ARCHIVES

OF

BIOCHEMISTRY

AND

173, 658-665 (1976)

BIOPHYSICS

Effect of Dichloroacetate

on Carbohydrate and Lipid Metabolism Isolated Hepatocytesl

of

DAVID W. CRABB,2 JAMES P. MAPES,3 RICHARD W. BOERSMA, AND ROBERT A. HARRIS4 Department

of Biochemistry,

Indiana

University

School of Medicine,

Received August

Indianapolis,

Indiana

46202

7, 1975

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 [l-‘%]oleate oxidation to “COZ without decreasing ketone body formation. With hepatocytes isolated from 4&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.

Dichloroacetate was reported in 1962 both to increase the respiratory quotient and to decrease the glycosuria and hyperglycemia in diabetic rats without affecting liver or muscle glycogen levels (1). Dichloroacetate has also been shown to increase glucose oxidation, to inhibit fatty acid oxidation in skeletal muscle of diabetic rats (2-4), and to increase glucose, pyruvate, and lactate extraction but to decrease fatty acid oxidation in the perfused dog heart (5). The hypoglycemic effect of dichloroace1 This work was supported by grants from the American Diabetes Association, American Heart Association (Indiana Affiliate), and the Showalter Residuary Trust. 2 This work was conducted during the tenure of a Public Health Service fellowship for medical students. s This work was conducted during the tenures of National Defense Education Act (Title IV and Title IXB) fellowships. Present address: Metabolic Research Laboratory, Nuffleld Department of Clinical Medicine, Radcliffe Infirmary, Oxford, OX2 6HE, England. 4 To whom requests for reprints should be sent.

tate can be explained partially by improved extrahepatic lactate and pyruvate oxidation, which impairs hepatic gluconeogenesis by interruption of the Cori and alanine cycles (6). Dichloroacetate appears to exert its effects by inhibiting pyruvate dehydrogenase kinase (5,7,8). This action allows pyruvate dehydrogenase phosphatase to convert the inactive, phosphorylated form of pyruvate dehydrogenase (EC 1.2.4.1) to the active, dephosphorylated form. In this study we examined the effects of dichloroacetate on liver metabolism with isolated hepatocytes. MATERIALS

658 Copyright 8 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND

METHODS

Preparation of liver cells. Isolated hepatocytes were prepared from rat liver by the method of Berry and Friend (9) with the modifications described previously (10). Male Wistar rats weighing about 200 g and either meal-fed 7 days on a high carbohydrate (“lipogenic”) diet (10) or starved for 48 h were used. The hepatocytes were washed three times and sus. pended in Krebs-Henseleit buffer. Dry weights were multiplied by 3.7 to convert to wet weight (11). Incubations of liver cells and analysis. Incuba-

