Inhibition of sterol synthesis by citrinin in a cell-free system from rat liver and yeast

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Biochimica et Biophysics @ Elsevier/North-Holland

Acta, 486 (1977) Biomedical

254-259

Press

BBA 56930

INHIBITION OF STEROL SYNTHESIS BY CITRININ IN A CELL-FREE SYSTEM FROM RAT LIVER AND YEAST

MASAO

KURODA,

YOKO

Fermentation Research Tokyo, 140 (Japan) (Received

August

HAZAMA-SHIMADA

Laboratories,

Sankyo

and AKIRA

Co. Ltd., l-2-58

END0

*

Hiromachi,

Shinagawa-ku,

2nd, 1976)

Summary Citrinin, a fungal metabolite known as an antibiotic, strongly inhibited the labeled acetate incorporation into nonsaponifiable lipids by a cell-free system from rat liver but not the labeled mevalonate incorporation. Of the enzymes involved in cholesterol synthesis, two enzymes, acetoacetyl-CoA thiolase (EC 2.3.1.9) and 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), were specifically inhibited by the antibiotic. The concentration required for 50% inhibition was 0.2 mM for the former enzyme and 0.5 mM for the latter. Essentially the same results were obtained with a cell-free system from yeast, although higher concentrations of the antibiotic were required for inhibition.

Introduction Citrinin, a fungal metabolite, was first isolated from cultures of the fungus citrinum in 1931 [1], which was then found to exhibit antibiotic activity toward several gram-positive bacteria, including Bacillus subtilis and Staphylococcus aureus [2]. Recently, citrinin was reported to be one of the toxic principles contained in grains infected with fungi [ 31. In a previous paper [4], we have demonstrated that citrinin, isolated from the culture filtrate of the fungus Pythium ultimum, strongly inhibits cholesterol synthesis in vitro by the rat liver enzyme system, and that administration of the inhibitor to rats causes a reduction in serum cholesterol levels. The present study was undertaken for the purpose of determining the site or sites of action of citrinin in the sterol biosynthetic pathway in a cell-free system from rat liver and yeast. The results indicate that the antibiotic specifically inhibits two enzymes, acetoacetyl-CoA thiolase (EC 2.3.1.9) and 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34).

Penicillium

* Author to whom correspondence should be addressed.

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Materials and Methods

[l=‘4C]Acetate (54.9 Ci/mol) was obtained from Daiichi Pure Chemicals Co, (Tokyo), and [l=‘4C]acetyl-CoA (493 Ci/mol), DL-[ 3-14C]3-hydroxy-3-methylglutaryl-CoA (18.8 Ci/mol) and DL-[2-‘4C]mevalonic acid lactone (27.3 Ci/ mol) were purchased from New England Nuclear Co. These labeled compounds were tested for the radiopurifies before use, which were over 98%- Acetyl-CoA, CoA and glucose Gphospbate dehydrogenase were obtained from Boehlinger, and acetoacetyl=CoA, ATP, NAD, NADPH, gtutathione (reduced), glucose l-phosphate, digitonin, lanosterol, cholesterol, DL-meValOniC acid lactone and dithiothreitol were purchased from Sigma Chemical Co. DL-3=Hydroxy-3=methylglutaryl-CoA was obtained from P-I., Biochemicals. Citrinin was obtained as previously described f4f. Experiments with rut liver enzyme preparations Rat liver microsomes and cytosolic enzyme fraction were isolated as described previously [ 5 J. For incorporation experiments, each incubation tube contained in a tot,al volume of 0.2 ml: 1 mM ATF; 10 mM glucose l-phosphate; 6 mM glutathione; 6 mM MgCIZ; 40 ,&I CoA; 0.25 mM NAD; 0.25 mM NADP; 100 mM potassium phosphate buffer, pH 7.4; microsomes (0.2 mg protein); cytosolic enzyme fraction (2.0 mg protein); and 1.0 mM [1-14C]acetate (sodium salt) (3.0 Ci/mol). In some experiments where indicated, 0.03 mM fl=14C]acetyl-CoA (1.0 CiJmol) or 0.44 mM DL-[2-‘4C]mevalonate (0.59 Cij mof) was used in the incorporation experiments in place of [1=14C]acetate= The tubes were incubated at 37°C for 60 min. Incubation was terminated by addition of 1 ml of 15% alcoholic KOH to the tubes. The synthesized nonsaponifiable products, digitonin-precipitable sterols, and fatty acids were isolated and determined as described previously [ 51. The sequential reaction of acetoacetyl-CoA thiolase and 3-hydroxg-3-methylglut~l-CoA synthase, and 3=hydro~y-3-methylglut~l=CoA synthase were assayed by the method of Sugiyama et al. [6]. 3=Hydroxy=3-methylglut~lCoA reductase was assayed as described previously [5]. Experiments with cell-free yeast extracts ~~ce~ar~rnyee~ cerevisiae ATCC 12341 (kindly supplied by Dr. A- Kawaguchi, tfniversity of Tokyo) was grown under partially anaerubic conditions, and the cell-free extracts were prepared as described by Kawaguchi [ 7f. The incubation mixture for incorporation experiments contained in a total volume of 0.2 ml: 5 mM ATP; 2 mM glutathione; 0.1 mM CoA; 1 mM NADP; 10 mM glucose B-phosphate; 2 mM MgS04; 1 mM MnS04; 100 mM potassium phosphate buffer, pH 7.0; X.0 mg (protein) of cell-free extracts; and 1.0 mM [f=‘4CJacetate (3.0 Cijmol). Where indicated, 0.03 mM fl=‘4C]acetyl=CoA fl.0 Cijmol), 0.65 mM ~~-[3-~~C]3=hydroxy-3-methylglut~l=CoA (l-57 Cilmol) or 0.44 mM DL-[%‘4C]meVa10nate (0.59 Ci/mol) was used instead of labeled acetate. The mixture was incubated at 37OC for 30 min, and the resultant nonsaponifiable fraction was obtained and counted as described previously [5]. Under these conditions, the incorporations of labeled compounds into non-

