ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS’ Vol. 189, No. 2, August, pp. 364-371, 1978

Inhibition

of Hepatic ROBERT

Department

of Biochemistry,

Gluconeogenesis

A. HARRIS” Indiana

AND

University

Received November

School

by Dichloroacetate’

DAVID

W. CRABB

of Medicine,

Indianapolis,

Indiana

46202

10, 1977; revised March 30, 1978

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 hypoglycemic agent is best explained by stimulation of peripheral tissue utilization of glucose and potential precursors for hepatic gluconeogenesis rather than by direct inhibition of hepatic gluconeogenesis.

tocytes and thus concluded that its hypoDichloroacetate decreases the glycosuria glycemic action is mainly a result of the and hyperglycemia in diabetic rats (l-3) and causes hypoglycemia in fasted rats (4, above mentioned peripheral effects. In conof 5). It increases glucose oxidation and in- trast, Stacpoole (10) found inhibition hibits fatty acid oxidation in skeletal muscle gluconeogenesis and concluded that direct of diabetic rats (6, 7) and increases glucose, effects of dichloroacetate on the liver are lactate and pyruvate extraction but de- important. The reason for the apparent difcreases fatty acid oxidation in perfused ference in sensitivity of gluconeogenesis to heart (8). Hence, the hypoglycemic effect dichloroacetate in these studies is reported of dichloroacetate can be explained at least here. in part by stimulation of peripheral utiliMATERIALS AND METHODS zation of glucose (1) and in part by imLiver ceils were prepared from 48-h fasted male proved extrahepatic lactate and pyruvate Wistar rats (180 to 220 g) by the method of Berry and oxidation which impairs hepatic gluconeogenesis by interruption of the Cori and al- Friend (11) with the modifications described previously (12). Incubations were carried out at 37°C for 60 anine cycles (4). Studies have also been conducted to de- min with 40 to 60 mg wet weight of cells in 25ml Erlenmeyer flasks in a final volume of 2 ml of incutermine whether more direct effects upon bation medium. Two incubation media were used: (a) hepatic gluconeogenesis are involved in the Krebs-Helseleit saline supplemented with 2.5% albuaction of dichloroacetate. Crabb et al. (9) min (charcoal-treated and dialyzed, Fraction V, Sigma found little or no inhibition of gluconeogenChemical Co.) with an atmosphere of 95% O2 and 5% esis by dichloroacetate with isolated hepa- CO?; and (b) low bicarbonate saline (modified Hank’s ’ This work was supported by grants from the U. S. Public Health Service (Grants No. AM19259 and AM21178), the Marion County Heart Association (Indiana Affiliate of the American Heart Association), the Grace M. Showalter Residuary Trust, and the Lilly Research Laboratory. ’ To whom requests for reprints should be sent.

,,.

3b4

0003-9861/78/1892-0364$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved

solution) as described by Stacpoole (10) which consisted of 0.14 M NaCI, 5.4 mM KCI, 0.8 mM MgCb, 1.0 rnM CaCh, and 10 mM sodium phosphate, pH 7.4, with an atmosphere of 100% O2 and a cen!er well containing 0.15 ml of 10% KOH. Metabolite assays were conducted on KOH-neutralized HClO.* extracts of the cell suspensions by enzymatic methods according to Hohorst et al. (13) for lactate and pyruvate, Williamson

