De novo fatty acid synthesis in eel-liver cytosol

Comp. Biochem. Physiol. Vol. 95B, No. 1, pp. 153 158, 1990

0305-0491/90 $3.00 + 0.00 © 1989 Pergamon Press plc

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D E N O VO F A T T Y A C I D SYNTHESIS IN EEL-LIVER CYTOSOL GABRIELE V. GNONI* and MARIA R. MUCI Laboratory of Biochemistry, Department of Biology, University of Lecce, 73100 Lecce, Italy (Tel: 0832 620602) (Received 31 May 1989)

Abstract--1. Some of the characteristics and cofactor requirements of acetyl-CoA carboxylase and fatty acid synthetase, both of which show remarkable activity in eel-liver cell sap, were investigated. 2. For optimal activity, acetyl-CoA carboxylase needed preincubation with citrate and was inhibited by avidin and palmityl-CoA. The latter inhibition was reversed by increasing citrate concentrations. 3. Fatty acid synthetase required NADPH as hydrogen donor. Among NADPH furnishing enzymes, the highest activity was associated with glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase. 4. The ratio of total radioactivity to radioactivity in carboxyl carbon of the synthesized fatty acids indicated that a de novo mechanism of fatty acid synthesis is operative in eel-liver cytosol.

INTRODUCTION De novo fatty acid synthesis is catalyzed by two enzymatic systems which function in sequence: acetylC o A carboxylase and the multienzyme complex fatty acid synthetase, this last converting malonyl-CoA produced by the former enzyme to long chain fatty acid. Much accumulated evidence concerning mammalian and avian lipogenesis suggests that acetylC o A carboxylase (EC 6.4.1.2), which catalyzes the ATP- and bicarbonate-dependent carboxylation of acetyl-CoA to form malonyl-CoA, plays a critical role in the regulation of this synthetic process (for review see Volpe and Vagelos, 1976). Acetyl-CoA carboxylase from mammalian tissues is activated by tricarboxylic acids such as citrate, while long-chain acyl-CoA thioesters inhibit this enzyme (Volpe and Vagelos, 1976). Furthermore the tissue contents of both these compounds vary under different metabolic and hormonal conditions, accompanied by changes in the rate of lipogenesis. This has been well documented with acetyl-CoA carboxylase from rat liver (Witters et al., 1979) and adipose tissue (Halestrap and Denton, 1974), rabbit mammary gland (Easter and Dils, 1968), as well as chicken liver (Goodridge, 1972). Lipogenesis in fish, on the other hand, has been much less investigated (for review see Walton and Cowey, 1982; Henderson and Tocher, 1987). However, the dynamics of both membrane structure and function point to the complex role of fatty acids in environmental adaptation (Bell et al., 1986). In fact it has been shown (Farkas and Csengeri, 1976) that fish very rapidly adjust the pattern of synthesized fatty acids to the prevailing temperature in order to

*Author to whom correspondence should be addressed at: Dipartimento di Biologia, Laboratorio di Chimica Biologica, Universitfi di Lecce, Via Monteroni, 73100 Lecce, Italy. caP,B)

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assure in this way the proper physico-chemical properties of membranes. The liver has been indicated as the major site of fatty acid synthesis in fish (Lin et al., 1977; Henderson and Sargent, 1981). To date, no study on the characteristics of eel de novo fatty acid synthesis has been carried out, in spite of the fact that the eel retains at least some of its ability to synthesize lipids from endogenous sources, even during a prolonged period of fasting (Abraham et al., 1984). Therefore the aim of the present investigation was to study some properties of both acetylC o A carboxylase and fatty acid synthetase in eel-liver cell sap. MATERIALS A N D M E T H O D S

