Effect of fatty acids on the synthesis and secretion of apolipoprotein Β by rat hepatocytes

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1 J. Biosci., Vol. 17, Number 4, December 1992, pp Printed in India. Effect of fatty acids on the synthesis and secretion of apolipoprotein Β by rat hepatocytes Ν SURESH KUMAR, RITA ABRAHAM, G SURESH KUMAR, Ρ R SUDHAKARAN* and Ρ A KURUP Department of Biochemistry, University of Kerala, Kariavattom, Trivandrum , India MS received 27 January 1992; revised 29 June 1992 Abstract. The modulation of apolipoprotein Β synthesis and secretion by fatty acids in rat hepatocytes was studied Maximum apolipoprotein Β production was obtained in the case of oleic acid followed by linoleic, stearic and palmitic/linolenic acid when compared to control which was not supplemented with any fatty acids. Oleic acid was found to exert a concentration dependent increase in the secretion of [ 3 H] apolipoprotein Β into the medium while that associated with the cell layer was not affected. Pulse chase experiments in the presence of oleic acid showed that it caused an increase in the secretion of apolipoprotein Β into the medium. 14 C-acetate incorporation into cholesterol and cholesteryl ester associated with the cell layer and secreted very low density lipoproteins also showed an increase in the presence of oleic acid indicating an increase in cholesterogenesis. The effect of oleic acid on [ 3 H] apolipoprotein Β and very low density lipoproteins secretion appeared to be mediated through cholesterol as (i) ketoconazole, an inhibitor of cholesterol synthesis caused significant reduction in the stimulatory effect of oleic acid on apolipoprotein secretion and (ii) mevinolin, another inhibitor of cholesterol synthesis also reversed the stimulatory effect of oleic acid on apolipoprotein Β secretion. These results indicated that oleic acid may influence apolipoprotein Β synthesis and secretion in hepatocytes probably by affecting cholesterol/cholesteryl ester formation which may be a critical component in the secretion of apolipoprotein Β as lipoproteins. Keywords. Hepatocytes; oleic acid; apolipoprotein Β synthesis; modulation by cholesterol. 1. Introduction The very low density lipoproteins (VLDL) synthesised and secreted by liver are primarily involved in the transport of triacylglycerol of endogenous origin from liver to other tissues. Isolated hepatocytes maintained in primary culture are a useful system to study the synthesis and secretion of triglyceride rich VLDL (Davis et al 1979). The availability of fatty acids, the primary substrate for triglyceride (TG) synthesis has been shown to increase the synthesis and secretion of TG as VLDL (Kohout et al 1971; Davis and Boogaerts 1982; Field et al 1988). Though different fatty acids exert different degrees of stimulation, it has been shown that oleic acid has the maximum stimulatory effect (Field et al 1988). While Pullinger et al (1989) and Moberly et al (1990) reported that oleic acid in addition to its effect on TG synthesis has also stimulated the synthesis of apolipoprotein Β (apob), the major apo protein of VLDL, Davis and Boogaerts (1982) did not observe any effect for oleic acid on apob synthesis. However, it is not known how the synthesis of apob that is taking place on the rough endoplasmic reticulum is coordinated with the synthesis of TG which is occurring in the smooth endoplasmic reticulum surface. *Corresponding author. 473