GLUCONEOGENESIS

AND

et al. (14) for acetoacetate and 3-hydroxybutyrate, Bernt and Bergmeyer (15, 16) for glutamate and (Yketoglutarate, Crow et al. (17) for aspartate, Gutmann and Wahlefeld (18) for malate, and Slein (19) for glucose, except NADP’ was replaced with NAD’ and the NAD’linked glucose 6-phosphate dehydrogenase (Type XXI) was used. The active form of the pyruvate dehydrogenase complex (E.C. 1.2.4.1) was assayed by the modification of previously published methods (20-22). An aliquot (0.2 ml) of the cell suspension incubated for 30 min as described above was mixed with 1.4 ml of an ice-cold solution containing 350 pmol of sucrose, 28 nmol of morpholinopropane sulfonic acid (pH 7.0), 4.2 nmol of EDTA, and 5.6 mg of digitonin. This solution was left on ice for 30 set and then centrifuged in an Eppendorf centrifuge for 10 sec. The supernatant was discarded, the inside of the tube wiped dry with a gauze sponge, and the pellet frozen and stored in liquid nitrogen. The pellet was thawed and suspended in 0.2 ml of a solution 20 mM in potassium phosphate (pH 7.0), 5% in rat serum, and 1% in Triton X-100. This extract was assayed for pyruvate dehydrogenase activity by the release of ?ZOs from [l-‘4C]pyruvate. Shell vials (1 dram) stoppered with serum caps were used. The incubation medium (0.2 ml) was 15 mM in potassium phosphate, pH 8.0, 0.5 rnM in MgCls, 0.25 mM in EDTA, 1 mM in coenzyme A, 0.5 mM in dithiothreitol, 1.2 mM in NAD’, 0.1 mM in thiamine pyrophosphate, 10 mM in mercaptoethanol, and 1 mM in [1-“‘Clpyruvate (250 cpm/nmol). The assay was conducted for 5 min at 37°C. The reaction was stopped with an injection through the serum cap of 0.5 ml of 6 N HCl and the “C02 released was collected in hanging cups con-

EFFECT

OF DICHLOROACETATE

365

DICHLOROACETATE

taining phenethylamine. Radioactivity was measured with OCS scintillation fluid (Amersham). Collagenase was obtained from Worthington Biochemical Corp.; other enzymes from Sigma Chemical Company; [1-“‘Clpyruvate from New England Nuclear. Dichloroacetate was from Fischer Scientific Company, and was used as the sodium salt, pH 7.4. RESULTS

Comparison of the Rates of Glucose Synthesis from Lactate by Hepatocytes Suspended in Low Bicarbonate Saline and Krebs-Henseleit Saline In previous studies on the effect of dichloroacetate on hepatic gluconeogenesis, Crabb et al. (9) used Krebs-Henseleit saline as the incubation medium, whereas Stacpool (10) used a modified Hank’s medium, called here low bicarbonate saline. The latter was very low in bicarbonate because it contained no added bicarbonate, was bubbled for 15 min with 100% oxygen, and the incubation flasks contained KOH-center wells to collect metabolic CO,. As shown in Table I, Krebs-Henseleit saline supported faster rates of gluconeogenesis from lactate than low bicarbonate saline. Lysine stimulation of glucose synthesis (23) was apparent in both buffers but was only 29% in low bicarbonate saline compared to 60% in Krebs-Henseleit saline (Table I). Signifi-

TABLE I (DCA) AND LYSINE ON GLUCONEOGENESIS

FROM LACTATE

BY ISOLATED

HEPATOCYTES”

Incubation

medium

Krebs-Henseleit

Low bicarbonate

Additions

(mM)

None DCA (1) DCA (7.5) Lysine (2) Lysine + DCA Lysine + DCA None DCA (1) DCA (7.5) Lysine (2) Lysine + DCA Lysine + DCA

(1) (7.5)

(1) (7.5)

Rate of glucose synthesis (pmol/min/g wet wt) 0.70 + 0.01 0.61 + 0.03* 0.49 f 0.04* 1.12 + 0.01: 1.14 + 0.01; 1.22 + 0.02* 0.52 + 0.04 0.13 + 0.06; 0.12 +- 0.06* 0.67 + O.OB* 0.13 f 0.07: 0.11 t- 0.058

Final concentration Lactate

(Ltmol/ml) 7.3 f 0.1 7.3 + 0.2 8.4 f 0.4* 5.8 f 0.2* 5.6 -c 0.3’ 5.1 + 0.2’ 8.1 f 0.6 10.3 + 0.4* 10.7 + 0.1* 7.9 + 0.3 10.6 f O.l* 10.5 + 0.3*

of:

Ratio of lactate/pyruvate

Pyruvate (umol/ml) 0.89 0.58 0.37 0.81 0.54 0.31 0.60 0.11 0.13 0.79 0.11 0.10

+ 0.07 + 0.05* r 0.03’ + 0.03 + 0.02; + 0.02’ + 0.07 + 0.04* -c 0.02: f 0.04 + 0.04; + 0.01:

8.4 + 0.7 13 + 1+ 24 f3* 7.3 + 0.4* 10.4 f 0.1; 16.3 f 0.6* 13.8 + 0.6 129 f 30* 90 -c 13* 10.1 -c 0.7* 136 -c40* 109 f IOf

n Results are expressed as means + SEM with at least four liver celI preparations prepared from 48-h fasted rats in each group. Values which are significantly different from the control (none) by the paired t-test are indicated by (*): P < 0.05. All incubations were for 66 min with 50 to 60 mg wet weight of liver cells. Lactate was present in ah flasks at an initial concentration of 11.8 mM.