Preparation of enzyme systems

Yellow eels (Anguilla anguilla), grown in sea water, were purchased from a local commercial source, Ittica-UgentoLecce, and kept in sea-water aquariums. All preparatory procedures were carried out at 0~4°C. As soon as the eels (150-300 g) were killed, the liver was removed, finely minced and washed several times in cold 0.25 M sucrose containing 3mM EDTA, 15mM fl-mercaptoethanol and 20mM Tris-HC1 (pH 7.0). Liver homogenate, prepared in the same medium (l g liver in 2 ml), was centrifuged at 20,000g for 15 min. The resultant supernatant was again centrifuged at 105,000g for 60min (Spinco L-5 50, Beckman). The clear supernatant fraction (cytosol), removed with a Pasteur pipette from beneath the upper fat layer, was used for the enzymatic assays. Protein was determined according to Lowry et al. (1951) using defatted albumin as the standard. Enzyme assays were carried out immediately. Determination o f enzyme activities

Acetyl-CoA carboxylase was assayed essentially as previously described (Gnoni et al., 1983). Unless otherwise indicated, acetyl-CoA dependent CO 2 fixation was assayed in a reaction mixture (0.8 ml) containing: 75 mM Tris-HCl (pH 7.0), 6mM fl-mercaptoethanol, 10mM Na-citrate, 10 mM MgCI2, 2 mg defatted albumin and 1.3 mg cell sap protein. After 15 min of preincubation, the reaction was started by the addition of 2 mM ATP, 0.12 mM acetyl-CoA and 12.5mM NaWaCO3 (0.5mCi/mmol). After 3min, 153

154

GABRIELEV. GNONI and MARIA R. MUCI

the reaction was blocked by precipitation of the protein with 0.2 ml 7 M HCI. The precipitate was separated by centrifugation, an aliquot (0.4 ml) of the supernatant removed, dried in a scintillation vial at 6 0 C under N 2 and radioactivity counted. All assays were performed in triplicate. Blank values were determined by omitting acetylCoA from the reaction mixture. Specific activity was expressed as nmol of [1J4C]malonyl-CoA formed/rain x mg protein. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6phosphogluconate dehydrogenase (EC 1.1.1.44), malic enzyme (EC 1.1.1.40), isocitrate dehydrogenase (NADP) (EC 1.1.1.42) and ATP citrate lyase (EC 4.1.3.8) activities were assayed as previously indicated (Gnoni et al., 1983). Fatty acid synthetase (FAS) activity was determined as described (Landriscina et al., 1976). The incubation mixture (lml) was: [2J4C]malonyl-CoA 30pM (2~Ci//zmol), acetyl-CoA 12.5 #M, NADPH 0.5 mM, #-mercaptoethanol 10 raM, ATP 1 mM, MgCI 2 10 mM, Tris HC1 80 mM (pH 7.0) and 1.2 mg cell sap protein; incubation time was 10 min. The specific activity of fatty acid synthetase was expressed as nmoles of [2-NC]malonyl-CoA incorporated into fatty acids/min × nag protein. To determine the ratio: total radioactivity/radioactivity in carboxyl carbon (TR/CR), [I-t~C]acetyl-CoA instead of [2-14C]malonyl-CoA was used as labelled precursor for fatty acid synthesis. In this case the incubation medium was: [l-~4C]acetyl-CoA 75#M (2/~Ci/#mol), ATP 4mM, NADPH 0.5mM, MgC12 10mM, NaHCO 3 60mM, Na citrate 10mM, Tris HCI 60mM (pH 7.0) and 1.5mg protein. Reaction time was 10 min.

