S-propyl cysteine reduces the secretion of apolipoprotein B100 and triacylglycerol by HepG2 cells

BASIC NUTRITIONAL INVESTIGATION

S-Propyl Cysteine Reduces the Secretion of Apolipoprotein B100 and Triacylglycerol by HepG2 Cells Seo-young Han, PhD, Ying Hu, MS, Takahiko Anno, PhD, and Teruyoshi Yanagita, PhD From the Laboratory of Nutrition Biochemistry, Department of Applied Biological Sciences, Saga University, Saga, Japan; and Food Development Laboratories, Nippon Shinyaku, Co., Ltd., Kyoto, Japan A number of sulfur-containing amino acids and peptides are found in allium plants such as onion and garlic that have physiologic functions. In HepG2 cells, S-propyl cysteine decreased the secretion of apolipoprotein B100. The compound reduced the secretion of newly synthesized triacylglycerol and cholesterols from radiolabeled acetate. We associated the decrease of apolipoprotein B100 secretion to the length of the acyl-chain of the sulfur-containing amino acids. The present study suggests that foods containing S-propyl cysteine including onions have beneficial effects. Nutrition 2002;18:505–509. ©Elsevier Science Inc. 2002 KEY WORDS: S-propyl cysteine, apolipoprotein B100, triacylglycerol, HepG2 cells

INTRODUCTION

EXPERIMENTAL MATERIALS AND REAGENTS

Allium plants such as onion (Allium cepa L.) and garlic (Allium sativum L.) are used not only in food for their characteristic taste and smell but also for their medicinal properties.1–3 In some epidemiologic studies, a negative correlation was found between the intake of allium plants and the prevalence of coronary heart diseases.4 Babu and Srinivasan reported that feeding of allium powder significantly reduces plasma glucose and serum lipid levels in diabetic rats.5 In addition, our recent experiments showed that intake of onion extract causes a marked reduction of the plasma triacylglycerol (TAG) level in rats fed a semisynthetic diet,6 suggesting that physiologically active compounds are present in this vegetable. This observation led to the suggestion that onion may be effective in preventing hyperlipidemic-related diseases by normalizing abnormal lipid metabolism. However, the putative hypolipidemic effect of onion remains controversial. Onion contains a large number of sulfur-containing amino acids and peptides.7 These sulfur-compounds may exert hypolipidemic effect,8 prevent platelet aggregation,9 and reduce plasma fibrinogen levels.10 Liu and Yeh reported that water-soluble organosulfur compounds inhibit TAG synthesis in cultured rat hepatocytes,11 albeit at higher concentrations (50 to 4000 ␮M). High level of apolipoprotein B100 (apoB100) is atherogenic.12 We recently showed that cycloalliin, a cyclic sulfur compound in onion, can reduce apoB100 secretion from HepG2 cells, derived from the human hepatoblastoma cell line.13 This prompted us to study whether other sulfur compounds in onion also influence apoB100 secretion from the liver. Overproduction of apoB100containing lipoproteins in the liver induces hyperlipidemia. Decreased synthesis of apoB100-containing lipoproteins in the liver may be one mechanism by which onion reduces plasma lipoproteins. To gain further insight into the mechanism of action of onion sulfur components on lipid metabolism, we evaluated apoB100 secretion and lipid metabolism by using human liver model cells.

Materials

Correspondence to: Teruyoshi Yanagita, PhD, Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan. E-mail: [email protected] Nutrition 18:505–509, 2002 ©Elsevier Science Inc., 2002. Printed in the United States. All rights reserved.

HepG2 cells were obtained from Riken Cell Bank (Tsukuba, Japan). Dulbecco’s Modified Eagle Medium and trypsin were provided from Gibco (Grand Island, NY, USA). Cysteine derivatives were supplied by Nippon Shinyaku, Ltd. (Kyoto, Japan). Apparatus for cell culture was purchased from Falcon (Lincoln Park, NJ, USA). Fetal calf serum was obtained from Sigma Chemical Company (St. Louis, MO, USA), and penicillin and streptomycin were obtained from Meiji Seika Kaisha, Ltd. (Tokyo, Japan). [14C]Oleate (specific activity: 2.04 MBq/␮M) and [14C]acetate (specific activity: 2.07 MBq/␮M) were purchased from Amersham, Inc. (Buckinghamshire, UK). Scintillation cocktail (Sintisol Ex-H) was purchased from Dojin Chemical Company (Kumamoto, Japan).

