ARCHIVES OF BIOCHEMISTRY AND

BIOPHYSICS

Vol. 190, No. 1, September, pp. 8-16, 1978

Studies on the Regulation

of Leucine Catabolism’

II. Mechanism Responsible for Dichloroacetate

Stimulation of Leucine Oxidation

by the Liver

ROBERT A. HARRIS,2 DAVID W. CRABB,

AND

RONALD M. SANS

Department of Biochemistry, Indiana University School of Medicine, Indianapolis,

Indiana 46202

Received February 2, 1978; revised March 30,197s Dichloroacetate, an activator of the pyruvate dehydrogenase complex and a hypoglycemic agent, activates leucine oxidation by isolated liver cells. The cY-ketoisocaproate dehydrogenase complex, which catalyzes the second step of leucine catabolism and is believed analogous to the pyruvate dehydrogenase complex, did not respond to dichloroacetate. Rather, dechlorination of dichloroacetate by liver cells produces glyoxylate which promotes leucine catabolism by serving as an amino group acceptor for either direct or indirect transamination of leucine. Support for this mechanism includes the following: (a) amino acids which serve as substrates for glyoxylate aminotransferase block the stimulatory effect of dichloroacetate on leucine oxidation; (b) glyoxylate stimulates the oxidation of leucine; (c) both dichloroacetate and glyoxylate increase the glycine content of isolated hepatocytes; (d) [2-“‘Cldichloroacetate is converted by isolated hepatocytes to [‘%]glycine, [‘%]oxalate, and ‘%OZ; (e) 2-chloropropionate, which also activates the pyruvate dehydrogenase complex but is not converted to glyoxylate, does not stimulate leucine oxidation; and (f) ethylene glycol, another compound known to be converted to glyoxylate by the liver, also stimulates leucine oxidation. Thus, in contrast to its mechanism of action on pyruvate metabolism, dichloroacetate promotes leucine catabolism by formation of glyoxylate which serves as substrate for the transamination of leucine by glyoxylate aminotransfersse.

Previous studies from this laboratory (3) demonstrated a stimulation of L-leucine catabolism by isolated hepatocytes by pyruvate and dichloroacetate. Both compounds were found to increase the transamination of leucine to cw-ketoisocaproate, the rate limiting step of leucine catabolism by liver. The mechanism responsible for the action of pyruvate was shown to involve the provision of more keto acids for transarnihation

with leucine. In contrast, the mechanism of action on leucine catabolism of dichloroacetate, an established activator of the pyruvate dehydrogenase (EC 1.2.4.1) complex (4-6) and an effective hypoglycemic agent in the diabetic animal (7,8), was not established. The present study suggests that glyoxylate aminotransferase, known to be present in peroxisomes (9), may be involved in hepatic leucine catabolism and that dichloroacetate has its effects via this enzyme.

’ 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 Indiana University Human Genetics Center (USPHS GM21054), the Grace M. Showalter Residuary Trust, and the Lilly Research Laboratory. This study was presented in part at the April FASEB meeting in Atlantic City (1) and in part at the June FASEB meeting in Atlanta (2). ’ To whom requests for reprints should be sent.

MATERIALS

All rights of reproduction in any form reserved

METHODS

fasted male Wistar rats (180 to 220 g) by the method of Berry and Friend (10) with the modifications described previously (11). Conversion of L-[1-‘%]leucine to “C02 by isolated liver cells was measured as described previously (3). Radioactive a-ketoisocaproate was measured by quantitative decarboxylation with

8 0003-9861/78/1901-0008$02.00/O Copyright 0 1978by Academic Press, Inc.

AND

Isolation and incubation of liver cells and assay of metabolites. Liver cells were prepared from 48-h