366

HARRIS

AND

cantly lower lactate to pyruvate ratios (Table I) and 3-hydroxybutyrate to acetoacetate ratios (Table II) were obtained in Krebs-Henseleit saline. Lysine lowered the lactate to pyruvate ratio but was without effect on the 3-hydroxybutyrate to acetoacetate ratio in both media.

CRABB

Hepatocytes incubated in Krebs-Henseleit saline accumulated much more glutamate and cw-ketoglutarate and slightly more malate than hepatocytes incubated in low bicarbonate medium (Table III). Aspartate levels were similar. Lysine greatly increased glutamate in both media, but n-ketoglutar-

TABLE

II

EFFECT OF DICHLOROACETATE (DCA) AND LYSINE ON THE REDOX STATE OF THE MITOCHONDRIAL COMPARTMENT OF ISOLATED HEPATOCYTES” Incubation dium

me-

Additions

Final concentration

(mM)

3-Hydroxybutyrate (pmol/ml) Krebs-Henseleit

Low bicarbonate

None DCA (1) DCA (7.5) Lysine (2) Lysine + DCA Lysine + DCA None DCA (1) DCA (7.5) Lysine (2) lysine + DCA Lvsine + DCA

(1) (7.5)

(1) (7.5)

0.06 0.10 0.13 0.11 0.17 0.25 0.16 0.30 0.12 0.29 0.40 0.14

of:

Acetoacetate (pmol/ml)

+ 0.01 f 0.01* + 0.02* f 0.01* f 0.071 + 0.01’ rc_0.03 -c 0.02* + 0.02 f 0.01* f 0.05* f 0.03

0.14 0.15 0.15 0.26 0.29 0.35 0.21 0.52 0.56 0.36 0.52 0.57

+ 0.02 + 0.02 -t 0.02 -+ 0.02* f 0.02* f 0.03’ +- 0.03 k 0.04* + 0.03* f 0.03* f 0.03* f 0.03*

a Incubations were as described in Table I. Values which are significantly by the paired t-test are indicated by (*): P < 0.05. TABLE

Ratio of 3-hydroxybutyrate/acetoacetate 0.41 0.66 0.83 0.41 0.59 0.73 0.75 0.60 0.23 0.81 0.78 0.25

different

-c 0.01 k 0.02* f 0.02* f 0.02 f 0.02* f 0.04* + 0.06 z!z0.08* + 0.05* -c 0.05 -r- 0.12 + 0.06*

from the control

(none)

III

EFFECT OF DICHLOROACETATE (DCA) AND LYSINE ON THE CONCENTRATION OF THE COMPONENTS OF THE MALATE-ASPARTATE SHUTTLE OF ISOLATED HEPATOCYTES” “OxSum’ Final concentration of: Incubaa; meAdditions (mM) aloaceoc-KetogluGlutamate Aspartate Malate tate”” tarate (,amol/g wet wt) Krebs-Henseleit

Low bicarboante

4.1 + 0.5 None 4.5 f 1.0 DCA (1) 5.0 f 0.2* DCA (7.5) 7.0 f 0.1* Lysine (2) Lysine + DCA (1) 8.7 -C 1.0’ 9.9 + 0.8” Lysine + DCA (7.5) 2.3 -t 0.4 None 1.4 * 0.3* DCA (1) 1.1 f 0.2* DCA (7.5) 4.1 f 0.5* Lysine (2) Lysine + DCA (1) 1.9 f 0.4* 1.5 -c 0.2* Lysine + DCA (7.5)