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saponifiable lipids as proportional to time up to 30 min. Yeast 3-hydroxy-3-methylglutaryl-CoA reductase was isolated from baker’s yeast (Sankyo Co., Tokyo) by the method of Dugan and Porter [S], except that the final step, DEAE-cellulose chromatography, was replaced by ultracentrifugation at 100000 X g for 60 min as descibred by Retey et al. [9]. The enzyme was assayed using DL-[ s-l4 C] 3-hydroxy-3-methylglutaryl-CoA as a substrate by the method for rat liver enzymes except that 100 mM potassium phosphate buffer, pH 7.4 was replaced by 70 mM potassium phosphate buffer, pH 6.7. The purified reductase preparation had an activity of 12.4 nmol of mevalonate formedfmin per mg of protein under the standard conditions.

0 ther determinations Prokin was determined by the method of Lowry et al. IlO], using bovine serum albumin as a standard. Radioactivity was measured with a Packard Tricarb Model 3390 or a Beckman LS-250 spectrometer in a toluene-based counting fluid. Samples insoluble in toluene were counted in a toluene/dioxane (1 : 4) fluid. Results and Discussion

Studies with rat liver enzymes The effect of citrinin on the incorporation of labeled precursors into nonsaponifiable fraction by rat liver enzyme system is shown in Fig. 1. As previously indicated [ 41, the [ 14C]acetate incorporation was strongly inhibited by citrinin, being approximately 50% of the control at a concentration of 0,034 mM of the ~tibiotic. Cholesterol synthesis from [ “C]acetyl-CoA was also diminished by the antibiotic. The degree of inhibition of [‘4C]acetyl-CoA incorporation was the same as that for [ 14C]acetate conversion at various concentrations of citrinin, suggesting that the antibiotic has no inhibitory effect on the activity of acetyl-CoA synthetase (EC 6.2.1.1) which catalyzes the formation of acetylGoA from acetate. The [ 14C]mevalonate incorporation into nonsaponifiable fractions was, however, not inhibited by citrinin, even at higher concentrations over 0.5 mM (Fig. 1). These data indicate that citrinin has no inhibitory effect on the post-mevalonic sites in cholesterol synthetic pathway, but that it is inhibitory to the enzymatic reactions leading to mevalonate formation, possibly steps between acetyl-CoA and mevalonate. Under the standard conditions, two sterols (cholesterol and lanosterol) and squalene represented greater than 90% of the total radioactivity from [‘4C]acetate incorporated into nonsaponifiable lipid, and the cholesterol/lanosterol/ squalene ratio was approximately 4 : 1 : 5, as determined by the method described previously [ 51. This ratio remained constant in the same experiments in which the [‘4C]acetate incorporation was reduced by approximately 50% in the presence of 0.03 mM citrinin. The data also suggest that the antibiotic shows no remarkable effect on the post-mevalonic sites in cholesterogenesis. For detailed studies of the citrinin inhibition of cholesterol synthesis, three enzymes which are involved in the formation of mevalonate from acetyl-CoA, i.e. acetoacetyl-CoA thiolase (EC 2.3.1.9), 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) and 3-hydroxy-3-methylglutaryl-CoA reduetase were