2 474 Ν Suresh Kumar et al It was postulated that the availability of cholesteryl ester which is synthesised by enzymes located in the rough endoplasmic reticulum rather than TG is a critical component in apob synthesis and its assembly and secretion as VLDL (Cianflone et al 1990). Recent reports from our laboratory (Kumar et al 1992) using hepatocytes and by others using HepG2 cells suggested that cholesterol ester is an obligatory requirement for synthesis and secretion of apob as VLDL. Therefore the effect of different fatty acids on apob synthesis and secretion of VLDL and the possibility that change in VLDL production in response to fatty acid challenge is mediated through cholesterol was examined using isolated rat hepatocytes in primary culture. 2. Materials and methods Oleic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid and ketoconazole were purchased from Sigma chemicals (USA). Foetal bovine serum was a product of Gibco. Tissue culture plastic wares were from NUNC, Denmark. Protein A- sepharose was from Pharmacia, Sweden. L- [ 3 H] leucine and [ 14 C] acetate were purchased from the Bhabha Atomic Research Centre, Bombay. 2.1 Preparation of hepatocytes Normal male adult rats (Sprague Dawley strain) weighing g were used for the isolation of hepatocytes by collagenase perfusion according to the procedure of Seglen (1976) as described before (Kumar et al 1992). Hepatocytes suspended in Eagles MEM supplemented with 10% FBS, penicillin (100 µg/ml), streptomycin sulphate 100 µg/ml) and insulin (0 1 nm) were plated in 35mm plastic petri dishes and maintained at 37 C in a 95% air 5% CO 2 atmosphere for about 4 h. The unattached cells were removed and cell monolayer was washed with serum free medium and incubated in serum free medium. The effect of in vitro addition of various substances was studied by incorporating them into the medium. 2.2 Metabolic labelling and immunoprecipitation of apob The cells in the basal medium were metabolically labelled with [ 3 H] leucine. At the end of the incubation period the medium was collected and cells were harvested in lysis buffer (0 2% SDS, 2mM EDTA, 2mM PMSF in 0 1 Μ Tris, ph 8 1). The [ 3 H] leucine labelled total apob secreted into the medium and that associated with the cell layer were immunoprecipitated by adding antisera raised against rat apob. The antigen-antibody complex was treated with protein A-sepharose as described before (Sudhakaran et al 1986) and was solubilized in Laemmli buffer and electrophoresed by the method of Laemmli (1970); gel slices were dissolved in 30% H 2 O 2 and the total apob associated radioactivity was measured in the liquid scintillation counter. 2.3 [ 14 C] acetate incorporation into cellular lipids Hepatocytes were incubated with Eagles MEM containing 1, 2 [ 14 C] acetate in the presence or absence of fatty acids or drugs. At the end of the incubation period the

3 Effect of fatty acids on ApoB by rat hepatocytes 475 medium was collected and the cell layer was washed three times with phosphate buffered saline (PBS), ph 7 4. The secreted VLDL was isolated by floatation at d < in the presence of carrier serum in a Sorvall ultracentrifuge (Hatch and Lees 1968). The cells were harvested using PBS and delipidated according to Folch et al (1957). The [ 14 C] labelled cholesterol and cholesteryl esters associated with the cell layer and secreted VLDL were then separated by TLC (Silica gel G) in a solvent system of hexane-ether-acetic acid according to Nelson (1972). Cholesterol and cholesteryl esters were scrapped out and transferred to scintillation vials and the radioactivity was measured. The protein was also measured according to Lowry et al (1951). 2.4 Fatty acid Albumin preparation The sodium salt of fatty acid was dissolved in water and added to a small amount of Eagles MEM-containing HEPES-(10mM, ph 7 4) and the appropriate amount of albumin to maintain fatty acid albumin ratio of 3 : 1 3. Results 3.1 Effect of different fatty acids on the secretion of apob into the medium by hepatocytes All the fatty acids, namely palmitic, stearic, oleic, linoleic and linolenic caused increased secretion of [ 3 H] apob into the medium when compared to the controls which was not supplemented with any fatty acids (table 1). Maximum secretion of [ 3 H] apob was observed with oleic acid and minimum with palmitic acid. In descending order of the [ 3 H] apob secretion into the medium, the different fatty acids are 18:2, 18:0, 18:3 = 16:0. But there was no significant difference in cell associated [ 3 H] apob in the case of different fatty acids. The sum of the secreted and cell associated [ 3 H] apob was maximum in the case of oleic acid followed by linoleic acid, stearic acid, and palmitic/linolenic acid. Table 1. Incorporation of [ 3 H] leucine into apob Effect of different fatty acids The cells were incubated with different fatty acids (500 μμ) for 12 h in medium containing 20 μci of [ 3 H] leucine and the radioactivity associated with secreted and cell associated apob was measured. The values given are the average of four different experiments ± S D. In all cases P < *Not significant.