DICHLOROACETATE tions were carried out at 37°C in a shaking water bath in 25ml stoppered Erlenmeyer flasks under a 95% O.&% CO* atmosphere. The incubation medium consisted of 4 ml of Krebs-Henseleit buffer with 2.5% bovine serum albumin (charcoal treated and dialyzed, fraction V, Sigma Chemical Co.). The rate of fatty acid synthesis was determined by the incorporation of 3HOH into total lipid fatty acids (12) as described previously (10). Incubations for fatty acid synthesis were run for 30 min before 3HOH was added and then continued for another 30 min before termination with perchloric acid (6% final concentration). Incubations of cells from mealfed and 48-h-starved rats for metabolite analysis were terminated with perchloric acid after 45 and 60 min, respectively. Glucose (13), glycogen (lo), lactate and pyruvate (14), acetoacetate, and 3-hydroxybutyrate (15) were determined spectrophotometrically on KOH-neutralized perchloric acid extracts. Citrate and malate were determined fluorimetrically (16) on Florisil-treated extracts prepared with hepatocyte separation tubes (17). Oxidation of fatty acids to CO, was assayed by WO, production from ll-‘4Cloleate. The ‘*CO, was collected in Hyamine hydroxide (center wells from Kontes Glass Co., Vineland, N. J.) following acidification of the incubation medium. Radioactivity was counted in a toluene-based scintillation fluid (0.01% p-bis-[2-(5phenyloxazolyl)lbenzene and 0.4% 2,5-diphenyloxazole) with a Searle Isocap/300 liquid scintillation spectrometer. Pyruvate dehydrogenase was assayed after incubating the liver cells under conditions reported in the text. The cells were separated from the incubation medium by centrifugation in a clinical centrifuge (5OOg for 15 s). The supernatant fluid was discarded, and the cells were suspended in 2 ml of a solution 20 mM in potassium phosphate (pH 7.0) and 40% in glycerol (v/v), precooled to -5°C. The liver cells were homogenized on dry ice for 15 s at full speed with a Polytron homogenizer (Type PT-10, Brinkman Instruments). The homogenate was assayed for pyruvate dehydrogenase activity in ldram shell vials stoppered with serum caps. A final volume of 0.2 ml of incubation medium (pH 8.0) contained 4.1 pmol of potassium phosphate, 0.1 pmol of MgCl,, 0.24 pmol of NAD+, 2.6 pmol of coenzyme A, 0.1 pmol of dithiothreitol, 1.0 pmol of 2-oxoglutarate, 1.0 pm01 of [l-‘4Clalanine (5 x lo4 cpm), 640 milliunits of lactate dehydrogenase (EC 1.1.1.27), 192 milliunits of glutamate-pyruvate transaminase (EC 2.6.1.21, 0.43 mmol of glycerol, and 3-4 mg wet weight of cells. The assay was conducted for 30 min at 30°C. The reaction was stopped with an injection of 0.5 ml of 6 N HCl through the serum cap. The “C0, released was collected in hanging cups containing Hyamine hydroxide (New England Nuclear) and counted as described above. The assay was linear with cell con-

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centration up to 10 mg wet weight of cells per assay and linear with time up to 60 min. Application of this assay to the determination of the pyruvate dehydrogenase activity of mitochondria has been described previously (18). Dichloroacetate was obtained from Fischer Scientific Company and was used as the sodium salt, adjusted to pH 7.4. All enzymes and most chemicals were obtained from Sigma. RESULTS

Effect of dichloroacetate upon carbohydrate metabolism of hepatocytes prepared from meal-fed rats. Dichloroacetate dramatically decreased the concentrations of lactate and pyruvate in the cell suspension when these metabolites were generated endogenously from glycogen (Table I). Dichloroacetate did not affect glycogen utilization but caused an increase in the rate of glucose accumulation in the medium. This resulted in a decrease in the rate of net glucose utilization (defined as the difference between the amount of glycogen utilized and the amount of glucose shed into the medium). The rate of lactate accumulation in the suspension was decreased greatly by dichloroacetate, whereas ketone body formation was increased significantly. Thus, dichloroacetate accelerates the utilization of lactate and pyruvate by liver cells but, in contrast to diabetic muscle (241, does not increase net utilization of glucose. Lactate plus pyruvate added at initial concentrations of 10 and 1 mM, respectively, effected a significant reduction in net glucose utilization by the cells (Table I). Further addition of dichloroacetate may have caused a slight increase in glucose utilization, but the observed increase was not significant statistically. Effect of dichloroacetate upon pyruvate dehydrogenase activity and fatty acid synthesis. Dichloroacetate produced a dramatic increase in pyruvate dehydrogenase activity (Table I). The addition of lactate plus pyruvate also activated pyruvate dehydrogenase; however, the combination of lactate plus pyruvate and dichloroacetate did not have an additive effect upon the enzyme. Indeed, the activity was intermediate between that observed with dichloroacetate and that with lactate plus pyruvate

alone.

