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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:68-71, January 2004

Nutrient-Gene Interactions
Research Communication

Conjugated Linoleic Acid Upregulates LDL Receptor Gene Expression in HepG2 Cells

Shaomei Yu-Poth*, Dezhong Yin**, Guixiang Zhao*, Penny M. Kris-Etherton{dagger},1 and Terry D. Etherton*,**

* Graduate Program in Nutrition, {dagger} Department of Nutritional Sciences, and ** Department of Dairy and Animal Science, The Pennsylvania State University, University Park, PA 16802

1To whom correspondence should be addressed. E-mail: pmk3{at}psu.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Conjugated linoleic acid (CLA) exerts anticarcinogenic and antiatherosclerotic effects in animals. The present study was conducted to examine the effects of CLA on LDL receptor (LDLr) expression in HepG2 cells, and to evaluate whether the sterol response element binding protein 1 (SREBP-1) and acyl CoA:cholesterol acyltransferase (ACAT) were involved in the regulation of LDLr expression by CLA. When HepG2 cells were cultured with serum-free DMEM for 48 h, there was a three- to fivefold (P < 0.05) increase in LDLr protein and mRNA levels. Incubation of HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH, 5 mg/L) for 24 h decreased LDLr protein and mRNA by 50–70% (P < 0.05) and mature SREBP-1 by 20–40% (P < 0.05). CLA, but not linoleic acid, antagonized the depressive effects of 25OH and increased both LDLr protein and mRNA abundance twofold (P < 0.05). LDLr protein and mRNA abundance were not different when HepG2 cells were cultured with CLA (0.4 mmol/L) plus 25OH in the presence or absence of an ACAT inhibitor (58–035, 1 mg/L). Furthermore, CLA had no effect on SREBP-1 abundance. These results suggest that CLA upregulates LDLr expression via a mechanism that is independent of ACAT and SREBP-1.

KEY WORDS: • cholesterol • conjugated linoleic acid • LDL • LDL receptor mRNA • LDL receptor protein

Conjugated linoleic acid (CLA)1 is a term used to describe a group of 18-carbon fatty acid isomers with conjugated double bonds (1). Several studies have shown that CLA has beneficial effects on chronic diseases, such as cancer and atherosclerosis (2,3). For example, CLA supplementation (1.8–2.0 g/d for 8 wk) significantly decreased plasma VLDL cholesterol (-32%) and triglyceride (TG, -21%) levels in healthy human subjects (4); CLA also significantly reduced plasma total (-26%) and LDL cholesterol (-21%) and TG (-43%) levels in hamsters (5,6). Many studies have shown that dietary fatty acids regulate plasma LDL cholesterol levels by affecting LDL receptor (LDLr) activity, protein and mRNA abundance (7,8). Cholesterol-raising SFA (12:0, 14:0, 16:0) decrease LDLr activity, LDLr protein and mRNA abundance, whereas unsaturated fatty acids increase these variables (7,8). It has been suggested that dietary fatty acids and cholesterol regulate hepatic LDLr activity via a cholesteryl ester (CE) and the free cholesterol regulatory pool (9). This cholesterol regulatory pool is affected by acyl CoA:cholesterol acyltransferase (ACAT), the enzyme that catalyzes esterification of cholesterol to different fatty acids. A study with hamsters suggested that CLA decreased serum cholesterol levels via downregulation of intestinal ACAT activity and reducing cholesterol absorption (10).

Sterols exert their effects via a sterol regulatory element 1 (SRE-1) in the LDLr gene promoter. SRE-binding proteins (SREBP-1 and -2) bind to SRE-1 to activate LDLr gene transcription in sterol-depleted cells (11). SREBP-1 is synthesized as a 125-kDa precursor protein, which is cleaved by specific proteases to release a mature SREBP-1 (~68 kDa) in the absence of oxysterols (12). The mature SREBP-1 enters the nucleus and activates transcription of the LDLr gene.

The present study was conducted to examine how CLA affects LDLr gene expression using human hepatoma HepG2 cells as a model cell line. HepG2 cells were chosen because dietary fatty acids and cholesterol regulate hepatic LDLr activity in this cell line (9). The mechanism by which CLA exerts its regulatory effects was also evaluated. Specifically, ACAT and SREBP-1 were examined to determine whether they are involved in the regulation of LDLr expression by CLA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Cell culture and treatments. HepG2 cells were maintained in DMEM plus 10% fetal bovine serum under 10% CO2 at 37°C in 10-cm dishes. The medium was supplemented with 0.1 mmol/L nonessential amino acids and 1 mmol/L sodium pyruvate. When the cells were 60–70% confluent, the maintenance medium was removed and cells were treated with the defined DMEM [1.5% bovine serum albumin (BSA), 5 mg/L transferrin, 2 µg/L selenium, 10 µg/L {alpha}-tocopherol, and 1 nmol/L triiodothyronine) for 24 h to upregulate LDLr expression and then treated with 25OH (5 mg/L) and/or fatty acids (0.4 mmol/L) for another 24 h.