To measure the total radioactivity/carboxyl radioactivity ratio, another aliquot of the combined extracts of fatty acids was dried and then decarboxylated as reported (Landriscina et al., 1970). A known amount of [1-~C]palmitic acid was decarboxylated under identical conditions as a control. The radioactivity recovered in the trapped CO 2 was almost equal to the theoretical value. RESULTS

AND

DISCUSSION

In Fig. 1 the effect of protein c o n c e n t r a t i o n a n d time of i n c u b a t i o n on the activity of acetyl-CoA carboxylase is reported. Figure I A shows that the enzymatic activity increased linearly up to a b o u t 2.0 mg of protein a n d Fig. 1B indicates that after a relatively fast initial phase of 2-4 min, m a l o n y l - C o A f o r m a t i o n rate did not increase parallel with the time of incubation. Optimal assay t e m p e r a t u r e was found to be 30:C (data n o t shown). W h e n the reaction mixture was incubated under conditions where acetyl-CoA carboxylase was linear with respect to b o t h time (3 rain) a n d protein concentration (1.3 mg), the enzyme showed maximal activity at pH 7.0 (Fig. 2). This value is slightly lower t h a n the o p t i m u m (pH 7.5) of chicken (Gregolin et al., 1968) and rat-liver enzyme (pH 7.8) (Allred a n d Roehrig, 1980), but equal to that found for catfish-liver acetylC o A carboxylase ( W a r m a n and Bottino, 1978). It is generally accepted that acetyl-CoA carboxylase exists in two c o n f o r m a t i o n a l states, i.e. a protomeric inactive and a polymeric active form. Usually citrate was added to the preincubation m e d i u m since it has been reported that the enzyme from rat liver a n d m a m m a r y gland, as well as adipose tissue, requires lengthy exposure to citrate to become fully active (Volpe and Vagelos, 1976). Figure 3 shows that when acetyl-CoA carboxylase was preincubated for 15 min with the indicated dose of citrate, maximal activation was o b t a i n e d at concentrations a r o u n d 5-10 m M . It is worth underlining that the preincub a t i o n with citrate increased carboxylase activity

Extraction and decarboxylation ~/'/btty acids

Fatty acid synthetase activity was blocked by the addition of 0.7 ml 10 M KOH and 2 ml ethanol. The mixture was then saponified at 9(YC for 1 hr under N2. After cooling, the non-saponifiable compounds were extracted with two successive additions of 5 ml petroleum either (b.p. 4 0 ~ 0 ' C ) and discarded. The aqueous phase was acidified to pH 2 3 to liberate fatty acids and these were then extracted with five successive additions of 5 ml light petroleum. An aliquot of the combined extracts of fatty acids was evaporated under N 2 and the radioactivity counted after the addition of scintillation mixture.

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Fig. 4. Effect of increasing Mg :+ concentrations on eel-liver acetyl-CoA carboxylase activity.

almost twice compared to the addition of citrate at the time of incubation (data not shown). Our data on citrate activation of acetyl-CoA carboxylase largely agree with those generally accepted from mammalian and chicken-liver enzyme (Volpe and Vagelos, 1976) and, as concerns fish, with those reported for catfish carboxylase (Warman and Bottino, 1978). On the other hand, they are different from results obtained by Iritani et al, (1984), who found that sea-bream Pagrus major, liver acetyl-CoA carboxylase was not activated by citrate and showed very little activity both in the absence and presence of citrate. In many fish species, particularly the eel, the liver is an important store of fat, triacylglycerols accounting for as much as 46% of dry wt of eel hepatocytes (Jankowsky et al., 1984). Moreover, lactate has been shown to be actively incorporated into

the glycerol moiety of triacylglycerols in eel hepatocytes (Renaud and Moon, 1980). Data reported here, indicate that acetyl-CoA carboxylase activity from eel liver is comparable with that measured in rat liver and higher than that observed in other rat tissues under similar experimental conditions (Gnoni et al., 1980, 1983). Furthermore, the remarkable acetylCoA carboxylase activity we found could account for the active lipogenesis from acetate observed both in vitro and in vivo experiments in liver of European eel (Hansen and Abraham, 1983). A dose-response curve for Mg 2+ is shown in Fig. 4, where it can be seen that eel-liver acetyl-CoA carboxylase, inactive in the absence of Mg 2÷, showed optimal activity in the presence of 10 mM of the ion. ATP is a substrate for acetyl-CoA carboxylase as well as an effector. Figure 5 shows the effect of