660

CRABB ET AL. TABLE I

EFFECT OF DICHLOROACETATE (1 m&f) ON METABOLIC INTERMEDIATES, PYRUVATE DEHYDROGENASE, AND FATI-Y ACID SYNTHEBI@

No additions

Lactate Pyruvate Lactatelpyruvate 3-Hydroxybutyrate/acetoacetate Glucose accumulation Glycogen utilization Net glucose utilizationb Lactate accumulation Ketone body formation Fatty acid synthesis’ Citrate Malate Pyruvate dehydrogenase

Dichloroacetate (1 mhi)

Lactate (10 mM) + pyruvate (1 mM)

Dichloroacetate (1 mM) + lactate (10 rnM) + pyruvate (1 mM)

Concentration of metabolite in medium (m@ or ratio 1.09 f 0.28 0.28 k 0.16* 7.4 + 0.6* 6.3 2 0.4* 0.30 -+ 0.06 0.06 r 0.02* 0.88 + 0.12* 0.56 f 0.07t 3.5 f 0.9 3.8 + 1.1 8.8 + 0.9* 11.5 k 1.0*t 0.28 in 0.05 0.55 2 0.11 0.42 + 0.03* 0.58 k 0.07* Rates of metabolic change (pm01 min-‘g-l) 1.01 k 0.11 1.19 2 0.13* 1.58 +- O.lO* 1.50 2 0.13* 2.16 2 0.21 2.06 r 0.19 1.73 k 0.15* 1.78 f 0.04* 1.14 + 0.12 0.87 2 0.12* 0.16 k 0.08* 0.28 2 O.lO* 0.72 + 0.12 0.16 k 0.08* -0.79 rf: 0.41* -1.46 k 0.44* 0.09 * 0.01 0.16 f O.Ol* 0.09 f 0.01 0.13 + 0.01 0.35 k 0.09 0.50 2 0.11* 0.62 f 0.16* 0.67 k 0.20* Concentration of components of cells (pmol/g) 0.20 k 0.03 0.19 * 0.04 0.37 ” 0.07* 0.33 c 0.09 0.29 f 0.04 0.48 + 0.09* 2.03 2 0.50” 2.39 + 0.52*t Enzyme activity (pm01 min-‘g-l) 0.41 k 0.08 2.04 + 0.24* 0.83 k 0.16* 1.17 k 0.24*

a Results are expressed as means 2 SEM with at least four liver cell preparations prepared from meal-fed rats in each group. Values which are significantly different from the control (No additions) by the paired t test are indicated by *: P < 0.05. The symbol t indicates whether values obtained with dichloroacetate + lactate + pyruvate were significantly different from those obtained with lactate plus pyruvate. * Glycogen utilization minus net glucose accumulation. c Expressed as micromoles of C, units per minute per gram.

Since direct measurements showed that the activity of pyruvate dehydrogenase in the cells was increased and since lactate and pyruvate levels were decreased much more than the decrease in net glucose utilization, it follows that dichloroacetate increased the flux of carbon through pyruvate dehydrogenase. This prompted an investigation of the effect of dichloroacetate upon the rate of fatty acid synthesis. Earlier studies with a reconstituted soluble liver system (18) and with intact adipocytes (19) demonstrated that the activity of pyruvate dehydrogenase and the rate of fatty acid synthesis correlate under certain experimental conditions. A study with varying concentrations of dichloroacetate gave a biphasic response with a maximum stimulation of fatty acid synthesis near 1 mM (Fig. 1). Table I shows that at this concentration dichloroacetate caused a significant increase in the rate of fatty acid synthesis. The rate of this proc-

?-rm-LDICHmRa4CET*TE w.Q FIG. 1. Effect of varying concentrations of dichloroacetate on the synthesis of fatty acids. Isolated hepatocytes were incubated with 3HOH as described in Methods and Materials. Rates of fatty acid synthesis are expressed as micromoles of C, units per minute per gram wet weight.

ess was also increased by lactate plus pyruvate. However, the stimulatory actions of lactate plus pyruvate and dichloroacetate on fatty acid synthesis were not additive. The percentage of increase in fatty acid synthesis caused by dichloroacetate