(pmoA$ wet

(pmobl

wet

(pmo;$

wet

2.6 2.8 3.1 2.5 3.0 2.7

+ f + + + +

0.4 0.1 0.1* 0.4 0.4 0.1

0.62 0.57 0.60 0.65 0.99 1.3

+ + f k f f

0.05 0.14 0.22 0.07 0.11* 0.1*

0.9 rt_0.1 1.2 f 0.1* 1.7 -c 0.1* 1.0 rt_0.1 1.3 f 0.1* 1.5 f 0.1:

0.027 0.022 0.017 0.033 0.030 0.022

a.2 9.1 10.4 11.1 14.0 15.4

0.9 0.5 0.6 1.5 0.5 0.5

f f f + f +

0.1 0.1* O.l* 0.3; 0.1* 0.11

0.60 0.19 0.23 0.70 0.16 0.17

+ + + + f f

0.02 0.01* 0.02* 0.09 0.02* 0.03;

0.7 0.5 0.4 0.7 0.6 0.6

0.012 0.001 0.001 0.017 0.001 0.001

4.5 2.6 2.3 6.7 3.2 2.8

from the control

(none)

n Incubations were as described in Table I. Values which are significantly different by the paired t-test are indicated by (*): P < 0.05. ’ Calculated concentrations of oxaloacetate for the cytoplasmic compartment. ’ Sum of the concentrations of the metabolites listed in the table.

+ k f. * + +

0.1 0.1’ 0.1* 0.1 0.1 0.1

GLUCONEOGENESIS

AND

ate only in the low bicarbonate saline. With or without lysine, the sum of the components of the aspartate-malate shuttle was much greater with Krebs-Henseleit saline. Effect of Dichloroacetate on Glucose Synthesis from Lactate in Low Bicarbonate Saline and Krebs-Henseleit Saline As reported previously from this laboratory with hepatocytes incubated in KrebsHenseleit saline (9), 1 mM dichloroacetate caused a slight inhibition (13%, Table I) of glucose synthesis from lactate. Greater inhibition (30%) of gluconeogenesis was caused by 7.5 mM dichloroacetate. Dichloroacetate was much more effective in low-bicarbonate saline, causing 75% inhibition of glucose synthesis at 1 mM and only slightly greater inhibition at 7.5 mM (Table I). These results are comparable to those of Stacpoole (10) who reported 50 percent inhibition at 0.1 mM and 88% inhibition at 8.7 mM. Lysine completely prevented dichloroacetate inhibition of glucose synthesis in Krebs-Henseleit saline (Table I). In contrast, lysine was without this effect in the low-bicarbonate saline.

DICHLOHOACETATE

367

and the lactate to pyruvate ratio as described by Williamson et al. (24). With one exception, the calculated values of oxaloacetate directly correlated with the rates of gluconeogenesis given in Table I. Thus, dichloroacetate produced a slight inhibition of glucose synthesis in Krebs-Henseleit saline and a slight decrease in the steady state cytoplasmic concentration of oxaloacetate. In low bicarbonate saline, dichloroacetate almost completely inhibited gluconeogenesis and greatly diminished the oxaloacetate concentration. The exception to this correlation was seen in Krebs-Henseleit saline with lysine at 7.5 mru dichloroacetate where a stimulation of glucose synthesis was observed together with a slight decrease in oxaloacetate. Effect of Dichloroacetate on Pyruvate Dehydrogenase Activity in Krebs-Henseleit Saline and Low Bicarbonate Saline

Hepatocytes incubated in low bicarbonate saline have a significantly lower pyruvate dehydrogenase activity than hepatocytes incubated in Krebs-Henseleit saline (Fig. 1). Nevertheless, the response to dichloroacetate is almost identical in the two media. Incubation of hepatocytes with high Effect of Dichloroacetate on the Redox concentrations of pyruvate, known to alState of the Cytoplasmic and Mitochonmost completely activate the pyruvate dedrial Compartments hydrogenase complex (25), was used to esDichloroacetate increased the lactate to tablish the relative percentage of the compyruvate ratio with hepatocytes incubated plex in the active form as a function of in Krebs-Henseleit saline but increased it dichloroacetate concentration. With remuch more in low bicarbonate saline (Table spect to the concentrations used in the I). Lysine blunted this increase in the cy- studies described above, 1 mu dichloroactoplasmic redox state only in Krebs-Henetate is a suboptimal concentration but 7.5 seleit saline. mu produces complete activation of the pyruvate dehydrogenase complex in both Effect of Dichloroacetate on the Compo- media. In experiments not shown, 2 mu nents of the Malate-Aspartate Shuttle lysine did not significantly alter the reDichloroacetate had opposite effects in sponse of the pyruvate dehydrogenase comthe two buffer systems upon the hepatocyte plex to dichloroacetate in either media. The content of glutamate, a-ketoglutarate, as- results shown in Figure 1 are somewhat partate, and malate. In general, dichloroacdifferent from those reported by Claus and etate caused these components of the mal- Pilkis (25). Almost complete activation ocate-aspartate shuttle to increase in Krebs- curred at 2 mM dichloroacetate under their Henseleit saline and to decrease in low bi- incubation conditions whereas 5 mM was carbonate saline (Table III). necessary in this study. Furthermore, a Oxaloacetate concentrations were calcu- greater percentage of the pyruvate dehylated for the cytoplasmic compartment drogenase was in the active form without (Table III) from the malate concentration dichloroacetate in this study. The reason

