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Regulation of Acyl-coenzyme ACholesterol Acyltransferase 2 Expression

Chin Med Sci J Vol. 25, No. 4 December2010 P.222-227

CHINESE

MEDICAL SCIENCES

JOURNAL

ORIGINAL ARTICLE

Regulation of Acyl-coenzyme A:Cholesterol Acyltransferase

2 Expression by Saturated Fatty Acids?

Zhu-qin Zhang, Hou-zao Chen, Rui-feng Yang, Ran Zhang,

Yu-yan Jia, Yang Xi, De-pei Liu*, and Chih-chuan Liang#

National Laboratory of Medical Molecular Biology, Institute of Basic

Medical Science, Chinese Academy of Medical Sciences &

Peking Union Medical College, Beijing 100005, China

Key words:acyl-coenzyme A:cholesterol acyltransferase 2; gene expression; saturated

fatty acid

Objective T o verify the regulation of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT 2), which is associated with cholesterol metabolism, by saturated fatty acids (SFAs).

Methods Palmitic acid (PA), the most abundant saturated fatty acid in plasma, and oleic acid (OA), a widely distributed unsaturated fatty acid, were used to treat hepatic cells HepG2, HuH7, and mouse primary

hepatocytes. In addition, PA at different concentrations and PA treatment at different durations were applied

in HepG2 cells. In in vivo experiment, three-month male C57/BL6 mice were fed with control diet and SFA

diet containing hydrogenated coconut oil rich of SFAs. The mRNA level of ACAT2 in those hepatic cells and

the mouse livers was detected with real-time polymerase chain reaction (PCR).

Results In the three types of hepatic cells treated with PA, that SFA induced significant increase of ACAT2 expression (P?0.01), whereas treatment with OA showed no significant effect. That effect of PA was

noticed gradually rising along with the increase of PA concentration and the extension of PA treatment dura-

tion (both P?0.05). SFA diet feeding in mice resulted in a short-term and transient increase of ACAT2 ex-

pression in vivo, with a peak level appearing in the mice fed with SFA diet for two days (P?0.05).

Conclusion SFA may regulate ACAT2 expression in human and mouse hepatic cells and in mouse livers.

Chin Med Sci J 2010; 25(4):222-227.

Received for publication October 29, 2010.

*Corresponding author Tel: 86-10-65296415, Fax: 86-10-65105093, E-mail: liudp@http://www.wendangku.net/doc/eb8be53043323968011c927a.html

#Deceased on June 14, 2006.

?Supported by National Natural Science Foundation of China (30721063), National High Technology Research and Development Program of China (863 Program) (2006AA02A406), National Basic Research Program of China (973 Program) (2006CB503801), and Special Fund of the National Laboratory of China (2060204).

CYL-coenzyme A:cholesterol acyltransferase

(ACAT) is an important enzyme family involved

in cholesterol metabolism. ACAT converts cho-

lesterol and fatty acyl coenzyme A to choles-terol esters, which are then assembled into very low den-sity lipoprotein (VLDL) or stored in lipid droplet. ACAT family has two members, ACAT1 and ACAT2, the latter being expressed mainly in liver and intestine. Liver ACAT2 catalyzes formation of cholesterol esters, which are as-sembled into VLDL together with ApoB and secreted into blood.1-3 Overexpression of ACAT2 in cells increases cho-lesterol ester synthesis and ApoB secretion.4,5 In contrast, liver-specific downregulation of ACAT2 in mice leads to reduced packaging of cholesterol into ApoB-containing lipoproteins.6 ACAT2 deficiency in mice also compromises ApoB secretion and leads to a dramatic decrease of VLDL cholesterol content.7 Thus ACAT2 and the regulation of its expression are significant in ApoB secretion and VLDL cholesterol level.