DICHLOROACETATE

was much less than the increase in pyruvate dehydrogenase activity. In contrast, the increase in fatty acid synthesis produced by lactate plus pyruvate was matched by a corresponding increase in pyruvate dehydrogenase activity. This result may be explained simply on the basis of the quantity of substrate (pyruvate) available for conversion to lipid precursors under the two different experimental conditions. In spite of the greater rate of fatty acid synthesis, dichloroacetate had no effect upon the citrate concentration of the cells (Table I). In contrast, the increased rate of fatty acid synthesis caused by lactate plus pyruvate correlated with an increase in the citrate content of the cells. Both dichloroacetate and lactate plus pyruvate caused an increase in the malate content of the cell. The increase caused by dichloroacetate was considerably less than that produced by lactate plus pyruvate. Effect of dichloroacetate on fatty acid oxidation by hepatocytes prepared from meal-fed rats. Dichloroacetate has been reported in other studies (2-8) to inhibit the oxidation of fatty acids and ketone bodies in muscle tissue. The action of dichloroacetate on hepatic fatty acid oxidation is shown in Table II. A significant inhibition of [1-14Cloleate oxidation to 14C0, was observed along with an elevation of the 3hydroxybutyrate to acetoacetate ratio. There was no effect of dichloroacetate upon the rate of ketogenesis. Effect of dichloroacetate on gluconeogenesis and glucose utilization by hepatocytes prepared from starved rats. Dichloroacetate (1 mM) caused a three- to fivefold activation of the pyruvate dehydrogenase activity of hepatocytes prepared from starved rats and incubated with 10 mM lactate (results not shown but very similar to those of Table I). Higher concentrations of dichloroacetate did not further increase the activity of pyruvate dehydrogenase. Dichloroacetate caused rather small but nevertheless statistically significant changes in the rate of gluconeogenesis from a number of substrates. Gluconeogenesis was slightly inhibited by dichloroacetate with lactate and with lactate plus oleate as substrates (Table III). Di-

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chloroacetate also lowered the pyruvate concentration and increased the lactate to pyruvate ratio of the cell suspension. The amino acid lysine is known to stimulate gluconeogenesis from lactate and to lower the lactate to pyruvate ratio (20). In addition to these effects (Table III), lysine was observed to overcome the tendency of dichloroacetate to inhibit glucoenogenesis from lactate and to increase the lactate to pyruvate ratio. In contrast to the results obtained with lactate, dichloroacetate slightly stimulated glucose synthesis with alanine as substrate. As expected (21), oleate also stimulated glucose synthesis from alanine. The stimulatory effects of dichloroacetate and oleate were not additive (Table III). With these substrates dichloroacetate produced a significant reduction of both lactate and pyruvate levels of the cell suspensions. This effect was more pronounced without oleate. Dichloroacetate had no effect upon gluconeogenesis or pyruvate levels with either pyruvate or glutamine as substrate (Table III).5 Net glucose utilization by cells from 48h-starved rats incubated with 5 mM glucose was not influenced significantly by dichloroacetate (control, 0.17 ~fr0.13; 1 mM dichloroacetate, 0.01 + 0.07 pmol min-’ g-l wet weight; mean +- SEM of five liver cell preparations; P > 0.05). DISCUSSION

The central position of pyruvate dehydrogenase in the metabolism of the liver is obvious: Its activity determines the flux of pyruvate derived from glycolysis into acetyl-CoA for citric acid cycle activity, lipogenesis, ketogenesis, and cholesterogenesis. The complex mode of regulation of pyruvate dehydrogenase, via both phosphorylation (22-24) and end-product inhibition (25), suggests further that the activity of pyruvate dehydrogenase must be finely controlled for optimal metabolic function. An activator of the enzyme such 5 Much higher concentrations (8 mM) of diisopropylammonium dichloroacetate have been reported to inhibit gluconeogenesis by isolated rat liver cells from a wide variety of substrates [Stacpoole, P. W., and Berry, M. N. (1971) Fed. Proc. 30, 767 (abstract)].