Fatty acids were added to the culture medium as a fatty acid-BSA complex. Briefly, CLA (41% of 9-cis, 11-trans and 9-trans, 11-cis; 44% of 10-trans, 12-cis; 10% of 10-cis, 12-cis, and other isomers; Nu-Chek-Prep, Elysian, MN) and linoleic acid (LA) (Sigma Chemical, St. Louis, MO) were dissolved in the serum-free defined DMEM described above. Fatty acid concentration in the DMEM was verified using GC analysis as described below.

Fatty acid-BSA complexes were added to culture dishes at a concentration of 0.4 mmol/L of fatty acid, a physiologic level, and 1.5% of BSA (fatty acid:BSA = 2:1 mol/L ratio) with 25OH (5 mg/L) in 0.05% ethanol or 0.05% ethanol alone. Control cells were exposed to the defined DMEM (Ctrl). Cells were also treated with a fatty acid mixture that was selected to approximate the fatty acid profile of plasma (13). The fatty acid mixture (Mix) was comprised of 3% 14:0, 22% 16:0, 12% 18:0, 34% 18:1, 26% 18:2(n-6), 1% 18:3(n-3), 1% 20:4(n-6) (arachidonic acid; AA), 0.5% 20:5(n-3) (eicosapentaenoic acid; EPA), and 0.5% 22:6(n-3) (docosahexaenoic acid); all fatty acids were from Sigma Chemical. ACAT inhibitor (1 mg/L, 58–035, kindly provided by Sandoz, East Hanover, NJ) was added to the medium to determine whether fatty acid regulation of LDLr gene expression was due to a change in the CE/free cholesterol pool. Cells were cultured for 24 h with test fatty acids with or without 25OH and with or without an ACAT inhibitor.

    Western blot analysis of LDLr and SREBP-1. HepG2 Cells were washed three times with PBS at 4°C and harvested. The cell pellet was resuspended and homogenized in solubilizing buffer containing 1.6% Triton X-100, 5 mol/L urea, 0.1 mmol/L leupeptin, and 1.5 mmol/L phenylmethylsulfonyl fluoride (14). After centrifugation at 12,000 x g for 30 min, the supernatant was removed and stored at -70°C. Protein concentration was measured using the Pierce bicinchoninic acid microprotein assay kit (Pierce, Rockford, IL). Cell lysates (25 µg) were separated on a 7.5% SDS-PAGE gel and then transferred to a nitrocellulose membrane. LDLr was detected with anti-LDLr IgG (generously provided by Dr. Allen Cooper, Stanford University, Palo Alto, CA) using the ECL-Western blotting protocol (Amersham Life Sciences, Boston, MA). A polyclonal antibody against SREBP-1 (K10, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect precursor and mature SREBP-1. Signals were quantified using the ImageQuant Image Analysis Software (Amersham Biosciences, Piscataway, NJ).

    Northern blot analysis of LDLr mRNA levels. LDLr mRNA expression was measured by Northern Blot analysis as described by Mustad et al. (8). Total RNA (15 µg) was separated on a 1% agarose gel and transferred onto a Genescreen membrane. The blot was probed with 32P-labeled human LDLr cDNA (1.2-kb cDNA was generously provided by Dr. Allen Cooper) and then exposed to a Kodak (Rochester, NY) X-Omat AR film at -70°C. To assess variation in loading or transfer of RNA, the blot was reprobed with a cDNA for 18S rRNA. The blots were scanned using the ImageQuant Image Analysis Software and normalized using the rat 18S rRNA to account for differences in loading.

    Quantification of cellular fatty acids. After culture, cells were washed three times with PBS and harvested. Lipids were extracted using the method of Folch et al. (15). The fatty acids were measured by GC. Briefly, fatty acids in the lipid extract were methylated using boron triflouride/methanol (16), and then were analyzed using a Hewlett-Packard (Palo Alto, CA) 5890 gas chromatograph equipped with a Supelco (Bellefonte, PA) SP-2330 capillary column with a helium carrier flow rate 30 mL/min, hydrogen 30 mL/min and air 300 mL/min. The temperature was set at 150°C for 8 min, and then increased to 190°C at 2°C/ min; the temperature was maintained at 190°C for 20 min. Both injector and detector temperature were 250°C. For quantitative estimation of LDL fatty acids, 70 µg of 17:0 internal standard was added to each sample.

