Diurnal variations of lipogenic enzyme mRNA quantities in rat liver

Biochimica et Biophysica Acta. 1086(1991) 261-264

26 l

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Diurnal variations of lipogenic enzyme mRNA quantities in rat liver Hitomi Fukuda and Nobuko lritani Tezukayama Gakuin College. Hanlmidai. Sakai. Osaka (Japan)

(Received5 February 1991) (Revisedmanuscriptreceived23 July Iqql ) Key words: Lipogenicenzyme:mRNAquantity:Diurnalvariation;(Rat liver) The diurnal variations in mRNA quantities of lipogenic enzymes (acetyI-CoA carboxylase, fatty acid s-ynthase, malic enzyme and glucose-6-phosphate debydrogenase) in rat livers were detected. When the rats began feeding actively after lights out at 1900 h, the mRNA quantities were high from 0500 h to 0900 h in the morning. The variation in fatty acid synthase mRNA quantities was the most dramatic. However, no measurable variation in any enzyme levels including fatty acid synthase was detected. It may be because the half-lives of the enzymes are too long to be effected by the mRNAs which were high for several hours.

Introduction

Materials and Methods

We previously reported that, when rats began feeding actively after lights out at 1900 h, the in vivo incorporation of tritiated water into fatty acids in the liver were high from 2200 h to 0100 h [1]. Kimura et al. [2] also found a similar result that, when the incorporation of intraperitoneally injected [l-t4C]acetate into total lipids of rat liver was measured, the dirnal variation of the lipogenesis was essentially similar to that of food intake. Other similar results were observed by Cornish et al. [3] in the incorooration of tritiated water into fatty acids in livers and adipose tissues of mice. However, we previously found that the enzyme activities of lipogenic enzymes did not vary over a 24 h period [1]. Since the hall-lives of the lipogenic enzymes were 1-3 days in various metabolic conditions [4-8], diurnal variation of the enzyme levels does not seem to be detected. On the other hand, as the half-lives of the m R N A s were 5 - 6 h [5-8], it is possible that the m R N A quantities have diurnal variation. Thus, in the present experiment, we have examined the diurnal variation in the m R N A quantities of acetyI-CoA carboxylase (EC 6.4.1.2), fatty acid synthase (EC 2.3.1.85), malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49).

Animals

Correspondence: N. Iritani, Tezukayama Gakuin College, 4-cho, Harumidai, Sakai, Osaka, 590-01,Japan.

Male Wistar rats (Japan SLC), 5 weeks old, were used for the experiment. The rats were fed a fatfree/high-carbohydrate diet for 1 week before the experiment. The fat-free diet ccatained 67.4% sucrose, 18% casein, 9.5% cellulose, 5% salt mixture, 0.1% choline chloride and vitamins [9]. The animals were allowed to take the diet and water ad libitum, and kept under an automatic lighting schedule from 0700 h to 19G0 h at 24°C. The rats were killed at 1900, 2300, 0500, 0900, 1500 and 1700 h. An aliquot of each liver was quickly removed and homogenized with 3 vol. of 0.25 M sucrose to measure the activities of lipogenic enzymes in 105000×g supernatant. Another aliquot of the liver was immediately frozen and total RNA was extracted from the frozen liver. Dot-blot hybridization assay

AcetyI-CoA carbozylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase eDNA inserts were cloned as described in our previous reports [5,6,8,10]. The cDNA labeling was carried out with ~2P-dCTP to a specific activity of 0.7-1.0-10 s cpm/p,g by using multiprimer DNA labeling system kit (Amersham). Total RNA was isolated from the liver with guanidine thioeyanate [I I]. To measure the m R N A quantities of lipogenic enzymes, the total RNA (5-10 /,~g) was denatured with formamide at 65°C for 15 min,