662

CRABB ET AL. TABLE II EFFECT

OF

DICHL~ROACETATE ON [~JT]OLEATE

Additions

OXIDATION TO “CO, AND KETONE BoDIEe”

Production of (pm01 min-‘g-l) wo*

None Dichloroacetate. 2 mM

Ratio of 3-hydroxybutyrate to acetoacetate

Ketone bodies

0.029 k 0.002 0.021 + 0.001*

0.45 + 0.10 0.50 2 0.06

0.53 k 0.17 0.92 + 0.12*

a Results are expressed as means f SEM with four liver cell preparations prepared from meal-fed rats in each group. The WO, data are calculated on the basis of micromoles of [l-*4Cloleate converted to CO*. Values significantly different from the control by the paired t test are indicated by *: P < 0.05. TABLE III EFFECTS OF DICHU)ROACETATE ON GLUCONEOCENESM

Additions (rnM)

Final concentzti: Pyruvate

None Dichloroacetate (1) Lactate (10) Lactate + dichloroacetate Lactate + oleate (0.75) Lactate + oleate + dichloroacetate Lactate + lysine (2) Lactate + lysine + dichloroacetate Alanine (10) Alanine + dichloroacetate Alanine + oleate Alanine + oleate + dichloroacetate Pyruvate (10) Pyruvate + dichloroacetate Glutamine (10) Glutamine + dichloroacetate

0.015 ‘- 0.002 0.023 + O.OOl* 0.41 f 0.06 0.22 k 0.02* 0.36 + 0.04 0.27 + 0.03* 0.54 + 0.05 0.47 r 0.06 0.24 k 0.01 0.10 k 0.04* 0.11 * 0.02 0.06 k O.Ol* 1.56 2 0.26 1.50 ?Y0.41 0.04 k 0.01 0.02 f 0.01

(mM) in meLactate 0.07 + 0.02 0.03 2 0.01 7.0 + 0.3 7.7 ?z 0.3* 6.7 k 0.4 6.9 +- 0.4 5.9 2 0.3 5.7 -+ 0.4 0.63 +- 0.06 0.22 2 0.02* 0.42 +- 0.05 0.24 k 0.02’ 1.6 ” 0.1 1.7 +- 0.1 0.17 f 0.03 0.08 f O.Ol*

Ratio of lactate to pyruvate

Rate of glucase synthesi&

5.1 f 2.0 1.4 2 0.2 19 54 37 2 5* 20 k4 27 k 4* 11 *2 13 +3 2.6 t 0.2 3.3 + 0.1* 4.4 k 1.2 3.8 k 0.2 1.2 2 0.2 1.0 * 0.2 4.8 zt 0.3 3.8? 0.5

0.09 k 0.01 0.08 r 0.01* 0.70 f 0.07 0.62 k 0.07* 1.09 ” 0.14 0.85 + 0.11* 1.12 + 0.13 1.04 f 0.13 0.29 f 0.04 0.39 2 0.05* 0.62 + 0.12 0.60 2 0.11 0.55 k 0.02 0.54 2 0.02 0.38 2 0.08 0.37 k 0.08

a Results obtained after 60 min of incubation are expressed as means k SEM with at least four liver cell preparations in each group. Values obtained with dichloroacetate which were significantly different by the paired t test from the values obtained without dichloroacetate are indicated by *: P < 0.05. b Glucose synthesis calculated as micromoles of glucose produced per minute per gram wet weight of cells and corrected for zero-time glucose.

as dichloroacetate provides a means by lactate and pyruvate. However, exogenous which to dissect some of these control lactate plus pyruvate decrease net glucose mechanisms. utilization, which is accompanied by an increase in the citrate content of the cells. St;&yRsfh Cells Prepared from Mealc i t ra t e is known to potentiate the inhibition of phosphofructokinase (EC 2.7.1.11) In contrast to heart and diabetic skeletal by ATP in several tissues (26, 27), but this muscle, in which both pyruvate and glu- effect is less well established for liver (27, case oxidation are stimulated by dichlo- 28). Dichloroacetate does not lower these roacetate (24, dichloroacetate causes a high levels of citrate, as it does in diabetic decrease in the lactate and pyruvate con- muscle (2,3), and does not relieve the @hicent,rations of liver suspensions without bition of glucose utilization by lactate plus increasing the utilization of glucose. This pyruvate. Lactate. plus pyruvate and disuggests that hepatic glycolysis is not reg- chloroacetate both activate pyruvate dehyulated by decreases in the end products drogenase, but only lactate plus pyruvate