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156

GABR1ELEV. GNONIand MARIAR. Mucl

increasing ATP concentrations on eel-liver acetylCoA carboxylase activity; the optimal ATP concentration was found to be 2 mM. The sensitivity of eel-liver acetyl-CoA carboxylase to palmityl-CoA is reported in Fig. 6, where an approx. 50% inhibition of enzyme activity at 25 #M of the drug was found. This concentration was higher than that which inhibited acetyl-CoA carboxylase from mammalian liver (Nikawa et al., 1979), adipose tissue (Halestrap and Denton, 1974) and lung (Maniscalco et al., 1982), probably because more purified enzymes were used in the above-mentioned studies. Several investigators have questioned the physiological role for the regulation of enzymes that fatty acyl-CoA thioester can play, since this metabolite at high concentrations can inhibit many enzymes irreversibly, because of its detergent properties (Volpe and Vagelos, 1976). The results reported in Fig. 6 indicate that palmityl-CoA (100#M) inhibition of acetyl-CoA carboxylase was gradually removed by simultaneous preincubation with increasing citrate concentrations. At this point it is worth recalling that fasting, which should enhance the level of acyl-CoAs in liver cell, strongly inhibited eel-liver acetyl-CoA carboxylase as well as total fatty acid synthesis from acetate (Hansen and Abraham, 1983). In Coho salmon, too, a reduced lipogenesis after both fasting or high fat diet was observed by Lin et al. (1977). In addition, they noted that refeeding fasted fish with a high carbohydrate diet, a state which in mammals and birds is linked to a rise in citrate level and a fall in long-chain acyl-CoA derivative concentrations (Volpe and Vagelos, 1976), increased the in vitro and in vivo rates of fatty acid synthesis in fish liver. To our knowledge, our data show for the first time that an in vitro regulation of fish acetyl-CoA carboxylase activity by these two allosteric modulators can occur. The opposite effect of citrate and palmityl-CoA on this enzyme could therefore represent a mechanism of eel-liver lipogenesis regulation. Avidin can readily and irreversibly bind to the biotinyl prosthetic group of most carboxylases, thereby blocking catalytic acitvity. Figure 7 shows that avidin, when preincubated with the enzyme at

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Fig. 7. Inhibition of acetyl-CoA carboxylase activity as a function of avidin concentration. ~ ~ Avidin preincubated with the enzyme for 5 min before the assay. © © Avidin added after activation of the enzyme were preincubated for 15 min with 10mM of citrate. 0"C for 5 min before starting the assay, strongly inhibited acetyl-CoA carboxylase activity. A 50% inhibition was already reached in the presence of 3-4 #g of the drug, the reaction being almost completely inhibited at avidin content of 50 #g. On the other hand, at all the tested avidin concentrations (5-100 #g), no inhibition was observed when the drug was added to the enzyme previously activated by citrate, suggesting that, in its polymeric form, fish acetyl-CoA carboxylase was resistant to avidin inactivation. Eel-liver fatty acid synthetase From 1 up to 3mg protein were required for optimal activity of eel-liver fatty acid synthetase (FAS) as shown in Fig. IA; the incorporation of labelled malonyl-CoA into fatty acids increased with the incubation time up to 15 rain (Fig. 1B). The enzyme showed optimal activity at pH 7.0, a value similar to that found for FAS purified from liver of plaice, Pleuronectes platessa (Wilson and Williamson, 1970). Figure 8 shows the rate of [2-14C]malonyl-CoA incorporation into fatty acids by eel-liver cell sap in the presence of various concentrations of NADH, NADPH, or NADH plus NADPH as hydrogen donors. Eel-liver FAS was dependent on the presence of NADPH in the reaction medium; the maximum stimulation was observed at a coenzyme concentration of 0.25 raM. When both NADPH and NADH were added, FAS activity was very close to that observed in the presence of only NADPH. Practically no effect of NADH on FAS activity could be detected at all the tested concentrations. Other enzymes related to fat O, acid synthesis In Table 1 the specific activities of other enzymes associated with lipogenesis, i.e. ATP-citrate lyase (responsible for the generation of extramitochondrial acetyl-CoA) and those which furnish NADPH for fatty acid synthesis [glucose-6-phosphate