262 spotted on nylon filter. To measure fl-actin mRNA quantity, 4 p.g of the RNA was used. The filter was radiated with ultraviolet light for 5 rain, and then prehybridized at 50°C for at least 4 h in a reaction mixture of 50 mM Tris-HC[ (pH 7.5), 6 × SSC(I × SSC: 0.15 M NaCI, 0.015 M sodium citrate), 5 × Denhardt's solution, 1% SDS and 100 tzg/ml of denatured salmon DNA (medium A). 5 x Denhardt's solution contained 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin. The hybridization was carried out for 36 h at 50°C in medium A with each riP-labeled lipogenic enzyme eDNA or /3-actin ,;DNA. The filter was then washed with 0.1 × SSC and 0.1% SDS at room temperature, and three times at 55°C. The filters were exposed for varying lengths of times at - 70°C to Kodak X-Omat AR film with Du pont Lighting-Plus intensifying screens. Relative densities of the hybridization signals were determined by scanning the autoradiograms at 525 nm (Model CS-9000, Simadzu, Japan). Lipogenic enzyme acticities Malic enzyme was assayed according to Ochoa [12] and glucose-6-phosphate dehydrogenase activity was assayed according to Glock and McLean [13]. AcetylCoA carboxylase was assayed by the HI4CO3 fixation method [14,15]. When liver was homogenized with the inhibitors of phosphatase and proteinase (0.02 M TrisHCI pH 7.4, 0.25 M sucrose, 0.1 M sodium fluoride, 0.01 M 2-mercaptoethanol, 2 mM EDTA and 0.5 mM phenylmethylsulfonyl fluoride) for the preparation of acetyl-CoA carboxylase, the enzyme activity was assayed using 0.5 mM citrate [14]. In addition, another aliquot of the liver was homogenized without the inhibitors and then acetyl-CoA carboxylase activity was assayed under optimum condition using 10 mM citrate [15]. Fatty acid synthase activity was assayed according to Hsu [16]. Enzyme activities were exnressed as nmol substrate utilized for glucose-6-phosphate dehydrogenase and malic enzyme, or product formed for acetyl-CoA earboxylase and fatty acid synthase per rain at 37 o C per mg protein of ~he 105 000 × g supernatant of liver homogenate. Results and Discussion When rats were fed a fat-free/high-carbohydrate diet ad libitum and maintained in equal periods of light (0700-1900 h) and dark (1900-0700 h), they ate most actively from 1900 h to 0500 h (Fig. 1). The amount of food in the stomach was the largest from 2300 h to 0900 h. These data essentially coincided with uur plevious results [1]. The diurnal variations in mRNA quantities of acetyI-CoA carboxylase, fatty acid synthase, glucose-6phosphate dehydrogenase and malic enzyme in rat

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0700 19oo clock ttme (h) Fig. I. Diurnal variationsof mRNA quanlities of liposenieenzymes in rat liver. The animals,adapted to a fat-free/high carbohydrate diet, were killed at the times indicated in the figure. The mRNA quantities are normalized to the values at 1900 h. The experiment was repeated four timesand one of the typicalresultsis shownin the figure. Values are mean:l:S.D. (,-3). Meanswith different superscript letters are significantly different at P < 0.05 at least (by one-way analysisof variance). The densitometric units at I in fold change were 4.69, 3.48, 4.13, 11.5 and 2.35, respectively, for aceWICoA carbo~lase, fatty acid synthase,malic enzyme,glucme-6-phosphale dehydrogenaseand/]-actin. Food intake (g/h) is shownin the bottom.

livers are shown in Fig. 1. The experiments were repeated four times. Typical data for one experiment is shown in Fig. 1. The mRNA quantities were low at 1900 h and high during 0500-0900 h. The variation of fatty acid synthase mRNA was the most dramatic. Fatty acid synthase mRNA quantities varied 5-fold in one day and the others 2-3-fold. In addition, the data from all four experiments are shown in Table 1. As standard deviations of means for the four experiments tended to be large, minor differences were observed between the typical data for one experiment (Fig. 1) and the means of all four experiments (Table 1). There was no diurnal variation in the quantities of /3-actin mRNA. Thus, the diurnal variations in lipogenic enzyme mRNAs were demonstrated.