DICHLOROACETATE

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663

hydrogenase (EC 1.1.1.35) compete for, affects the cell citrate content; obviously and are limited by, the supply of mitochonthe activity of pyruvate dehydrogenase drial NAD+. With a relatively fixed NAD+ alone does not regulate citrate levels. would make pyruDichloroacetate has been reported to de- pool, dichloroacetate crease fatty acid synthesis in adipose tis- vate dehydrogenase more effective in comsue (8) and to have no effect in muscle (2). peting for NAD+, thereby decreasing the However, Jungas has shown in adipose amount of labeled acetyl-CoA produced tissue that by activating pyruvate dehy- without changing the acetyl-CoA pool size. This results in decreased 14C0, production drogenase with high pyruvate levels, fatty with no change in ketogenesis, as seen acid synthesis increases in parallel with Uncoupling oxidative pyruvate dehydrogenase activity (19). Pre- experimentally. should increase the availvious work from this laboratory (18) with a phosphorylation reconstituted system also indicates that ability of NAD+, reduce the competition pyruvate dehydrogenase is important in between pyruvate dehydrogenase and 3the regulation of fatty acid synthesis. Cit- hydroxyacyl-CoA dehydrogenase, and rate is both the form in which carbon is minimize the effects of dichloroacetate on experiments transported from the mitochondrion for oleate oxidation. Preliminary suggest that 2,4-dinitrophenol does in fact fatty acid synthesis (29, 30) and an activator of acetyl-CoA carboxylase (EC 6.4.1.2) relieve the inhibition of oleate oxidation to CO, caused by dichloroacetate. (29, 31). The increase in fatty acid synthesis caused by dichloroacetate is not accompanied by an increase in citrate content of Studies with Cells Prepared from 48-hStarved Rats the whole cells, which unfortunately says nothing about the intracellular distribuThe hypoglycemic effect of dichloroacetion of citrate. Dichloroacetate, by increastate in diabetic and starved rats has been to the activation of pyruvate ing the malate content, may have in- attributed creased the flux of citrate into the cyto- dehydrogenase in peripheral tissues and increased pyruvate utilization (5), which plasm on the malate-citrate antiport withincreases glucose utilization and decreases out increasing the overall citrate content. the flux of substrates (lactate, alanine, It is also possible that dichloroacetate increases lipogenesis by making the and pyruvate) to the liver for gluconeogenesis (6). Dichloroacetate has rather small NADP+ couple more reduced, as evidenced by the increase in the malate/pyruvate ra- but significant effects directly on the liver. tio (32). This seems unlikely in view of the It does not increase glucose utilization, but slightly inhibits gluconeogenesis from lacnormally very reduced NADP+ couple, which should be capable of reducing 3- tate and lactate plus oleate, and stimuoxoacyl-CoA during fatty acid synthesis. lates gluconeogenesis from alanine, but not Figure 1 shows a biphasic response of lipo- in the presence of oleate. The hypoglygenesis to increasing dichloroacetate con- cemic action of dichloroacetate therefore centrations. This may be the result of an appears to be mainly a result of the aboveeffect on the mitochondrial pyruvate mentioned peripheral effects; however, the transporter, which dichloroacetate com- effects on liver are of interest with respect petitively inhibits (33, 341, or of an unrecto the possible role of pyruvate dehydroognized effect. genase in the regulation of gluconeogenesis. Dichloroacetate stimulates ketogenesis from endogenous carbon. This is consistent The inhibition of gluconeogenesis from with a greater flux of pyruvate through lactate and without oleate can perhaps be pyruvate dehydrogenase to increase the rationalized by the reversal of this inhibiacetyl-CoA pool. However, dichloroacetate tion by lysine. Dichloroacetate presumdoes not affect ketogenesis from oleate but ably increases the mitochondrial NADH/ decreases the oxidation of [l-14C]oleate to NAD+ ratio by activating pyruvate dehy14C0,. This is understandable if pyruvate drogenase. This may slow transport of redehydrogenase and 3-hydroxyacyl-CoA de- ducing equivalents into the mitochondrion