368

HARRIS 25

AN11 CRABB

II

z ouo 0 DICHLOROACETATE 2 1 (rnM1 6 8

FIG. 1. Effect of varying concentrations of dichloroacetate on the activity of the pyruvate dehydrogenase complex in Krebs-Henseleit saline and low-bicarbonate saline. Results are expressed as means +- SEM for three liver cell preparations from 48-h fasted rats. Circles represent experiments conducted in KrebsHenseleit saline; triangles in low bicarbonate saline. Incubations were conducted for 30 min with 40 to 50 mg wet weight of liver cells. Lactate was present in all flasks at an initial concentration of 10 mM. The relative percentage of the enzyme in the active form was calculated from the activity of the enzyme of cells incubated with 10 mM pyruvate. Such conditions are known to completely activate the pyruvate dehydrogenase complex (25).

for these discrepancies is not known but may relate to differences in incubation conditions or methods used in isolating the hepatocytes. Effect of Dichloroacetate on Ethanol datkon in Krebs-Henseleit Saline Low Bicarbonate Saline

Oxiand

Lactate is known to stimulate ethanol oxidation by isolated hepatocytes (26, 27) by providing a source of carbon to restore physiological concentrations of the components of the malate-aspartate shuttle (17, 27). Figure 2 shows this effect of lactate with cells suspended in Krebs-Henseleit saline and low bicarbonate saline. Ethanol oxidation is much less in the low bicarbonate saline than in Krebs-Henseleit saline, presumably because reducing equivalents can not be transported as rapidly into the mitochondria because of a shortage of shuttle components. Since lactate plus lysine more readily restores shuttle components in the presence of high concentrations of bicarbonate (Table III), lactate has a

0

2

4

b

8

10

FIG. 2. Effect of varying concentrations of lactate on the oxidation of ethanol in Krebs-Henseleit saline and low bicarbonate saline. Circles represent experiments conducted in Krebs-Henseleit saline; triangles in low bicarbonate saline. Closed symbols indicate 1 mM dichloroacetate was present in the incubation medium. Ethanol was present in all flasks at an initial concentration of 10 mM. A representative experiment is presented but it was reproduced with two additional preparations of liver cells from 48-h fasted rats.

greater effect on ethanol oxidation in Krebs-Henseleit saline than in low bicarbonate saline (Fig. 2). Dichloroacetate does cause significant inhibition of ethanol oxidation in both media at high concentrations of lactate. A paradoxical stimulation of ethanol oxidation by dichloroacetate in both media was noted in the absence of lactate. DISCUSSION

Stacpoole (10) observed inhibition of lactate gluconeogenesis by dichloroacetate with isolated hepatocytes and concluded that the blood-glucose-lowering effect of dichloroacetate is explained in part by inhibition of hepatic gluconeogenesis. Crabb et al. (9) independently observed little or no inhibition of gluconeogenesis from lactate and, therefore, reached the opposite conclusion. The basic experimental observations of both Stacpoole (10) and Crabb et al. (9) have been reproduced in this study. The explanation for the difference in results lies in the sensitivity of lactate gluconeogenesis to dichloroacetate in the two different incubation media used in these studies. Dichloroacetate is a very effective