263 T h e acetyI-CoA carboxylase activity m e a s u r e d u n d e r o p t i m u m conditions using l0 m M citrate should be a c c o m p a n i e d by p r o p o r t i o n a t e c h a n g e in q u a n t i t y of i m m u n o c h e m i c a l l y rezctive p r o t e i n [15]. However, the aeetyI-CoA carboxyla~c activity, which w a s extracted with inhibitors of p h o s p h a t a s e a n d p r o t e i n a s e , a n d m e a s u r e d using 0.5 m M citrate, s h o u l d be close to the physiological activity [14,17]. N o diurnal variation in activities o f acetyI-CoA carboxylase w a s o b s e r v e d in e i t h e r case (Fig. 2). N o r w e r e a n y significant variations in e n z y m e activities o f fatty acid synthase, malie enzyme a n d g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e observed in f o u r experiments. T h e half-lives o f hepatic lipogenic enzymes w e r e 2 - 3 days in fed states [4]. H o w e v e r , the m R N A s w e r e in high levels for only 4 h o r so in a o n e d a y period. O n the o t h e r h a n d , in f a s t e d - r e f e d rats, the half-lives o f the m R N A s w e r e c a l c u l a t e d to b e 4 - 6 h a n d those o f the enzymes 2 2 - 2 3 h [5-8]. It a p p e a r e d t h a t the half-lives of the enzymes w e r e too long for the e n z y m e s to be i n c r e a s e d by the m R N A s . If the p e r i o d o f high m R N A c o n c e n t r a t i o n s lasted longer, the e n z y m e levels m a y be influenced. In addition, the m R N A q u a n t i t i e s o f acetyI-CoA carboxylase, fatty acid synthase, malic e n z y m e a n d glucose-6p h o s p h a t e d e h y d r o g e n a s e in polysomes o f the livers w e r e a p p r o x i m a t e l y p r o p o r t i o n a l to the total m R N A quantities ( d a t a not shown). A s the polysomal m R N A q u a n t i t y m a y be a n index o f t r a n s l a t i o n a l activity, the t r a n s l a t i o n a l activities o f the lipogenic e n z y m e s d o not a p p e a r to vary over 24 h period. W e previously r e p o r t e d that, w h e n r a t s b e g a n feeding actively a f t e r lights o u t at 1900 h, fatty acid synthesis r e a c h e d m a x i m u m at 2200 h a n d w a s high until 0530 h [1]. T h e e n z y m e c o n t e n t s c a n not c h a n g e so rapidly [ 4 - 8 ] , b u t the catalytic activity c a n c h a n g e rapidly. T h e catalytic activity o f a c e t y I - C o A carboxylase is k n o w n to be activated by citrate [18]. W h e n f a s t e d rats w e r e r e f e d a h i g h - c a r b o h y d r a t e diet, the h e p a t i c c o n c e n t r a tion o f citrate w a s rapidly i n c r e a s e d [19]. T h e r e f o r e , it is s u g g e s t e d t h a t the r a p i d rise in h e p a t i c fatty acid

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Fig. 2. D i u r n a l variations of enzyme activities o f lipogenie enzymes in rat liver. The enzyme activities are shown as m U / m g protein at

3"PC. I mU is defin,:d as l nmol product formed per rain for acetyI-CoA earboxylase and fatty acid s~nthase, ol substrate utilized for malic enzyme and glucose-6-phosphate dehydrogenase. For extraction of acctyI-CoA carbozylase, liver was homogenized with (broken line) or without (solid line) inhibitors of phospha)ase and proteinase, as de~ribed in Materials and Methods. The acetyI-CoA cadx)zylase act±riB' was assayed using 0.5 mM (broken line) or 10 mM (~,olid line) citrate. The experiment was repeated four times and one of the typical results is shown in the figure. Values are mean± S.D. (n = 3). The animals were the ~me as those shown in Fig. 1. synthesis o b s e r v e d a f t e r s h o r t - t e r m r e f e e d i n g could be principally d u e to the m o d u l a t i o n o f the catalytic efficiency [19]. O n the o t h e r h a n d , we previously suggested t h a t even the maximal c o n c e n t r a t i o n s o f acetyI-CoA

TABLE I Diurnal rariations of mRNA quantities of lipogenic enzymes in rat lirer (mean rah.'x for all four experiments) The animals, adapted to a fat-free/high-carbohydrate diet, were killed at the times indicated. The mRNA quantities ~re normalized to the values at 1900 h. Numbers of animals used for four experiments were 12, expect 9 at 1500h. Mean + S.D. Means with different superscript letters are significantly different at P < 0.05 at least (by one-way analysis of variance). Clock time (h) (h) 1900 2300 0500 0900 1500 1700