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Fig. 1. Effect of eitrinin on the incorpora~on of labeled precursors into nonsaponifiable lipids by rat liver enzymes. Experiments were carried out as described in Materials and Methods. The radioactivity incorporated was 0.022 nmol/min per mg protein for [ * 4C1acetate. 0.049 nmol/min per mg protein for 1’ 4Clacetyl-CoA and 0.124 nmol/min per mg protein for [ 14Clmevalonate, respectively. +----4, [14C1acetate incorporation: o------o, [14C1acetyl-CoA incorporation; n----0, [ 14C1mevalonate incorporation. Fig. 2. Effect of citrinin on rat liver 3-hydroxy-3-methyl~u~ryl-CoA synthase, on the sequential reaction of rat liver acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl~oA synthease, and on 3-hydroxy-3methylglutaryl-CoA reductase. Reaction mixture for the assay of 3-hydroxy-3-methylglutaryl-CoA synthaw contained in a total volume of 0.1 ml: 50 mM Tris . HCl, PH 7.4; 0.05 mM EDTA; 0.3 mM [14Clacetyl-CoA (1.0 Ci/mol); 0.1 mM acetoacetyl-CoA: and cytosolic enzyme fraction (0.02 mg protein). After incubation for 30 min at 37’C, 0.2 ml of 6 N HCl was added to the mixture, and the non-volatile radioactivity (as [ * 4C13-hydroxy-3-methylglutaryl-CoA) was determined. In the control experiments carried out without added citrinin, 5.9 nmol/min per mg protein of radioactivity were incorporated. Reaction mixture for the sequential reaction of acetoacetyl-CoA thiolase and 3-hydroxy-3-methyl~u~ylCoA synthase contained (total volume, 0.1 ml): 50 mM Tris . HCl, pH 7.4; 0.03 mM EDTA; 0.15 mM [ 14C]acetyl-CoA (1.0 Ci/mol): and cytosolic enzyme fraction (0.02 mg protein). Following a lo-min incubation at 37’C, radioactivity incorporated into 3-hydroxy-3-methylglutaryl-CoA was determined as described above, which averaged 13.8 nmol/min per mg protein in the control experiments carried out in the absence of citrinin. Reaction mixture for the assay of 3-hydroxy-3-mebhylglutaryl-CoA reductase contained in a total volume of 0.05 ml: 0.11 mM DL-t14C13-hydroxy-3-methylglutaryl-CoA (1.57 Ci/ mol); 100 mM potassium phosphate buffer, pH 7.4; 10 mM EDTA; 10 mM dithiothreitol; 5 mM NADPH: and 0.03 mg microsomal protein. After incubation for 30 min at 37’C, the reaction was terminated by addition of 0.01 ml of 2 N HCl to the mixture, and the resultant mevalonolactone was isolated and counted as described previously 141. In the control experiments, 0.75 nmol mevalonate were formed under these conditions: o -0, thiolasesynthase system; a -a, synthase; op n, reductase.

assayed for inhibition by the antibiotic. As shown in Fig, 2, the sequenti~ reaction of acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglut~l-CoA synthase, i.e. the conversion of acetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA in the absence of acetoacetyl-CoA, was found to be inhibited by citrinin. The concentration required for 50% inhibition (0.2 mM) was, however, nearly 6-times greater than that obtained for [14C]acetate incorporation into nonsaponifiable lipid (Fig. 1). 3-Hydroxy-3-methylglut~yl-CoA synthase was diminished only slightly by the antibiotic, by less than 20% at higher concentrations up to 1.0 mM (Fig. 2). These data indicate that citrinin inhibits acetoacetyl-CoA thiolase but not 3-hydroxy-3-methylglutaryl-CoA synthase. The CoA dependent cleavage of acetoacetyl-CoA to form acetyl-CoA, which was assayed spectrometrically by measuring absorbancy at 300 nm [ 111, could not

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be determined in the presence of higher concentrations of citrinin because of disturbance of the assay due to strong absorbancy at this wave length of the antibiotic. Citrinin was also inhibitory to 3-hydroxy-3-methylglut~yl-CoA reductase, the rate-controlling enzyme in cholesterogenesis (Fig. 2). At a concentration of 0.5 mM, the inhibition was approximately 5076, a value comparable to that obtained for the coupled reaction of thiolase and synthase (Fig. 2).