4 476 Ν Suresh Kumar et al 3.2 Effect of different concentrations of oleic acid on the secretion of apob The secretion of [ 3 H] apob increased up to the highest concentration of oleic acid tested i.e. 1 mm (figure 1). However, there was no significant increase in the cell associated [ 3 H] apob. But there was significant increase in the apob in the medium plus cell associated apob indicating increased production of apob. Figure 1. Effect of oleic acid on the secretion of [ 3 H] apob. Hepatocytes were labelled with 30 μci of [ 3 H] leucine for 12 h in the presence of different amounts of oleic acid. [ 3 H] apob secreted into the medium was immunoprecipitated and determined as described in the text. The results of a typical experiment are given. Values are the average of four experiments. The variation for different experiment was less than 5% 3.3 Pulse chase experiment The increase in the level of [ 3 H] apob in the medium in the presence of oleic acid can be due to an increased rate of synthesis or increased rate of secretion or due to both. In order to examine this, cells were pulse labelled with radioactive amino acids and chased in non radioactive medium in the presence of oleic acid. In the presence of oleic acid the content of [ 3 H] apob secreted into the medium was found to be significantly more (figure 2), probably indicating that the rate of secretion is higher. 3.4 Effect of oleic acid on the incorporation of [1, 2 14 C] acetate into the cholesterol of secreted VLDL and cell associated cholesterol There was significant increase in the incorporation of [ 14 C] acetate both into the

5 Effect of fatty acids on ApoB by rat hepatocytes 477 Figure 2. Pulse-chase analysis of apob in the presence of oleic acid. Hepatocytes were pulse labelled with 100 μci of [ 3 H] leucine for 3 h. The media was removed, cells were washed and reincubated in medium containing non-radioactive leucine in the presence (test) or absence (control) of oleic acid (500 μμ) for 6 h. The apob secreted into the medium was immunoprecipitated and quantitated as described in the text. The results of a typical experiment are given. The values are the average of four experiments. The variation for different set of experiments was less than 5%. cholesterol of VLDL and that associated with the cell in the presence of oleic acid (250 μμ) when compared with the control (table 2). Table 2. Incorporation of [1, 2 14 C] acetate into cholesterol Effect of oleic acid. The cells were incubated with 10 μci of [ 14 C] acetate in the presence of oleic acid (500 μm) for 12 h. [ 14 C] cholesterol was separated from cell extract as well as from isolated secreted VLDL and estimated radioactivity. The values given are the average of four experiments ± S D P < 0 01.

6 478 Ν Suresh Kumar et al 3.5 Effect of inhibitors of cholesterogenesis on the stimulation of apob secretion by oleic acid In order to examine whether the increased apob and VLDL production in response to oleic acid challenge is related to cholesterol synthesis, the effect of ketoconazole and mevinolin, two drugs, which inhibit cholesterol synthesis on apob synthesis were studied. Ketoconazole at a concentration of 1 μμ caused significant inhibition in the incorporation of labelled acetate into total and esterified cholesterol when compared with the control (figure 3). The drug alone caused significant inhibition of the secretion of [ 3 H] apob into the medium and also decreased the cell associated [ 3 H] apob when compared to control cells. The stimulation of apob secretion into the medium by oleic acid was significantly reduced in the presence of the drug (figure 4). Addition of mevinolin (20 μμ) to cultures in the presence of oleic acid also reversed the stimulatory effect of oleic acid on apob production (figure 4). Similar effect of mevinolin was observed in short term experiments where [ 3 H] leucine incorporation into apob was studied in the presence of mevinolin alone or with oleic acid (data not given). Figure 3. Effect of oleic acid and inhibitors of cholesterol synthesis on the incorporation of [ 14 C] acetate into cholesterol and cholesteryl ester by hepatocytes. Hepatocytes were incubated with 10 μci of [ 14 C] acetate in the presence or absence of oleic acid, mevinolin and ketoconazole for 12 h. [ 14 C] cholesterol were separated from cell extracts and radioactivity was measured as described in the text. The values given are the mean of four experiments. *Not significant; C, control; O, oleic acid; K, ketoconazole; Μ, mevinolin. 3.6 Effect of mevinolin on the stimulation of [ 14 C] acetate incorporation by oleic acid The stimulation of the synthesis of cholesterol and cholesteryl ester by oleic acid was studied in the presence of mevinolin, by measuring the incorporation of [ 14 C] acetate. The results are given in figure 3. The stimulatory effect of oleic acid on [ 14 C] acetate incorporation into free and ester cholesterol was reduced by mevinolin.