HepG2 Cell Culture The HepG2 cells were precultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum, penicillin (100 ␮g/mL), and streptomycin (100 ␮g/mL) at 37°C in air containing 5% (v/v) CO2. Medium was replaced every 2 d. Before the experiments, semiconfluent cells were preincubated in 0.1 M phosphate-buffered saline (PBS) and Dulbecco’s Modified Eagle Medium containing 1% bovine serum albumin and 0.5 mM oleate (experimental medium, pH 7.0) on flat-bottomed tissue culture plate for 24 h.

Experiment 1 The cells were cultured in experimental medium supplemented with 10 ␮M of S-propyl cysteine or without (control) for 3 or 24 h; 18.5 kBq of [14C]acetate or [14C]oleate was added into the experimental medium at the beginning of the experiment. After incubation, the cells and the supernatant were harvested separately with a rubber policeman and frozen at ⫺80°C until analysis. 0899-9007/02/$22.00 PII S0899-9007(02)00749-9

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Experiment 2 The cells were cultured in experimental medium supplemented with one cysteine derivative (S-propyl cysteine, S-ethyl cysteine, or S-methyl cysteine) at the concentration of 10 ␮M or without (control) cysteine derivatives for 24 h; 18.5 kBq of [14C]acetate was added to the experimental medium at the beginning of experiment, as reported previously.14 After incubation, the cells and the supernatant were harvested separately with a rubber policeman and frozen at ⫺80°C until analysis. Preparation of Experimental Medium Cysteine derivatives, dissolved in PBS, were diluted in Dulbecco’s Modified Eagle Medium containing 1% bovine serum albumin. All experimental media were sterilized with 0.45-␮m Millipore R filter before use. Determination of Cellular Protein Cells were homogenized with sonicator (Sonifier 250, Branson Ultradonid Company, CT, USA) before analysis and the cellular protein concentration was determined with the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA).15 Cytotoxicity Assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) assay was performed as a parameter of cytotoxicity, as published previously.16 The cells were precultured in basal medium on a 96-well plate. After cell culture with experimental medium, 20 ␮L of MTT solution (5 mg/mL of PBS) was added to each well. The cells were incubated for 3 h. After that, the medium was removed, and 150 ␮L of mixture (dimetyl sulfoxide:ethanol ⫽ 1:1, v/v) was added to terminate the reaction. After shaking for 15 min, the absorbance was determined at 570 nm by the 96-well plate reader (CS-9300 PC, Simadzu Company, Ltd., Kyoto, Japan). Determination of apoB100 Secretion Into the Medium of HepG2 Cells ApoB100 concentration was measured by a double antibody enzyme-linked immunosorbent assay according to the method of Young et al.17 After cell culture with experimental medium, the supernatant was collected and diluted 200-fold in PBS. Ninetysix–well plates were coated with coating buffer (15 mM Na2CO3, 31 mM NaHCO3, pH 9.6) and 17.3 mg/L of rabbit antihuman immunoglobulin G at 37°C for 2 h; each well was then incubated with 0.1 mL of bovine serum albumin (0.5% sodium azide) for another 2 h. The wells were washed three times with PBS containing 0.5 ␮L/mL of Tween 20. For the standard curve, the apoB standard and sample were added to the wells and incubated at 4°C for 16 h. After washing, 0.1 mL of phenylenediamine substrate solution was added to each well and developed at 25°C for 30 min. The reaction was stopped by adding 50 ␮L of 4.5 M H2SO4; the absorbance was determined in individual wells at 482 nm by the 96-well plate reader (CS-9300 PC, Simadzu Company, Ltd.). Lipid Synthesis and Secretion The incorporation of radioactivity into cellular lipids and their secretion were measured as reported previously.18 Total lipid was extracted and purified by the method of Bligh and Dyer19 and measured by liquid scintillation counter (Wallic System 1410, Pharmacia, Uppsala, Sweden). Neutral lipid subclasses were separated by thin-layer chromatography precoated with silica gel type G in the solvent mixtures of petroleum ether:diethylether:acetate