664

CRABB

and shift the equilibrium of lactate dehydrogenase (EC 1.1.1.27) to give a higher free NADH level. The result is the increased cytoplasmic NADH/NAD+ ratio seen as a rise in the lactate/pyruvate ratio.6 This would tend to reduce the oxaloacetate concentration and inhibit gluconeogenesis in a manner similar to the effect of ethanol, which also increases the cytoplasmic NADH/NAD+ ratio via alcohol dehydrogenase (EC 1.1.1.1). Lysine, by donating amino groups to the components of the aspartate shuttle (201, increases the transport of reducing equivalents into the mitochondria, lowers the cytoplasmic NADH/ NAD+ ratio, as evidenced by the decrease in the lactate/pyruvate ratio,6 and perhaps relieves the inhibition of gluconeogenesis by dichloroacetate. Determination of the intracellular levels of metabolites might refute this rationalization of the experimental observations but was beyond the scope of this study. The stimulation of gluconeogenesis by dichloroacetate with alanine as substrate was not seen when oleate was also present, suggesting a similar mechanism of stimulation for dichloroacetate and oleate. Oleate stimulation of gluconeogenesis requires active P-oxidation (21), which provides NADH, needed to convert oxaloacetate to malate for transport out of the mitochondrion, and acetyl-CoA, an absolute requirement for pyruvate carboxylase (EC 6.4.1.1) activity (35). Dichloroacetate also increases acetyl-CoA and NADH by activating pyruvate dehydrogenase and, therefore, may stimulate alanine glucone6 The assumption is made here that the cell membrane is readily permeable to lactate and pyruvate; therefore, their ratio in the cell suspension reflects their ratio in the cytoplasmic compartment. This was tested by conducting incubations with different substrates for 60 min, followed by separation of the cells from the suspending medium with the hepatocyte separation tubes described previously (17). With three liver cell preparations incubated with 10 rnM lactate plus 2 mM lysine, the ratio of lactate to pyruvate for the suspending medium was 9.1 ? 1.0; for the separated cells, 8.0 +- 1.3 (P > 0.05); with cells incubated with 10 mM pyruvate, the ratio of lactate to pyruvate for the suspending medium was 0.8 * 0.3; for the separated cells, 0.8 + 0.2.

ET AL.

ogenesis by a mechanism similar to that of oleate. Dichloroacetate has no effect on gluconeogenesis from pyruvate, which itself activates pyruvate dehydrogenase (36), or glutamine, which does not have pyruvate as an intermediate in its conversion to glucose. These effects, or lack of effects, are therefore expected. The study presented above emphasizes the number of metabolic processes in the liver which are affected by alterations in the activity of pyruvate dehydrogenase. It also shows that the changes in metabolism caused by changes in enzyme activity can be quite different in different tissues, viz., liver, muscle, and adipose tissue. Whitehouse et al. (8) suggest that, on the basis of studies with dichloroacetate, a genetic deficiency of pyruvate dehydrogenase kinase might be manifested by low fasting blood glucose and lactate, to which one might now add the possibility of increased hepatic fatty acid synthesis perhaps with characteristic changes in the plasma lipid profile. ACKNOWLEDGMENT We thank Ms. P. Jenkins for technical during the course of this work.

assistance

REFERENCES 1. LORINI, M., AND OMAN, M. (1962) Biochem. Pharmacol. 11, 823-827. 2. STACPOOLE, P. W., AND FELTS, J. M. (1970) Metab. Clin. Exp. 19, 71-78. 3. STACPOOLE, P. W., AND FELTS, J. M. (1971) Metab. Clin. Ezp. 20, 830-834. 4. STACPOOLE, P. W. (1969) J. Clin. Pharmacol. 9, 282-291. 5. MCALLISTER, A., ALLI~QN, S. P., AND RANDLE, P. J. (1973) Biochem. J. 134, 1067-1081. 6. BLACKSHEAR, P. J., HOLLOWAY, P. A. H., AND ALBERTI, K. G. M. M. (1974)Biochem. J. 142, 279-286. 7. WHITEHOUSE, S., AND RANDLE, P. J. (1973) Biothem. J. 134, 651-653. 8. WHITEHOUSE, S., COOPER,R. H., AND RANDLE, P. J. (1974) Biochem. J. 141, 761-774. 9. BERRY, M. N., AND FRIEND, D. S. (1969) J. Cell BioE. 43, 506-620. 10. HARRIS, R. A. (1975) Arch. Biochem. Biophys. 169, 168-180. 11. KREBS, H. A., CORNELL, N. W., LUND, P., AND HEMS, R. (1973) in Regulation of Hepatic Me-