GLUCONEOGENESIS

AND

inhibitor in low bicarbonate saline but not in Krebs-Henseleit saline. Lysine has no effect in low bicarbonate saline but completely prevents dichloroacetate inhibition in Krebs-Henseleit saline. The greatest difference between the two buffers is seen when both lactate and lysine are included. Dichloroacetate at 7.5 mM caused 84% inhibition in low bicarbonate saline but 9% stimulation in Krebs-Henseleit saline (Table I). Crabb et al. (9) previously reported stimulation of gluconeogenesis from alanine by dichloroacetate. This was confirmed by Claus and Pilkis (25) who also observed slight inhibition with lactate as substrate and slight stimulation with lactate plus NH&l. Thus, dichloroacetate inhibition of gluconeogenesis is only observed when hepatocytes are incubated in a nonphysiological medium, i.e., a medium deficient in bicarbonate and/or amino acids. An explanation was offered previously (9) for the slight inhibition of glucose synthesis by dichloroacetate in Krebs-Henseleit saline and its reversal by lysine. In this study the determination of the mitochondrial redox state from the 3-hydroxybutyrate to acetoacetate ratio as well as the levels of the components of the malate-aspartate shuttle provides more evidence which is still consistent with that explanation. Dichloroacetate increases the mitochondrial NADH/NAD’ ratio (Table II) by its known (8) activating effect upon pyruvate dehydrogenase. A concentration of 7.5 mM dichloroacetate had a greater effect than 1 mM on both the activity of the pyruvate dehydrogenase complex and the mitochondrial NADH,/NAD+ ratio. This action of dichloroacetate may decrease the transport of reducing equivalents into the mitochondrion, causing the cytoplasmic compartment to assume a more reduced state. The resu!t is an increase in the cytoplasmic NADH/NAD+ ratio. This reduces the oxaloacetate concentration (see calculated values, Table III) and inhibits gluconeogenesis in a manner analogous to the effect of ethanol, which increases the cytoplasmic NADH/NAD’ ratio via alcohol dehydrogenase. Lysine, by donating amino groups to the components of the malate-aspartate shuttle (23), increases the relative concen-

DICHLOROACETATE

369

tration of these components (Table III) and promotes the transport of reducing equivalents into the mitochondria. This lowers the cytoplasmic NADH/NAD’ ratio, as evidenced by the decrease in lactate/pyruvate ratio, and relieves dichloroacetate inhibition of gluconeogenesis (Table I). The results also suggest an explanation for the greater sensitivity of lactate gluconeogenesis to dichloroacetate in the low bicarbonate medium. The major effect of dichloroacetate is to prevent the accumulation of the components of the malateaspartate shuttle (Table III). This does not occur in the Krebs-Henseleit saline buffer but may be a natural consequence of activation of the pyruvate dehydrogenase complex in the low bicarbonate saline. Pyruvate carboxylase may not be able to compete effectively with pyruvate dehydrogenase for pyruvate in the low bicarbonate medium for want of bicarbonate. Furthermore, the pyruvate concentration is known to affect the affinity of pyruvate carboxylase for bicarbonate (28). The Michaelis constant for bicarbonate is 3 mM at infinite pyruvate but approaches 25 mM at zero pyruvate (28). This could not be considered an important factor under physiological conditions or with incubations conducted with Krebs-Henseleit saline where the bicarbonate concentration is approximately 25 IIIM. However, it is a potentially important factor in low bicarbonate saline when the pyruvate concentration is decreased by dichloroacetate activation of the pyruvate dehydrogenase complex. Regardless of whether pyruvate or bicarbonate is limiting, a decreased rate of pyruvate carboxylation would suppress the accumulation of the components of the malate-aspartate shuttle. The decrease in shuttle components would limit the transfer of reducing equivalents from the cytoplasmic space to the mitochondrial matrix resulting in the elevated lactate to pyruvate ratios and decreased 3-hydroxybutyrate to acetoacetate ratios observed in these experiments. Gluconeogenesis then becomes limited at the level of phosphoenolpyruvate carboxykinase because of a low steady state concentration of oxaloacetate. Stacpoole (10) suggested that dichloroacetate may inhibit glu-