FIG. 1. Effect of S-propyl cysteine on the section of apolipoprotein B100 from HepG2 cells. Semiconfluent HepG2 cells were preincubated in Dulbecco’s Modified Eagle Medium containing 1% bovine serum albumin and 0.5 mM of oleate for 24 h. Cells were then incubated in the medium containing 10 ␮M of S-propyl cysteine for 3 and 24 h. After harvest, the secreted apolipoprotein B100 was measured by the double-antibody method, enzyme-linked immunosorbent assay. Data represent means ⫾ standard errors of four samples. *P ⬍ 0.001 versus control, significant.

(80:20:1, v/v/v). The radioactivity was measured with an imaging plate and BAS 1000 film (Fuji Photograph Film Company, Ltd. Kanagawa, Japan). Statistical Analyses Each value is presented as means ⫾ standard error. Data were analyzed by one-way analysis of variance, and all differences were examined with Duncan’s new multiple-range test.20 P ⬍ 0.05 was considered statistically significant. Student’s t test21 was also used in some comparisons.

RESULTS Effect of S-Propyl Cysteine on Viability and Protein Content of HepG2 Cells MTT activity correlates with the number of cells, so it can be used as an index of cytotoxicity. S-propyl cysteine did not have any effect on MTT activity when treated at 10 to 1000 ␮M (data not shown). Similarly, no change in protein content between control cells and cells cultured with S-propyl cysteine was observed (data not shown). These results suggested that S-propyl cysteine at the concentrations used in this study is not cytotoxic to HepG2 cells. Effect of S-Propyl Cysteine on ApoB100 Secretion Figure 1 shows the apoB accumulation in the medium. S-propyl cysteine induced an 18% reduction in apoB100 secretion by HepG2 cells at the end of 3 and 24 h of incubation when compared with controls. Effect of S-Propyl Cysteine on Incorporation of [14C]Acetate Into TAG and Cholesterol The incorporation of [14C]acetate into newly synthesized fatty acid and then into TAG was similar between control cells and cells exposed to S-propyl cysteine for 24 h (Fig. 2). However, S-propyl cysteine significantly reduced by 38% the secretion of labeled TAG into the culture medium (Fig. 2). A similar result was observed for phospholipids (data not shown). Cells treated with

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FIG. 3. Effect of S-propyl cysteine on the incorporation of [14C]oleate into TAG and CE. Semiconfluent HepG2 cells were preincubated in Dulbecco’s Modified Eagle Medium containing 1% bovine serum albumin and 0.5 mM of oleate for 24 h. After preincubation, cells were incubated in medium containing 10 ␮M of S-propyl cysteine and 18.5 kBq of [14C]oleate for 24 h. The value of the incorporation of [14C]oleate into TAG in untreated cells (422 ⫾ 27.8 ⫻ 10⫺3 dpm/mg cell protein) was arbitarily defined as 100. The value of the incorporation of [14C]oleate into cholesterol in untreated cells (5.71 ⫾ 0.34 ⫻ 10⫺3 dpm/mg cell protein) was arbitarily defined as 100. Data represent means ⫾ standard error of four samples. *P ⬍ 0.05 versus controls, significant. CE, cholesterol ester; TAG, triacylglycerol.

significantly reduced by S-propyl cysteine treatment. This suggests that S-propyl cysteine inhibits the secretion of lipids from the cells. Effect of S-Propyl Cysteine on Esterification of [14C]Oleate to Form TAG and Cholesterol Ester Incorporation of [14C]oleate into TAG and cholesterol ester by HepG2 cells was reduced by 42% and 34%, respectively, after exposure to S-propyl cysteine (Fig. 3). Effect of Cysteine Derivatives on ApoB100 and Lipid Secretion