DICHLOROACETATE tabolism (Lundquist, F., and Tygstrup, N., eds.), Alfred Benzon Symposium 6, pp. ‘718743, Munksgaard, Copenhagen. 12. JUNGAS,R. L. (1968)Biochemistry 7, 37083717. 13. SLEIN, M. W. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 117123, Academic Press, New York. 14. HOHORST,H. J., KREUTZ, F. H., AND BOCHER,T. (1959) Biochem. 2. 332, 18-46. 15. WILLIAMSON,P. H., MELLANBY, J., AND KREBS, H. A. (1962) Biochem. ,I. 82, 90-96. 16. WILLIAMSON, J., AND CORKEY,B. E. (1969) in. Methods in Enzymology (Lowenstein, J. M., ed.), vol. 14, pp. 434-515, Academic Press, New York. 17. MAPES, J. P., AND HARRIS, R. A. (1975) FEBS Lett. 51, 80-83. 18. MAPES, J. P., and HARRIS, R. A. (1975) &ids, 10, 757-764. 19. TAYLOR, S. I., AND JUNGAS, R. L. (1974) Arch. Biochem. Biophys. 164, 12-19. 20. CORNELL,N. W., LUND, P., AND KREBS, H. A. (1974) Biochem. J. 142, 327-337. 21. WILLIAMSON,J. R., BROWNING,E. T., SCHOLZ,R. A., KREISBERC,2. A., AND FRITZ, F. B. (1968) Diabetes 17, 194-208. 22. LINN, T. C., PETTIT,F. H., AND REED,L. J. (1969) Proc. Nat. Acad. Sci. USA 62, 234-241.

23. LINN, T. C., PELLEY,J. W., PETTIT, F. H., HUN-

24. 25. 26. 27. 28. 29. 30.

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CHO, F., RANDELL, D. D., AND REED, L. J. (1972) AFC~. Biochem. Biophys. 148, 327-342. SIESS,E. A., AND WIELAND, 0. (1975)FEE3 Lett. 52, 226-230. GARLAND,P. B., AND RANDLE, P. J. (1964) Biothem. J. 91, 6C-7C. NEWSHOLME,E. A., AND GEVERS,W. (1967) Vitam. Harm. 25, l-87. RANDLE,P. J., DENTON,R. M., AND ENGLAND,P. J. (1968) &o&em. Sot. Symp. 27, 87-103. KEMP, R. G. (1971) J. Biol. Chem. 246, 245-252. LOWENSTEIN,J. M. (1968) Biochem. Sot. Symp. 27, 61-86. WATSON,J. A., AND LOWENSTEIN,J. R. (1970)J. Biol. Chem. 245, 5993-6002.

31. MARTIN, D. B., AND VAGELOS,P. R. (1962) J. Biol. Chem. 237, 1787-1792.

32. KREBS, H. A., AND VEECH, R. L. (1970) in Pyridine Nucleotide Dependent Dehydrogenases (Sund, H., ed.), pp. 413-434, Springer-Verlag, Berlin. 33. HALESTRAP,A. P. (1975)Biochem. J. 148, 85-96. 34. HALESTRAP,A. P., AND DENTON, R. M. (1975) Biochem. J. 148, 97-106. 35. SCRUTTON,M. C., AND UTTER, M. F. (1967) J. Biol. Chem. 242, 1723-1735.

36. PATZELT,C., LOFFLER,G., AND WIELAND, 0. H. (1973) Eur. J. Biochem. 33, 117-122.

Effect of dichloroacetate on carbohydrate and lipid metabolism of isolated hepatocytes.

ARCHIVES OF BIOCHEMISTRY AND 173, 658-665 (1976) BIOPHYSICS Effect of Dichloroacetate on Carbohydrate and Lipid Metabolism Isolated Hepatocytes...
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