Saturated fatty acids (SFAs) have been regarded as “bad” for its deteriorating effects in increasing low den-sity lipoprotein (LDL) cholesterol and decreasing high density lipoprotein (HDL) cholesterol,8-10 in which the former effect is known to be the result of reduced uptake of LDL from plasma.11SFAs were also reported to stimulate ApoB secretion and to increase plasma VLDL cholesterol in the presence of high cholesterol level.12-14 The mechanism for SFA regulation of ApoB and VLDL metabolism, however, is still not clear. Given the im-portance of ACAT2 in ApoB and VLDL metabolism, we speculate that one possible mechanism for SFAs might be to regulate ACAT2, therefore we performed this study to verify this hypothesis.

MATERIALS AND METHODS

Cells and cell cultures

Human liver carcinoma cells HepG2 and HuH7 obtained from American Type Culture Collection were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). Mouse primary hepatocytes (mPH) were isolated from male C57 mice with two-step collagenase perfusion method.15 The perfusion was per-formed through inferior vena cava. After examined by means of trypan blue exclusion, the cells with more than 80% viability were used in the following experiments.

Fatty acid preparation and cell treatment

To observe whether ACAT2 expression was regulated by SFAs, palmitic acid (PA) and oleic acid (OA) were used, in which the former is the most abundant saturated fatty acid in plasma, and the latter is the most widely distributed and most plentiful unsaturated fatty acid in nature, used as control.16-18 Plasma free fatty acids (FFAs) concentration was suggested to be 0.25-3.0 mmol/L.19 FFA solutions were prepared as previously described.20,21 Briefly, PA at 100 mmol/L (Sigma-Aldrich, St. Louis, MO, USA) and OA at 100 mmol/L (Sigma-Aldrich) were prepared in 0.1 mol/L NaOH at 70°C and diluted with bovine serum albumin (BSA) solution, producing 5 mmol/L FFA/5% BSA mixture. The cells were washed twice with phosphate-buffered saline (PBS) before treatment. The 5mmol/L FFA/5% BSA mix-ture was mixed with serum-free culture medium at dif-ferent ratios to reach the needed final concentrations of FFA, to be specific, at the ratio of 1?4 to the concentration of 1 mmol/L, 1?24 to the concentration of 0.2 mmol/L, and 1?9 to the concentration of 0.5 mmol/L. In the assay in-dicated in Figure 1, the cells were treated with BSA, 1 mmol/L OA, or 1 mmol/L PA for 14 hours. In Figure 2A, cells were treated with BSA, 0.2 mmol/L, 0.5 mmol/L, or 1 mmol/L PA for 14 hours. In Figture 2B, cells were treated with 1 mmol/L PA for 0, 6, 12, and 18 hours.

SFA diet in mice

Three-month-old male C57/BL6 mice (Vital River Labo-ratory Animal Technology Co. Ltd., Beijing, China) were subjected to experiments. In initial experiment, 16 mice were divided into two groups according to body weight (n=8) and were given SFA or control diet for 3 months. Mice were sacrificed after 3 months and livers were iso-lated for reverse transcription-polymerase chain reaction (RT-PCR). In another experiment, 21 mice were divided into 7 groups according to body weight (n=3), and given SFA diet for 0, 1, 2, 3, 5, 7, and 14 days, respectively. SFA diets of all the 7 groups were stopped at the same end point, and control diet was given before the start of SFA diet, which was different among the groups. All the mice were sacrificed at the same end point to isolate livers for RT-PCR.