FIG. 2. Effect of S-propyl cysteine on the incorporation of [14C]acetate into TAG, FC, and CE and secretions into the medium. After preincubation, the cells were incubated in medium containing 10 ␮M of S-propyl cysteine and 18.5 kBq of [14C]acetate for 24 h. The value of the incorporation of [14C]acetate into TAG in untreated cells (685.6 ⫾ 27.5 ⫻ 10⫺3 dpm/mg cell protein) and the secretion of the labeled TAG into the medium (16.1 ⫾ 0.20 ⫻ 10⫺3 dpm/mg cell protein) were arbitarily defined as 100, respectively. The value of the incorporation of [14C]acetate into FC in untreated cells (66.1 ⫾ 2.41 ⫻ 10⫺3 dpm/mg cell protein) and the secretion of the labeled FC into the medium (4.40 ⫾ 0.24 ⫻ 10⫺3 dpm/mg cell protein) were arbitarily defined as 100, respectively. The value of the incorporation of [14C]acetate into CE in untreated cells (16.1 ⫾ 0.25 ⫻ 10⫺3 dpm/mg cell protein) and the secretion of the labeled CE into the medium (0.44 ⫾ 0.04 ⫻ 10⫺3 dpm/mg cell protein) were arbitarily defined as 100, respectively. Data represent means ⫾ standard error of four samples. *P ⬍ 0.05 versus controls, significant. CE, cholesterol ester; FC, free cholesterol; TAG, triacylglycerol.

S-propyl cysteine also tended to reduce incorporation of acetate into cellular cholesterol, although the decrease was not statistically significant (Fig. 2). However, secretion of labeled cholesterol was

HepG2 cells were incubated in medium containing 10 ␮M of one of the three cysteine derivatives with different chain lengths (i.e., S-propyl cysteine, S-ethyl cysteine, or S-methyl cysteine), and apoB100 content secreted in the medium was measured. ApoB100 content was reduced by all three cysteine derivatives, in the following order—S-propyl cysteine, S-ethyl cysteine, and S-methyl cysteine—suggesting that this action is more marked with an increasing carbon chain length (Fig. 4). Secretion of [14C]labeled TAG and cholesterols to the medium was reduced by all three cysteine derivatives (Fig. 5). The reduction of cellular TAG content also tended to increase with carbon chain length; the reductions as compared with control cells were 18% for S-propyl cysteine, 11% for S-ethyl cysteine, and 5% for S-methyl cysteine.

DISCUSSION The present study showed that S-propyl cysteine derived from onion reduces apoB100 secretion by the human liver cells HepG2. High level of apoB100 is a risk factor for atherosclerosis, so the reduction of apoB100 secretion by cysteine derivatives may be beneficial for health. Factors that regulate the synthesis and secretion of apoB100-containing lipoproteins by liver cells are the availability of apoB100 and lipids and the activity of microsomal triacylglyerol transfer protein (MTP).22 ApoB100, a 556-kDa hydrophobic protein, is synthesized in the endoplasmic reticulum and

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FIG. 4. Effect of cysteine derivatives on the secretion of apolipoprotein B100 from HepG2 cells. Semiconfluent HepG2 cells were preincubated in Dulbecco’s Modified Eagle Medium containing 1% bovine serum albumin and 0.5 mM of oleate for 24 h. After preincubation, cells were incubated in medium containing 10 ␮M of cysteine derivatives, S-methyl-, S-ethyl-, and S-propyl cysteines for 24 h. After harvest, the secreted apolipoprotein B100 was measured by enzyme-linked immunosorbent assay. The value of the secreted apolipoprotein B100 into the untreated medium (2.08 ⫾ 0.11 ␮g/mg cell protein) was arbitarily defined as 100. Data represent means ⫾ standard error of four samples. Values not sharing the same letters are significantly different at P ⬍ 0.05.