Fig. 3A shows the effect of citrinin on the incorporation of four labeled precursors into nonsaponifiable lipid by the cell-free extracts from yeast. The incorporation of both [ 14C]acetate and [ 14C]acetyl-CoA was diminished to the same extent, being approximately 50% of the control in the presence of 0.4 mM citrinin. The incorporation of [‘“Cl 3-hydroxy-3-methylglut~yl-CoA was also inhibited by the antibiotic but lesser sensitive than was that of labeled acetate and acetyl-CoA. Sterol synthesis from [‘4C]mevalonate was, however, not affected by citrinin at concentrations up to 0.8 mM (Fig. 3A). These results indicate that, as in the case of studies with rat liver enzymes, the antibiotic specifically inhibits enzymatic steps located between acetyl-CoA and mevalonate, without affecting the post-mev~onic steps in cholesterogenesis. Concentrations of citrinin required for inhibition of incorporation were, however, higher for yeast enzymes than for rat liver system. Fig. 3B shows the effect of increasing concentrations of citrinin on the activity of yeast 3-hydroxy-3-methylglutaryl-CoA reductase. The concentration required for 50% inhibition was approximately 2.4 mM, a value that is 5-times greater than that for rat liver reductase.

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Fig. 3. Effect of citrinin on the incorporation of labeled precursors into no~apo~i~able lipids by (A) a yeast cell-free system and (B) on yeast 3-hydroxy-3-methylgltaryl-CoA reductase. Experiments were carried out as described in Materials and Methods. In (A) radioackty incorporated was 0.15 nmol/min per mg protein for [14Clacetate, 0.41 nmol/min per mg protein for [ 1 4Clacetyl-CoA, 0.23 nmol/min per mg protein for DL-[~4C13-hydroxy-3-methylglutaryl-CoA and 0.40 nmol/min per mg protein for [14C1mevalonate. respectively. *A, [l 4Claeetate incorporation: o-----o, [14Clacetyl-CoA incorporation; a-1, DL-~14C13-hyd~oxy-3-methylglutaryl-CoA incorporation: P----TI, [ 14Clmevalonate incorporation.

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The results shown in this paper have indicated that the antibiotic citrinin inhibits sterol synthesis by a cell-free system from rat liver and yeast. In addition, two enzymes involved in cholesterol synthesis, acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA reductase, have been shown to be specifically inhibited by the antibiotic. The data that these two enzymes in rat liver are inhibited by citrinin suggest that hypocholesterolemic activity of the antibiotic in rats, which was shown in the previous paper [ 41, is mostly ascribed to the inhibition of these two enzymes in liver, the major organ supplying serum cholesterol. References 1 R&trick, H. and Hetherington. A.C. (1931) Phil. Trans. Roy. Sot. London B220, 279-296 2 R&trick, H. and Smith, G. (1941) Chem. Ind. 828-830 3 Ssito, M.. Emote, M. and Tatsuno, T. (1971) Microbial Toxins (Ciegler, A., Kadis. S. and Ajl. S.J., eds.). Vol. 5. pp. 357-367. Academic Press, New York 4 Endo, A. and Kuroda, M. (1976) J. Antibiotics, 29, 841443 5 Kuroda, M. and Endo. A. (1977) Biochim. Biophys. Acta, 486.70-81 6 Sugiyama, T.. Clinkenbeard, K.D., Moss, J. and Lane. M.D. (1972) Biochem. Biophys. Res. Commun. 48. 255-261 7 Kawaguchi. A. (1970) J. Biochem. 67.219-227 8 Dugan, R.E. and Porter. J.W. (1971) J. Biol. Chem. 246. 53616364 9 RBtey. J.. Stetten, E., Coy, U. and Lynen, F. (1970) Eur. J. Biochem. 15, 72-76 10 Lowry, O.H., Rosebrough. N.J., Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 11 Clinkenbeard. K.D.. Sugiyama, T., Moss, J., Reed, W.D. and Lane, M.D. (1973) J. Biol. Chem. 248, 2275-2284