7 Effect of fatty acids on ApoB by rat hepatocytes 479 Figure 4. Effect of oleic acid and inhibitors of cholesterol synthesis on the incorporation of [ 3 H] leucine into apob Hepatocytes were incubated with 30 μci of [ 3 H] leucine for 12 h in the presence or absence of oleic acid (50 μμ), mevinolin (20 μμ) and ketoconazole (1 μμ). The radioactivity associated with the apob secreted into the medium as well as that associated with the cell layer was measured as described in the text (A) Media; (B) Cell layer. *Not significant; C, control, K, ketoconazole; Ο, oleic acid; Μ, mevinolin. 4. Discussion The results now obtained indicate that exogenous fatty acids have significant effect in stimulating the secretion of apob into the medium. Of the different fatty acids studied, oleic acid exerted maximum effect. The results now obtained are generally similar to those reported by Field et al (1988) using CaCo-2 cells and Davis and Boogaerts (1982) using hepatocytes on the effect of different fatty acids on the secretion of triacylglycerol rich lipoproteins. However Field et al (1988) in their experiments did not study the secretion of apob but only triacylglycerol secretion. The increase in the secretion of apob containing lipoproteins in the presence of exogenous fatty acids now obtained was observed when compared to controls which is not supplemented with fatty acids. When stearate was compared with C 18 - unsaturated fatty acids for their effect on apob synthesis and secretion, it is seen that in the presence of linolenic acid, lower amounts of apob containing lipoproteins was secreted into the medium. Oleic acid significantly increased the output of apob containing lipoproteins, while with linoleate, the secretion was more, even though lower than that observed in the case of oleic acid. This is relevant in the light of the fact that the serum VLDL/LDL levels are higher in response to saturated fat in the diet and is significantly lower if the dietary fat is rich in polyunsaturated fatty acids particularly of the linolenic acid group containing n-3 fatty acids (Mölgaard et al 1990). Since oleic acid showed maximum stimulatory effect on apob secretion, its effect was studied in greater detail. Eventhough the cell associated apob was not

8 480 Ν Suresh Kumar et al significantly altered, the total of secreted and cell associated [ 3 H] apob showed a concentration dependent increase indicating increased synthesis of apob. These results together with pulse chase analysis indicate that oleic acid increases both synthesis and secretion of apob by hepatocytes. This does not appear to be due to an alteration in the total protein synthesis in response to oleic acid as it did not show any effect on general protein synthesis. The results reported by Moberly et al (1990) and Pullinger et al (1989) with HepG2 cells are in agreement with our findings. Cianflone et al (1990) demonstrated that oleic acid stimulated apob secretion in HepG2 cells and this apparently was shown to be a specific effect as apoa-i secretion was unaffected. However, Davis and Boogaerts (1982), did not find any effect for oleic acid on apoprotein synthesis. Exogenous fatty acids appear to cause parallel changes in cholesterol synthesis and apob production as evidenced by the results on cholesterogenesis. Oleic acid caused significant increase in the incorporation of [ 14 C] acetate into cellular cholesterol indicating increased cholesterogenesis. This is similar to the results reported by Goh and Heimberg (1977) in liver perfusion studies with free fatty acids where the activity of HMG CoA reductase was shown to be linearly proportional to the uptake of oleic acid suggesting increased cholesterogenesis. An inhibitor of cholesterol biosynthesis namely ketoconazole was observed in our experiment to counteract the stimulation of apob secretion caused by oleic acid. Ketoconazole is an antifungal drug which inhibits cholesterol synthesis by blocking demethylation of lanosterol (Miettinen 1988) and decreased incorporation of [ 14 C] acetate into total and esterified cholesterol indicating an inhibitory effect on cholesterol synthesis in hepatocytes also. The effect on free cholesterol appeared to be less significant probably because the radioactivity associated with the free cholesterol fraction in ketoconazole treated cells may also contain that of methylated intermediates. Mevinolin, a specific competitive inhibitor of HMG-CoA reductase (Alberts et al 1980) also reversed the stimulatory effect of oleic acid on the production of these lipoproteins by hepatocytes indicating that the stimulatory effect of oleic acid on the synthesis and secretion of apob containing lipoproteins is mediated through the availability of cholesterol. This is in line with the observation made by Koshyk et al (1988), Khan et al (1989) and Cianflone et al (1990). We have shown that any condition that results in increasing intracellular cholesterol results in increasing apob synthesis and secretion and where the cholesterol synthesis was inhibited there was a reduction in apob synthesis and secretion suggesting a critical role for cholesterol ester rather than triglyceride in apob synthesis and secretion (Kumar et al 1992). The results discussed above also suggested that the increase in the synthesis and secretion of apob containing lipoproteins in hepatocytes in response to fatty acid challenge is triggered through enhanced cholesterol synthesis. In our studies oleic acid caused more than two-fold increase in the incorporation of radioactive acetate into the cholesterol in the secreted VLDL. This newly synthesised cholesterol may be required in the packaging and assembly of VLDL. Acknowledgement Financial assistance received from the Department of Science and Technology, New Delhi, to carry out this work is gratefully acknowledged.