shows low-density lipoprotein receptor-binding activity.23,24 After synthesis, it is stabilized through binding with lipids and then secreted as apoB100-containing lipoproteins. If insufficient lipid is available, apoB100 is unstable and undergoes degradation via the ubiquitin–proteasome pathway.25 Therefore, the control of apoB100 expression in cells is considered posttranscriptional.26 In HepG2 cells, S-propyl cysteine reduced the secretion of apoB100 in comparison to untreated cells (Fig. 1). ApoB100 is a major constituent of the lipoproteins produced in the liver. Therefore, the decreased secretion of apoB100 by HepG2 cells suggests that hepatic secretion of apoB100-containing very low-density lipoprotein would be reduced. Exposure of HepG2 cells to S-propyl cysteine significantly reduced the secretion of [14C]TAG from [14C]acetate (Fig. 2), despite the fact that [14C]acetate incorporation into cellular lipids did not alter. These results suggest that S-propyl cysteine inhibits the process of lipoprotein assembly and secretion. Liu and Yeh reported that S-propyl cysteine at high concentration (50 to 4000 ␮M) inhibits fatty acid synthesis and thus TAG synthesis in cultured rat hepatocytes.11 The discrepancy between the present study and that by Liu and Yeh with regard to fatty acid synthesis by S-propyl cysteine may be due to the difference of concentrations and cells used (rat hepatocytes versus HepG2 cells). Cholesterol metabolism also affects lipoprotein synthesis.14 In the present study, incubation with S-propyl cysteine markedly reduced the secretion of [14C]cholesterol from [14C]acetate from the cells. However, the incorporation rate of [14C]acetate into cellular cholesterol did not change, although it was slightly lower with S-propyl cysteine treatment. It is well recognized that MTP is an important factor in lipoprotein synthesis by the liver.27 MTP is a heterodimer consisting of a 97-kDa subunit with lipid transfer activity and a 58-kDa subunit with protein disulfide isomerase activity, and is essential for the transfer of lipid to nascent lipoproteins.28 Complete absence of MTP caused abetalipoproteinemia,29,30 whereas MTP inhibitors inhibited the development of atherosclerosis in WHHL rabbits.31 We previously found that cycloalliin, a cyclic imino compound in onion, markedly reduces plasma TAG concentration in rats.32

FIG. 5. Effect of cysteine derivatives on the incorporation of [14C]acetate into TAG, FC, and CE and secretions into the medium. After preincubation, cells were incubated in medium containing 10 ␮M of cysteine derivatives, S-methyl-, S-ethyl-, and S-propyl cysteines, and 18.5 kBq of [14C]acetate for 24 h. The value of the incorporation of [14C]acetate into TAG in untreated cells and the secretion of the labeled TAG into the medium (as control) were arbitarily defined as 100, respectively. The value of the incorporation of [14C]acetate into FC in untreated cells and the secretion of the labeled FC into medium (control) were arbitarily defined as 100, respectively. The value of the incorporation of [14C]acetate into CE in untreated cells and the secretion of the labeled CE into medium (control) were arbitarily defined as 100, respectively. Data represent means ⫾ standard error of four samples. Values not sharing the same letters are significantly different at P ⬍ 0.05. CE, cholesterol ester; FC, free cholesterol; TAG, triacylglycerol.

Therefore, it is necessary to investigate the effect of sulfur amino acids on MTP activity and mRNA expression. It is noteworthy that the inhibitory effect of the cysteine deriv-

Nutrition Volume 18, Number 6, 2002 atives on apoB100 secretion from the cells increased in the order of S-propyl cysteine, S-ethyl cysteine, and S-methyl cysteine, indicating that a longer carbon chain has a stronger inhibitory effect (Fig. 4). An interaction of these compounds with the cell membrane and their entry into the cells may be responsible for this difference in their activity. Because incubation with S-propyl cysteine reduced cellular TAG and cholesterol ester syntheses from [14C]oleate, one might suppose that the sulfur compound inhibits the esterification of exogenous fatty acid to cellular lipids. However, there was no difference in cellular TAG synthesis from [14C]acetate. For these reasons, we hypothesize that S-propyl cysteine reduces the transport of exogenous fatty acids into the cells. In conclusion, S-propyl cysteine from onion reduced apoB100 secretion by HepG2 cells derived from the human hepatoblastoma cell line. Accordingly, the beneficial actions of foods including onion can be attributed to the presence of cysteine derivatives.

ACKNOWLEDGMENT The authors are grateful to H. Yotsumoto, MD, and Y. M. Wang for their help in this project.

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