370

HARRIS

AND

coneogenesis at the level of glyceraldehyde phosphate dehydrogenase in low bicarbonate saline. This was based on the observation that glucose synthesis was inhibited by dichloroacetate only with those substrates (e.g., lactate, pyruvate, or glycerate) entering the gluconeogenic pathway before glyceraldehyde phosphate dehydrogenase. Indeed, the inhibition of glucose synthesis from glycerate would appear to rule out any effects at the mitochondrial level. However, the synthesis of glucose from glycerate is dependent on the transfer of reducing equivalents from the mitochondrial space to the cytoplasm. The only known pathway for transferring reducing equivalents under such conditions involves the conversion of glycerate to pyruvate, intramitochondrial carboxylation of pyruvate, intramitochondrial reduction of oxaloacetate, extramitochondrial oxidation of malate, formation of phosphoenolpyruvate from oxaloacetate, and regeneration of pyruvate from phosphoenolpyruvate (20, 29). Hence, competition between pyruvate dehydrogenase and pyruvate carboxylase for pyruvate could also lead to a limited availability of cytoplasmic NADH needed for gluconeogenesis from glycerate. As presented above, the known action of dichloroacetate on the pyruvate dehydrogenase complex may account for its inhibitory effects on gluconeogenesis. However, other effects of dichloroacetate are now being recognized and are probably as important or even more important to the inhibition of gluconeogenesis. For instance, the stimulation of ethanol oxidation by dichloroacetate shown in this study is not readily explained by activation of the pyruvate dehydrogenase complex. Furthermore, there is no correlation between the extent of inhibition of gluconeogenesis and the degree of activation of the pyruvate dehydrogenase complex by dichloroacetate. In low bicarbonate saline gluconeogenesis is abolished by concentrations of dichloroacetate that only marginally activate the pyruvate dehydrogenase complex. The opposite is the case in Krebs-Henseleit saline. In studies not shown, very high concentrations of dichloroacetate (50-100 mu) cause substantial inhibition of gluconeogenesis in

CRABB

Krebs-Henseleit saline in spite of the fact that full activation of the pyruvate dehydrogenase complex occurs at 5 mM dichloroacetate. These results argue that another mechanism of action exists for dichloroacetate inhibition of gluconeogenesis. Furthermore, this laboratory has demonstrated a stimulation of leucine oxidation by dichloroacetate which is not related to any effect on pyruvate dehydrogenase activity (30). Rather dichloroacetate is catabolized by hepat,ocytes to glyoxylate which promotes leucine oxidation by functioning as an amino group acceptor for leucine transamination (31). In studies to be reported elsewhere (32), we have found glyoxylate and its oxidation product oxalate to be potent inhibitors of gluconeogenesis by isolated hepatocytes. Thus, catabolism of dichloroacetate to compounds which directly inhibit gluconeogenesis may be more important than activation of the pyruvate dehydrogenase complex. In studies on the in viva effects of dichloroacetate on metabolites of the liver, Blackshear et al. (4) observed dichloroacetate to decrease both the lactate to pyruvate and the 3-hydroxybutyrate to acetoacetate ratios. This is opposite to the effects of dichloroacetate on isolated hepatocytes (Tables I and II). Furthermore, no accumulation of any gluconeogenic intermediate was observed that might indicate an inhibitory site of action of dichloroacetate (4) and the changes observed were more readily explained by a decrease in the availability of substrates for gluconeogenesis. Therefore, rather than any direct effect upon some step of the pathway, substrate supply appears to limit gluconeogenesis by the liver of animals treated with dichloroacetate. In conclusion, inhibition of lactate gluconeogenesis by dichloroacetate can be shown with isolated liver cells (9, 10, 25). However, it is marginal when physiological levels of bicarbonate are available and does not occur at all when lysine (9) or ammonia (25) are present to provide an adequate source of amino groups for the aspartatemalate shuttle. Furthermore, alanine is considered an important substrate for glucose synthesis by the liver and alanine glu-

GLUCONEOGENESIS

AND

coneogenesis is significantly stimulated by dichloroacetate (9. 25). Therefore. the nrimary mechanism responsible for the h;oglycemic action of dichloroacetate is decreased peripheral release of substrates than can be used for gluconeogenesis by the _. hver. ACKNOWLEDGMENT Technical Jenkins.

assistance was provided

DICHLOROACETATE

16.

17. 18.

by Ms. Patricia

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