DICHLOROACETATE

AND

half-saturated ceric sulfate in 3 N HzS04 (12) followed by the collection of 14COz in phenethylamine: methanol, 1:1, v/v. Radioactive amino acids formed from [2-‘4C]dichloroacetate were isolated from neutralized perchloric acid extracts of cell suspensions by ion exchange chromatography with Bio-Rad AG 5OWX8. The labeled amino acids (mainly glycine) were identified by two dimensional thin-layer chromatography on Silica Gel G plates (13). Essentially all of the radioactivity of the fraction obtained by ion exchange chromatography was established by thin-layer radioautography to be associated with glycine. Radioactive oxalate formed from [2-‘?]dichloroacetate was measured with oxalate decarboxylase (EC 4.1.1.2) by a modification of the procedure described by Rofe et al. (14). Radioactive CO? was released from the incubation medium by the addition of perchloric acid and denatured protein was removed by centrifugation. Samples were incubated in 0.2 M sodium citrate, pH 3.0, for 24 h with 0.1 unit of oxalate decarboxylase. Radioactive CO* was collected in phenethylamine. Ammo acids were measured in neutralized perchloric acid extracts with a Beckman model 121-M amino acid analyzer. a-Ketoglutarate was measured enzymatitally by the method of Bergmeyer and Bernt (15). Assay of a-ketoisocaproate dehydrogenase andpyruuate dehydrogenase. A modification of the method of Johnson and Cormelly (16) was used to assay for a-ketoisocaproate dehydrogenase. Hepatocytes were separated from the incubation medium by centrifugation in a clinical centrifuge (50Cg for 15 set). 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% glycerol (v/v), precooled to -5’C. The cells were homogenized on Dry Ice for 20 set at full speed with a polytron homogenizer (Type PT-10, Brinkman Instruments). The homogenate was assayed for a-ketoisocaproate dehydrogenase activity in l-dram shell vials stoppered with serum caps. A final volume of 0.25 ml of incubation medium (pH 7.2) contained 37.5 pmol of mannitol, 8.2 pmol of potassium phosphate, 0.25 pmol of NAD’, 0.15 ,umol of coenzyme A, 0.75 ~01 of dithiothreitol, 0.25 pmol of MgC12,0.73 pmol of [l-‘4C]a-ketoisocaproate, and 3-6 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 14C02 released was collected in hanging cups containing phenethylamine. The assay was linear with cell concentration up to 10 mg wet weight of cells per assay and linear with time up to 60 min. Pyruvate dehydrogenase was assayed with the same incubation conditions except that [1-‘?]pyruvate replaced the [I-‘?]a-ketoisocaproate. Sources of materials. Radioactive compounds were obtained from New England Nuclear, Research Products International Corp., ICN Pharmaceuticals, Inc., and Schwarz/Mann. L-[ l-‘4C]Leucine was diluted with

LEUCINE

9

CATABOLISM

L-leucine to a specific radioactivity of 200 cpm/nmol. Commercial preparations of L-[l-‘4C]leucine are always contaminated with ‘?Oz as well as [l-‘4C]aketoisocaproate, as determined by decarboxylation with ceric sulfate. New lots of L-[l-‘4C]leucine were always examined and discarded if the contamination was such that it would interfere with the studies to be conducted. [1-‘?]Lu-Ketoisocaproate was prepared from L-[l-‘4C]leucine by deamination with snake venom L-amino acid oxidase followed by purification by gel filtration and ion exchange chromatography (3). The product obtained had a specific radioactivity of 144 cpm/nmol, was ninhydrin negative, and was contaminated with negligible amounts of ‘?Oz. [2?Z]Dichloroacetate was prepared from [2-j4C]trichloroacetate by the method described by McAllister et aZ. (4). [2-‘4C]Trichloroacetate was obtained as a special order from Amersham Corp. The [2-Y!]dichloroacetate prepared had a specific radioactivity of 75.3 cpm/nmol and was established by the thin-layer chromatography procedure described by McAllister et al. (4) to be at least 95% pure. The nmr spectrum of the product was identical to that of commercial dichloroacetic acid. Unreacted starting material ([2“C]trichloroacetic acid) accounted for most of the contamination. Glyoxylate contamination, determined by oxidation of NADH with lactate dehydrogenase (EC 1.1.1.27), was found to be only 0.01% which is better than commercial dichloroacetic acid. No detectable [‘4C]oxalate, as measured by decarboxylation with oxalate decarboxylase, was detectable in the [2“C]dichloroacetate. Collagenase was obtained from Worthington Biochemical Corp., other enzymes from Sigma Chemical Company. Dichloroacetate was from Fischer Scientific Company, and was used as the SOdium salt, pH adjusted to 7.4.2~Chloropropionate was from Eastman Kodak Company and was also used as the sodium salt, pH 7.4. RESULTS

Effect of various amino acids and dichloroacetate on the oxidation of L-[I YJleucine by isolated hepatocytes. As presented previously (3), dichloroacetate increases the formation of [l-‘4C]cw-ketoisocaproate from L-[1-14C]leucine which, in turn, increases the rate of 14C02 production by isolated liver cells from L-[l-14C]leucine (Table I). The doubling of the rate of L-[l“C]leucine oxidation caused by dichloroacetate was not affected by valine, isoleucine, glutamate, lysine, tryptophan, proline, arginine, or threonine but was suppressed by serine, methionine, asparagine, glutamine, phenylalanine, and glycine. The most effective inhibitor of the stimulation by dichloroacetate was asparagine, followed by

10

HARRIS,

CRABB,

AND

TABLE EFFECTS Addition

OF AMINO

ACIDS

ON THE OXIDATION

Leucine Without dichloroacetate (nmol/min/g

None Vahne Isoleucine Glutamate Lysine Tryptophan Proline Arginine Serine Threonine Methionine Asparagine Glutamine Phenylalanine Glycine Histidine