Fold change AcetyI-CoA carboxylase

Fatty acid synthase

Malic enzyme

1.00+0.20 a 0.97+0.19 :~ 2.05+0.25 b 2.24 ± 0.19 b 1.77± 0.64 ~*' 0.96±0.09 ~

1.00±0.37 ~ 1.49±037 ~ 4.13±0.46 t, 4.58±0.84 b 2.18 ± 0.46 :' 1.74:[:0.57 ~

1.00±0.31 " 0.83 + 0.31 " 1.79+0.27 b 2.01 ::t:t1.34b 1.51+ 0.78 J.b 1.12+11.38~

Glucose-b-phosphate dehydrogenase 1.(10=1:0.31a 0.56±0.34 a 1.74+0.33 t, 1.35± 0.26 a.b 1.10± 0.16 ~ 0.91 ±0.33 ~

/3-Actin

I.I)0±0.28 a 0.98±0.07 ~ I.I;4+0.2,5 ° 0.97±0.03 " 0.92 ± 0.05 " 0.%±0.10 ~

264 and citrate in rat livers would be lower than the K m and K a values, respectively [1]. T h e hepatic levels of malonyI-CoA, a product of acetyl-CoA carboxylase increased after food intake and paralleled the fatty acid synthesis from tritiated water. T h e r e f o r e , the diurnal variation in hepatic fatty acid synthesis a p p e a r e d to be ascribed to a change in catalytic activity of acetyI-CoA carhoxylase. In the present study, we have found that, although the m R N A quantities of lipogenic enzymes vary in one day, the diurnal variations have no m e a s n r a b l e effects on the enzyme levels. It may be because the half-lives of the enzymes are too long to he affected by the m R N A s which were high for several hours. Thus, the diurnal variations in m R N A quantities of lipogenic enzymes should not contribute to the variation of fatty acid synthesis. References 1 Fukuda, H., Katsurada, A. and Iritani, N. (1985) Biochim. Biophys. Acta 835, 163-168. 2 Kimura, T., Maji, T. and Ashida, K. (1970) J. Nutr. 100, 691-697. 3 Cornish, S. and Cawthrone, M. A. (1978) Horm. Metab. Res. 10, 286-290. 4 Iritani, N., Fukuda, H. and Fukuda, E. (1981) Biochim. Biophys. Acta 665, 636-639.

5 Katsurada, A., lritani, N., Fukuda, H., Matsumura, Y., Nishimoto. N., Noguchi, T. and Tanaka, T. (19901 Eur. J. Biochem. 190, 435-441. 6 Katsurada, A.. Iritani, N., Fukuda. H., Matsumura, Y., Nishimoto, N., Noguchi, T. and Tanaka, T. (1990) Eur. J. Biochem. 190, 427-433. 7 Fukuda. H., Katsurada, A. and lritani, N. (1990) Eur. J. Biochem. 188, 517-522. 8 Katsurada, A.. Iritani, N., Fukuda. H., Noguchi. T. and Tanaka, T. (1990) Biochim. Biophys. Acta 1006. 104-110. 9 Iritani, N., Fukuda, E. and Inoguchi, K. (1979) Atherosclarosis 34. 41-47. 10 Katsurada, A., Iritani, N., Fukuda, H.. Noguchi, T. and Tanaka, T. 0987) Eur. J. Biochem. 168, 487-491. 11 Chirgwin,J.M., Przybyla.A.E., Macdonard, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. 12 Ochoa, S. (1955) Methods Enzymol. I, 739-753. 13 GIock, G.E. and McLean, P. (1953) Biocham. J. 55, 400-408. 14 Holland, R., Witters, L.A. and Hurdle, D.G. (1984) Eur. J. Biochem. 140, 325-333. 15 Nakanishi, S. and Numa, S. (1970) Eur. J. Biochem. 16, 161-173. 16 I-Isu,R.Y., Butterworth, P.H.W. and Porter J.W. (1969) Methods E:lzym01. 14, 33-39. 17 Wada, k. and Tanabe, T. (1983) Eur. J. Biochem. 135,17-23. 18 Numa, S., Nakanishi, S., Hashimoto, T., Iritani, N. and Okazaki, T. (1970) Vitam, Horm. 28, 213-243. 19 Nishikori, K., Iritani, N. and Numa, S. (1973) FEBS Lett. 32, 19-21.