitant acetyl-CoA levels were drastically decreased by valproate. From this it may be concluded that inhibition of fatty acid oxidation by valproate results in reduced production of two carbons units and a drop of NADH/NAD+ ratio in rat hepatocyte. This suggests that valproate seriously interferes with beta-oxidation of physiological long-chain fatty acids.     

Regulation of ketogenesis, gluconeogenesis and the mitochondrial redox state by dexamethasone in hepatocyte monolayer cultures.

The effects of the glucocorticoid dexamethasone on fatty acid and pyruvate metabolism were studied in rat hepatocyte cultures. Parenchymal hepatocytes were cultured for 24 h with nanomolar concentrations of dexamethasone in either the absence or the presence of insulin (10 nM) or dibutyryl cyclic AMP (1 microM BcAMP). Dexamethasone (1-100 nM) increased the rate of formation of ketone bodies from 0.5 mM-palmitate in both the absence and the presence of BcAMP, but inhibited ketogenesis in the presence of insulin. Dexamethasone increased the proportion of the palmitate metabolized that was partitioned towards oxidation to ketone bodies, and decreased the cellular [glycerol 3-phosphate]. The latter suggests that the increased partitioning of palmitate to ketone bodies may be associated with decreased esterification to glycerolipid. The Vmax. of carnitine palmitoyltransferase (CPT) and the affinity of CPT for palmitoyl-CoA were not affected by dexamethasone, indicating that the increased ketogenesis was not due to an increase in enzymic capacity for long-chain acylcarnitine formation. Dexamethasone and BcAMP, separately and in combination, increased gluconeogenesis. In the presence of insulin, however, dexamethasone inhibited gluconeogenesis. Changes in gluconeogenesis thus paralleled changes in ketogenesis. Dexamethasone decreased the [3-hydroxybutyrate]/[acetoacetate] ratio, despite increasing the rate of ketogenesis and presumably the mitochondrial production of reducing equivalents. The more oxidized mitochondrial NADH/NAD+ redox couple with dexamethasone is probably due either to an increased rate of electron transport or to increased transfer of mitochondrial reducing equivalents to the cytoplasm.