    Statistical analysis. Data were analyzed using SAS statistical analysis computer program (version 6.12; SAS Institute, Cary, NC) and expressed as means ± SEM. ANOVA was performed to determine treatment effects. When there was a significant effect of treatment, a t test (least significant differences) was used for the appropriate pair-wise comparisons. In addition, a paired Student’s t test was used to test the differences between treatments with and without ACAT inhibitor. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Effects of fatty acids on LDLr abundance. To confirm that fatty acids in the culture medium were incorporated into cell lipids, we measured the fatty acid profiles in HepG2 cells after exposure to the respective fatty acid. After each fatty acid treatment, the percentage of the targeted fatty acid increased >20% (Table 1). This suggested that the targeted fatty acid was incorporated into cell lipids. We studied the effects of 25OH and 25OH plus CLA, LA or a fatty acid mixture (Mix) on LDLr protein abundance in HepG2 cells by Western blotting analysis. Compared with control cells cultured in the defined medium (Ctrl), 25OH decreased LDLr protein abundance ~55% (P < 0.05) in HepG2 cells (Fig. 1). CLA completely antagonized the 25OH suppressive effects and increased LDLr abundance by more than twofold (P < 0.05) compared with HepG2 cells treated with 25OH (Fig. 1). Similarly, the fatty acid mixture (Mix) significantly increased LDLr protein (P < 0.05) (Fig. 1). However, LA did not antagonize the suppressive effects of 25OH on LDLr expression (Fig. 1). A previous study demonstrated that 1 mg/L of an ACAT inhibitor, 58–035, completely blocked the activity of ACAT in J774 macrophages (17). In the present study, 58–035 (1 mg/L) did not affect the stimulatory effects of CLA or the fatty acid mixture on LDLr abundance in HepG2 cells (Fig. 1). Therefore, ACAT appears not to be involved in the regulation of LDLr expression by CLA in HepG2 cells.


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  		TABLE 1 Fatty acid profiles in HepG2 cells treated with the defined DMEM for 24 h and then treated with 25-hydroxycholesterol (25OH; 5 mg/L) and/or fatty acids (0.4 mmol/L) for another 24 h1

 


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  		FIGURE 1 LDLr expression in HepG2 cells treated with the defined DMEM for 24 h and then treated with 25-hydroxycholesterol (25OH; 5 mg/L) and/or fatty acids (0.4 mmol/L) for another 24 h with (+) or without (-) the acyl CoA:cholesterol acyltransferase (ACAT) inhibitor. (Panel A) A representative Western blot. (Panel B) LDLr protein levels are presented as a percentage of the control in the absence of the ACAT inhibitor (100%). Values are means ± SEM, n = 3. Means without a common letter differ, P < 0.05. The ACAT inhibitor did not affect the abundance of LDLr.

 
    Effects of fatty acids on LDLr mRNA levels. In agreement with the changes in LDLr protein abundance, 25OH decreased LDLr mRNA by 70% (P < 0.05) compared with the control (Ctrl) (Fig. 2). Furthermore, CLA, but not LA, completely antagonized the 25OH suppressive effects and increased LDLr mRNA abundance by one- to twofold (P < 0.05) (Fig. 2). However, the fatty acid mixture (Mix) and LA did not affect LDLr mRNA levels compared with 25OH (Fig. 2), although the mix significantly increased LDLr protein abundance (Fig. 1). In addition, the ACAT inhibitor did not affect the CLA-stimulated increase in LDLr mRNA expression in HepG2 cells, although it affected the regulation of LDLr mRNA expression by 25OH and the fatty acid mixture plus 25OH (P < 0.05) (Fig. 2).



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  		FIGURE 2 LDLr expression in HepG2 cells treated with the defined DMEM for 24 h and then treated with 25-hydroxycholesterol (25OH; 5 mg/L) and/or fatty acids (0.4 mmol/L) for another 24 h with (+) or without (-) the acyl CoA:cholesterol acyltransferase (ACAT) inhibitor. (Panel A) A representative Northern blot. To assess variation in loading or transfer of RNA, the blot was reprobed with a cDNA for 18S rRNA (18S). (Panel B) LDLr protein levels are presented as a percentage of the control in the absence of the ACAT inhibitor (100%). Values are means ± SEM, n = 3. Means without a common letter differ, P < 0.05. *Different from the respective mean without the ACAT inhibitor, P < 0.05.

 
    Effects of fatty acids on SREBP-1 abundance. To determine whether CLA was involved in the regulation of SREBP-1 expression, Western blotting analysis was performed. 25OH decreased SREBP-1 levels by 20–40% (P < 0.05) in HepG2 cells compared with the control (Fig. 3). However, CLA, LA or the fatty acid mixture did not significantly affect the abundance of SREBP-1 (both precursor and mature) (Fig. 3). These results are consistent with a recent study with hamsters (18). In addition, the ACAT inhibitor did not affect SREBP-1 expression in HepG2 cells (Fig. 3).