Diet recipes were determined according to a previous report.22 Recipe for SFA diet: hydrogenated coconut oil (144 g/kg), corn oil (17 g/kg), corn starch (123 g/kg), sucrose (182 g/kg), and cellulose (146 g/kg). Recipe for control diet: hydrogenated coconut oil (33 g/kg), corn oil (21 g/kg), corn starch (245 g/kg), sucrose (292 g/kg), and cellulose (12 g/kg). The ingredients contained in both diets include casein (206 g/kg), maltodextrin (100 g/kg), cholesterol (2.0g/kg), methionine (3.0g/kg), vitamin mix (10 g/kg), choline bitartrate (2.3 g/kg), ethoxyquin (0.037 g/kg), mineral mix (37 g/kg), and calcium phos-

A

s phate (4.2 g/kg). The hydrogenated coconut oil (Fuji Oil, Shanghai, China) used in the experiments included caprylic acid (8.63%), undecylic acid (6.7%), lauric acid (49.47%), myristic acid (16.82%), palmitic acid (7.83%), stearic acid (9.97%), oleic acid (0.23%), and others (0.35%).

RNA isolation, reverse transcription, and real-time PCR

RNAs were extracted from HepG2, HuH7, mouse primary hepatocytes, and mouse livers with TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s in-structions, and reversely transcribed to cDNA as template for PCR. Real-time PCR was performed with an iQ5 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) to detect ACAT expression level. The primers used are as follows: human ACAT2, 5’-TCTATCCTGCATGCCACGTTG-3’ (for-ward), 5’-AGTTCCACCAGTCCCGGTAGAA-3’ (reverse); hu-man ACAT1 (negative control), 5’-TTAACTCCATCTTGCCA- GGTGTG-3’ (forward), 5’-TGTCACCAAAGCGTAACATCTCA- 3’ (reverse); human GAPDH (internal control), 5’-GCCTCA- AGATCATCAGCAATGC-3’ (forward), 5’-TCTTCTGGGTGGC- AGTGATGG-3’ (reverse); mouse ACAT2, 5’-CGCTGCGTGC- TGGTCTTT-3’ (forward), 5’-ATGCCCTTTCCTCCTCTGACA- 3’ (reverse); mouse actin (internal control), 5’-CCTTCCT- TCTTGGGTATGGAATCAG-3’ (forward), 5’-AGCACTGTGTT- GGCATAGAGGT-3’ (reverse).

Statistical analysis

All statistics analyses were performed with GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Data were expressed as means±SEM. Differences among groups were tested with one-way analysis of variance (ANOVA). Com-parisons between two groups were performed with un-paired t -test. The results were presented as means±SEM. A P lever under 0.05 was considered statistically signifi-cant.

RESULTS

PA treatment induced ACAT2 expression in hepatic cells

The results of real-time PCR showed that in HepG2 cells, 1 mmol/L PA treatment for 14 hours induced a significant increase in ACAT2 expression, while OA treatment caused no significant change (Fig. 1A, P ?0.01). Similar results were also observed in HuH7 cell line and mPH treated with 1 mmol/L PA or 1 mmol/L OA for 14 hours (Fig. 1A, both P ?0.01). We also observed ACAT1 expression in response to PA treatment since it is also expressed in liver. In con-trast to ACAT2, ACAT1 expression level nearly remained constant after PA treatment (Fig. 1B). ACAT1 and ACAT2 are two isoforms of ACAT gene family. SFA regulation of ACAT2, but not ACAT1, suggested that ACAT2 and ACAT1 have different regulation patterns. It is possible that ACAT1 is expressed constitutively whereas ACAT2 expression may be induced by SFA.

PA treatments led to dose- and time-dependent in-creases of ACAT2 expression

Treatment with PA at different concentrations in HepG2 cells resulted in a concentration-dependent increase of ACAT2 expression. Although PA at 0.2 mmol/L did not induce much change in ACAT2 expression after 14-hour treatment, a significant increase in ACAT2 expression was observed in HepG2 cell treated with 0.5 mmol/L PA (P ?0.05) and an even more dramatic one with 1 mmol/L PA (Fig. 2A, P ?0.01). To investigate the existence of time- dependent effect of PA, we treated HepG2 cells with 1 mmol/L PA for different time periods ranging from 0 to 18 hours. There was a sustained and gradual rise of ACAT2 expression along with the extension of treatment duration (Fig. 2B, all P ?0.05). Taken together, these results sug-gested that PA treatment has a dose- and time-dependent effect in inducing the increase of ACAT2 expression.