9 Effect of fatty acids on ApoB by rat hepatocytes 481 References Alberts A W, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez J Μ, Joshua Η, Harris Ε, Patchett A, Monaghan R, Currie S, Stapley E, Albers-Schonberg G, Hensens O, Hirshfiled J, Hoogsteen K, Liesch J and Springer J 1980 Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase and a cholesterol lowering agent; Proc. Natl. Acad. Sci. USA Cianflone Κ Μ, Yazrud Ζ, Rudriguez Μ A, Vas D A and Sniderman 1990 Regulation of apob secretion from HepG2 cells, evidence for a critical role for cholesterol ester synthesis in the responses to fatty acid challenge; J. Lipid Res Davis R A, Engelhorn S C, Pangburn S H, Weinstein D Β and Steinberg D 1979 Very low density lipoprotein synthesis and secretion by cultured rat hepatocytes; J. Biol. Chem Davis R A and Boogaerts J R 1982 Intrahepatic assembly of very low density lipoproteins; J. Biol. Chem Field F J, Albright Ε and Mathur S Ν 1988 Regulation of triglyceride-rich lipoprotein secretion by fatty acids in CaCo-2 cells; J. Lipid Res Folch J, Lees Μ and Sloane Stanley G Η 1957 A simple method for the isolation and purification of total lipids from animal tissues; J. Biol. Chem Goh Ε Η and Heimberg Μ 1977 Effect of free fatty acids on activity of hepatic microsomal 3-hydroxy-3- methyl glutaryl coenzyme A reductase and on secretion of Triglyceride and cholesterol by liver; J. Biol. Chem Hatch F Τ and Lees R S 1968 Practical methods for plasma lipoprotein analysis; Adv. Lipid Res Khan B, Wilcox Η G and Heimberg Μ 1989 Cholesterol is required for secretion of very-low-density lipoprotein by rat liver; Biochem. J Kohout Μ Β, Kohoutova Β and Heimberg Μ 1971 The regulation of hepatic triglyceride metabolism by free fatty acids; J. Biol. Chem Koshyk V A, Surguchov A P, Podres Ε A, Novikov D K, Sudarickov A B, Beresteskaya YU V, Repin V S and Smirnov V Ν 1988 VLDL apoprotein secretion and apob mrna level in primary culture of cholesterol loaded rabbit hepatocytes; FEBS Lett Kumar Ν S, Abraham R, Kumar G S, Sudhakaran Ρ R and Kurup Ρ A 1992 Synthesis and secretion of VLDL by rat hepatocytes Modulation by cholesterol and phospholipids; Indian J. Biochem. Biophys. (in press) Laemmli U Κ 1970 Cleavage of structural proteins during the assembly of the head of bactereophage T4 ; Nature (London) Lowry Ο Η, Rosebrough Ν J, Farr A L and Randall R J 1951 Protein measurement with the Folin phenol reagent; J. Biol. Chem Miettinen Τ A 1988 Cholesterol metabolism during Ketoconazole treatment in man; J. Lipid Res Moberly J Β, Cole Τ G, Alpers D Η and Schonfeld G 1990 Oleic acid stimulation of apolipoprotein Β secretion from HepG2 and CaCo-2 cells occur Post-transcriptionally; Biochim. Biophys. Acta Mölgaard J, Von Schenck Η, Lassivk C, Kuusi Τ and Olsson A G 1990 Effect of fish oil treatment on plasma lipoproteins in type III hyperlipoproteinemia; Atherosclerosis Nelson G J 1972 Quantitative analysis of blood lipids; in Blood lipids and lipoproteins: Quantitation, composition and metabolism (ed.) G J Nelson (New York: Wiley-Interscience) pp Pullinger C R, North J D, Teng Β Β, Rifici V A, Ronhild de Brito Α Ε and Scott J 1989 The apolipoprotein Β gene is constitutively expressed in HepG2 cells: regulation of secretion by oleic acid, albumin, and insulin, and measurement of the mrna halflife; J. Lipid Res Seglen Ρ Ο 1976 Preparation of isolated liver cells; Methods Cell Biol Sudhakaran Ρ R, Stamatoglou S C and Hughes R C 1986 Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes; Exp. Cell. Res

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