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  		FIGURE 3 Western blotting analysis of sterol response element-binding protein (SREBP)-1 expression in HepG2 cells treated with the defined DMEM for 24 h and then treated with 25-hydroxycholesterol (25OH; 5 mg/L) and/or fatty acids (0.4 mmol/L) for another 24 h with (+) or without (-) the acyl CoA:cholesterol acyltransferase (ACAT) inhibitor. A representative blot of three independent experiments is shown. The upper band is SREBP-1 precursor (125 kDa) and the lower bands are mature SREBP-1 (~68 kDa).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
LDL cholesterol levels are influenced by the amount and type of dietary fatty acids. One of the mechanisms involved in the metabolism of cholesterol is the LDLr-mediated endocytic uptake of cholesterol-carrying lipoproteins, thereby controlling cholesterol levels in cells and plasma. Both in vivo and in vitro studies have shown that dietary fatty acids differently regulate LDLr mRNA levels, thus affecting LDLr protein abundance. However, there is limited information about how CLA elicits its hypocholesterolemic effects. It has been hypothesized that CLA may be metabolized to one or more active products affecting various biological processes (19). For example, CLA can affect the metabolic interconversion of fatty acids in liver that may ultimately affect lipid metabolism (20). CLA (trans-10, cis-12) decreased stearoyl-CoA desaturase activity (21) and lowered liver lipid concentration by reducing TG synthesis and enhancing fatty acid oxidation in rats (22). In addition, CLA increased hepatic acyl-CoA oxidase and fatty acid-binding protein mRNA levels in rats (23). The present study found that CLA significantly increased both LDLr protein and mRNA abundance in HepG2 cells. These results suggest that CLA may decrease plasma LDL cholesterol by upregulation of LDLr gene expression to promote the clearance of LDL cholesterol from circulation. In contrast, in the presence of 25OH, LA did not affect LDLr protein and mRNA levels in the present study, which is in agreement with earlier findings with another unsaturated fatty acid, oleic acid (17). Thus, CLA is distinctively different from LA with respect to regulating LDLr expression. The present study also showed that the fatty acid mixture increased LDLr protein abundance, but not LDLr mRNA levels. The effect on LDLr protein abundance may be due to some of the fatty acids in the mixture, e.g., 18:0, AA and EPA. Furthermore, the fatty acid mixture might increase LDLr protein stability.

Studies have suggested that fatty acids affect LDLr activity via ACAT, a very important factor for cellular cholesterol distribution (9). An increase in ACAT activity results in cellular cholesteryl esters, which do not affect LDLr expression. In vitro studies showed that fatty acids, such as oleic acid, palmitic acid, AA and EPA increase ACAT activity (17), which then results in an increase in LDLr activity (9). In the present study, the ACAT inhibitor did not affect LDLr expression in HepG2 cells, suggesting that CLA regulatory effects on LDLr gene transcription may be via a pathway that is independent of ACAT. However, the ACAT inhibitor affected the regulation of LDLr mRNA levels by 25OH alone or with the fatty acid mixture (Fig. 2). It is unclear how the ACAT inhibitor decreased LDLr mRNA levels under these conditions.

One possible mechanism by which fatty acids exert their regulatory effects is via sterol-mediated feedback repression of LDLr gene transcription. Elevated levels of cellular sterols block the SREBP maturation process, thereby inhibiting LDLr gene expression (24). PUFA were shown to reduce the hepatic content of precursor and nuclear SREBP-1 60 and 85%, respectively (25). However, a recent study indicated that dietary fat suppressed fatty acid synthesis in hamster intestine independently of SREBP-1 expression (18). Dietary fat did not alter the abundance of SREBP-1 in hamsters (18). Similarly, the present study did not show significant differences in effects of CLA and the fatty acid mixture on SREBP-1 abundance. Therefore, CLA may regulate LDLr gene expression via a pathway that is independent of SREBP-1.

Another possible mechanism for fatty acid regulatory effects on LDLr expression is through peroxisome proliferator-activated receptors (PPAR). Some studies have shown that fatty acids could bind to PPAR and activate it directly (26). CLA also has been reported to be a ligand and an activator of PPAR{alpha} (23). Activated PPAR binds to the peroxisome proliferator response element located in promoters of target genes to mediate gene transcription (27). Further studies are required to determine whether PPAR are involved in the regulation of LDLr gene expression by CLA.


   FOOTNOTES
 
2 Abbreviations used: 25OH, 25-hydroxycholesterol; ACAT, acyl CoA:cholesterol acyltransferase; CE, cholesteryl ester; CLA, conjugated linoleic acid; EPA, eicosapentaenoic acid; LA, linoleic acid; LDLr, LDL receptor; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol response element-binding protein; TG, triglyceride. Back

Manuscript received 10 July 2003. Initial review completed 25 August 2003. Revision accepted 17 October 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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