15 f 14 f 12 f 13 f 12 f 13 +11+ 13 f 15 f 10 + 12 + 11 f 10 + 12 + 16 f 13 f

1 1 1” 2 1** 1 1** 1 1 1** 1** 1** 1** 1** 1 1**

SANS

I

OF [l-‘%]LEUCINE

BY ISOLATED

decarboxylation: With

dichloroacetate

Without

dichloroacetate

wet weight) 30 zk 29 -+ 27 + 28 + 25 f 25k 23 f 24 + 21+ 19f 16f 12 -t 16f 15f 19 f 18k

HEPATOCYTES~

a-Ketoisocaproate

accumulation: With

dichloroacetate

(mnol/mI) 2* 3* 2* 2* 2* I* 1* 1* 1* 1* I* 1 l* 1* 2’ l*

5.9 4.3 4.4 4.2 4.0 4.1 5.3 3.8 5.9 8.1 4.5 4.7 5.1 3.6 6.2 4.8

f 0.2 If: 0.7 + 0.2 + 0.2 f ox** r+- 0.4 f 0.2 + 0.7** f 1.2 +- 1.6** f 0.6 k 0.3 f 0.2 + 0.6** + 0.9 f 0.2**

11.7 13.4 12.4 10.2 12.2 12.7 12.2 10.2 8.2 18.2 7.4 4.2 8.2 5.0 7.9 7.6

f 0.9’ rf: 0.4* +- 0.5* + 0.7: f 1.0* f 0.2* 2 2.61 f 2.5’ k 1.4” f 1.8* + 0.3; f 0.4 + 0.3* f 0.9’ 2 1.2* z!z 0.4*

a Results are expressed as means f SEM with three liver ceil preparations from 48-h fasted rats. Values which are signitkantly different from corresponding controls without dichloroacetate are indicated by *: P < 0.05. Values which are significantly different from the no addition sample (none) are indicated by **: P -z 0.05. Student’s t-test for paired samples was used. AU incubations were for 30 min at 37°C with 51 to 59 mg wet weight of liver cells. Amino acids were added at an initial concentration of 5 mu; dichloroacetate, 1 mu; and L[l-‘4C]leucine, 1 mM.

phenylalanine and methionine. For the most part, the effects of these amino acids can be explained on the basis of their influence on the accumulation of a-ketoisocaproate, which in turn can be explained by the substrate specificity of glyoxylate aminotransferase as discussed below. Asparagine completely eliminated the increase in cy- ketoisocaproate by dichloroacetate. methionine and glycine Phenylalanine, were also quite effective, whereas amino acids such as valine and isoleucine were completely ineffective in preventing the dichloroacetate effect on either 14C02 production or a-ketoisocaproate accumulation. The amino acids which inhibited L-[l“C]leucine decarboxylation nearly all caused a decrease in a-ketoisocaproate levels. Threonine was an exception, being an effective inhibitor of 14C02 production while causing an accumulation of [1-14C]a-ketoisocaproate. As discussed below, this anomalous behavior of threonine is readily explained on the basis of the effects on leucine catabolism of a-ketobutyrate, which is pro-

duced by the action of threonine dehydratase (EC 4.2.1.16). Effect of dichloroacetate on the activity of the a-ketoisocaproate and pyruvate dehydrogenases. The increase in [1-14C]a-ketoisocaproate formation from L-Cl-14C]leutine caused by dichloroacetate argues for activation of the transamination of leucine rather than an effect upon the a-ketoisocaproate dehydrogenase. Nevertheless, a dichloroacetate effect upon the cu-ketoisocaproate dehydrogenase was conceptually difficult to abandon because of the known activation of the pyruvate dehydrogenase complex by dichloroacetate (4-6). However, direct assay of the a-ketoisocaproate dehydrogenase complex after incubation of hepatocytes for 30 min with 1 mu dichloroacetate failed to produce any activation of the enzyme. The enzyme activity without dichloroacetate was 0.73 + 0.05 pmol/min/g wet weight; with dichloroacetate, 0.70 + 0.05 pmol/min/g wet weight (values are means + SEM for experiments with 7 cell preparations). The pyruvate dehydrogen-

DICHLOROACETATE

AND

ase activity, assayed for the same cell preparations with [ l-‘4C]pyruvate replacing [l“C]a-ketoisocaproate, was increased 67% by dichloroacetate. Hence, dichloroacetate does not activate the a-ketoisocaproate dehydrogenase complex. Effect of glyoxylate and a-ketobutyrate on the oxidation of L-[l-‘4C]leucine. Previous studies from this laboratory (3) established the stimulatory effect of pyruvate on 14C02 formation and [l-14C]cw-ketoisocaproate accumulation from L-[l-‘4C]leutine shown in Table II. A search for other potential amino group acceptors revealed that glyoxylate and a-ketobutyrate were effective in increasing [l-‘4C]cu-ketoisocaproate accumulation but were inhibitors of 14C02formation (Table II). The concentration dependence of these effects was further investigated with glyoxylate being found to have a biphasic effect upon L-[l“C]leucine oxidation (Fig. 1). At low concentrations (