Eel-liver fatty acid synthesis

157

Table 2. Characteristics of fatty acid synthesis in eel-liver cytosol starting from a differently labelled precursor T.R./C.R. Reaction system ratio nmol [2J4C]malonyl-CoA incorporated/min × mg protein Complete system 1.8 ± 0.13 -minus ATP 1.4 + 0.11 -minus NADPH 0.7 + 0.09 -minus MgCI2 1.3 _+0.10 --

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dehydrogenase, 6 - p h o s p h o g l u c o n a t e dehydrogenase, malic enzyme a n d ( N A D P ) isocitrate dehydrogenase] are reported. While a n active ATP-citrate lyase was f o u n d in C o h o salmon liver (Lin et al., 1977), only very low activities were detected in liver from rainbow trout, c h a n n e l catfish or in tissues o f the A m e r i c a n eel (Anguilla rostrata) (for review see H e n d e r s o n a n d Tocher, 1987). D a t a in Table 1, in agreement with t h a t reported by A b r a h a m et al. (1984), show a substantial activity of A T P citrate lyase in the liver of E u r o p e a n eel. As regards the N A D P H furnishing enzymes, the highest activity we f o u n d was associated with glucose-6-phosphate dehydrogenase a n d ( N A D P ) isocitrate dehydrogenase.

Mechanism o f f a t t y acid biosynthesis Finally in Table 2 the cofactor requirements o f de novo fatty acid synthesis from a differently labelled precursor are summarized. Starting from [2J4C]malonyl-CoA, a r e m a r k a b l e reduction o f fatty acid synthesis was observed when ATP, M g 2÷ or, especially, N A D P H was omitted from the reaction mixture. In order to investigate the m e c h a n i s m of fatty acid biosynthesis in eel-liver cell sap, [1J4C]acetyl-CoA instead o f [2J4C]malonyl-CoA was used as labelled

Table 1. Activity of enzymes associated with fatty acid synthesis in eel-liver cytosol Enzymes Specific activity Glucose-6 phosphate dehydrogenase 330 ± 38* 6-Phosphogluconate dehydrogenase 123 ± 18" Malic enzyme 64 ± 9* (NADP) isocitrate dehydrogenase 366 ± 32* ATP citrate lyase 52 __+8.5t Specific activity expressed as: *nmol NADPH formed/min x mg protein. +nmol NADH ox/min × mg protein. Results are the mean of five experiments _+S.D.

nmol [1J4C]acetyl-CoA incorporated/min ×mg protein Complete system 0.52 ± 0.06 7.2 : 1 minus ATP 0.14 ± 0.03 6.8:1 minus NADPH 0.28 ± 0.02 7.5 : 1 minus MgCI2 0.11 ± 0.009 -minus NaHCO 3 0.07 ± 0.008 -Data are the mean of six experiments ±S.D.; T.R./C.R. =total radioactivity/carboxylradioactivity ratio of the synthesizedfatty acids.