Regulation of Acyl-coenzyme ACholesterol Acyltransferase 2 Expression

Figure 1. Palmitic acid (PA) treatment in hepatic cells HepG2, HuH7, and mouse primary hepatocytes (mPH) induces ACAT2 expression a

confirmed by the results of real-time polymerase chain reaction (PCR). The three types of cells were treated with bull serum albumin (BSA) (as control), 1 mmol/L oleic acid (OA), or 1 mmol/L PA for 14 hours in a serum-free medium (A). In contrast to ACAT2, ACAT1 expression is not promoted by PA treatment in HepG2 cells (B).

P ?0.05, P ?

0.01 compared with the results in BSA-treated cells.

Regulation of Acyl-coenzyme ACholesterol Acyltransferase 2 Expression

Regulation of Acyl-coenzyme ACholesterol Acyltransferase 2 Expression

Regulation of Acyl-coenzyme ACholesterol Acyltransferase 2 Expression

Figure 2.Dose- and time-dependent increases of ACAT2 expression induced by PA treatment in HepG2 cells.

A. ACAT2 expression in HepG2 cells treated for 14 hours with different concentrations of PA.

B. ACAT2 expression in HepG2 cells treated with 1 mmol/L PA for different time periods.

P?0.05, P?0.01 compared with the control.

Regulation of Acyl-coenzyme ACholesterol Acyltransferase 2 Expression

Regulation of Acyl-coenzyme ACholesterol Acyltransferase 2 Expression

f

C57/BL6 mice. Mice divided into seven groups (n=3)

were given SFA diet for time periods ranging from 0 to

14 days. SFA diet was started at different time points

among the groups to ensure the some end point.

Control diet was given before the initiation of SFA diet.

ACAT2 expression level in the livers of those mice was

analyzed with real-time PCR.

*P?0.05 compared with the level in the group not fed

with SFA diet.

major type of oil often used in animal diets. Another major type is palm oil, containing a high proportion of palmitic acid, yet also large quantities of unsaturated fatty acids including oleic acid and linoleic acid.25 In contrast, hy-drogenated coconut oil is primarily composed of SFA, making it more suitable than palm oil for our study. Al-though its major component is not palmitic acid, hydro-genated coconut oil presents a mixture of SFAs, and the impact of hydrogenated coconutoil on ACAT2 expression could be concluded as the effect of SFA.

In HepG2 cells, we observed that PA treatment led to a sustained and gradual increase of ACAT2 expression, but hydrogenated coconut oil feeding caused only a short-term rise of ACAT2 expression in mouse liver, with a peak in those fed with SFA diet for two days. This discrepancy was associated with different treatments, reflecting the dif-ference in strength and duration of the impact of different treatments on ACAT2 expression. The results of our study suggest that PA might have a strong and long effect on ACAT2 expression in hepatic cells, whereas hydrogenated coconut oil might have a weak and short influence on ACAT2 expression in liver in vivo.

ACAT2 is an important enzyme in lipid metabolism, the overexpression of which stimulates cholesterol ester syn-thesis and ApoB secretion.4,5 Given the changes observed in ACAT2 expression in our study, we inferred that PA and hydrogenated coconut oil might regulate cholesterol ester synthesis, ApoB secretion, and VLDL cholesterol ester through acting on ACAT2. Further work will be necessary to demonstrate detailed mechanism in the regulatory process of ACAT2 expression by SFA and to determine the possible role of SFA in regulating cholesterol ester biosynthesis and ApoB secretion.

ACKNOWLEDGEMENT

We thank Lei Li, Lu Lu, Guo-wei Zhao, Bei-bei Mao, Huan Gong, Zhen-ya Li, Li Li, Hui-na Zhang, and Shuang Zhou for their technical assistance in this study.

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