precursor. Obviously starting from acetyl-CoA, b o t h acetyl-CoA carboxylase a n d fatty acid synthetase were operative. The ratio total fatty acid [1-1ac] i n c o r p o r a t i o n / c a r b o x y l [1-14C] i n c o r p o r a t i o n ( T R / C R ) was utilized to indicate the p a t h w a y by which [1J4C]acetyl-CoA was incorporated into fatty acids. The high T R / C R ratios ( a b o u t 7.0:1) here reported, clearly indicated that a de novo m e c h a n i s m of fatty acid synthesis was operative in eel-liver cytosol. As expected, w h e n acetyl-CoA was used as the labelled precursor for fatty acid synthesis, an absolute dependence on bicarbonate, A T P a n d M g 2+ was observed. C o n t r a d i c t o r y results, p r o b a b l y depending o n fish species ( W a r m a n a n d Bottino, 1978; A b r a h a m et al., 1984; Iritani et al., 1984; F a r k a s a n d Csengeri, 1976) have so far been reported on the capacity o f fish to synthesize fatty acids from a n e n d o g e n o u s source. T a k e n together, o u r results indicate that in eel-liver cell sap an active de novo fatty acid synthesis occurs. M o r e o v e r this pathway shows a n in vitro regulation qualitatively similar to t h a t used by more evolved birds a n d m a m m a l s . REFERENCES

Abraham S., Hansen H. J. M. and Hansen F. N. (1984) The effect of prolonged fasting on total lipid synthesis and enzyme activities in the liver of European eel (Anguilla anguilla). Comp. Biochem. Physiol. 79B, 285-289. Allred J. B. and Roehrig K. L. (1980) Inhibition of rat liver acetyl-CoA carboxylase by chloride. J. Lipid Res. 21, 488-491. Bell M. V., Henderson J. R. and Sargent J. R. (1986) The role of polyunsaturated fatty acids in fish. Comp. Biochem. Physiol. 8311, 711-719. Easter D. J. and Dils R. (1968) Fatty acid biosynthesis IV. Properties of acetyl-CoA carboxylase in lactatingrabbit mammary gland. Biochim. biophys. Acta 152, 653-668. Farkas T. and Csengeri I. (1976) Biosynthesis of fatty acids by the carp, Cyprinus earpio L., in relation to environmental temperature. Lipids 11, 401-407. Gnoni G. V., Landriscina C. and Quagliariello E. (1980) Fatty acid biosynthesis in adipose tissue and lung subcellular fractions of thyrotoxic rats. FEBS Lett. 122, 37-40.

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Landriscina C., Gnoni G. V. and Quagliariello E. (1976) Effect of thyroid hormones on microsomal fatty acid chain elongation synthesis in rat liver. Eur. J. Biochem. 71, 135-143. Lin H., Romsos D. R., Tack P. I. and Leveille G. A. (1977) Influence of diet on in vitro and in vivo rates of fatty acid synthesis in Coho salmon Oncorhynchus kisutch (Walbaum). J. Nutr. 107, 1677-1682. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Maniscalco W. M., Finkelstein J. N. and Parkhurst A. B. (1982) De novo fatty acid synthesis in developing rat lung. Biochim. biophys. Acta 11, 49-58. Nikawa J., Tanabe T., Ogiwara H., Shiba T. and Numa S. (1979) Inhibitory effects of long-chain acyl Coenzyme A analogues on rat liver acetyl-Coenzyme A carboxylase. FEBS Lett. 102, 223-225. Renaud J. M. and Moon T. W. (1980) Characterization of gluconeogenesis in hepatocytes isolated from the American eel, Anguilla rostrata Le Sueur. J. Comp. Physiol. 135, 115-125. Volpe J. J. and Vagelos P. R. (1976) Mechanism and regulation of biosynthesis of saturated fatty acids. Physiol. Rev. 46, 339-417. Walton M. J. and Cowey C. B. (1982) Aspects of intermediary metabolism in Salmonid fish, Comp. Biochem. Physiol. 73B, 59-79. Warman A. W. III and Bottino N. R. (1978) Lipogenic activity of catfish liver. Lack of response to dietary changes and insulin administration. Comp. Biochem. Physiol. 59B, 153-161. Wilson A. C. and Williamson I. P. (1970) Fatty acid synthetase from the plaice, Pleuronectes platessa. Biochem. J. 117, 26P-27P. Witters L. O., Moriarity D. and Martin D. B. (1979) Regulation of hepatic acetyl-Coenzyme A carboxylase by insulin and glucagon. J. biol. Chem. 254, 6644-6649.