Onion peel extract increases hepatic low-density lipoprotein receptor and ATP-binding cassette transporter A1 messenger RNA expressions in Sprague-Dawley rats fed a high-fat diet

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Nutrition Research 32 (2012) 210 – 217 www.nrjournal.com

Onion peel extract increases hepatic low-density lipoprotein receptor and ATP-binding cassette transporter A1 messenger RNA expressions in Sprague-Dawley rats fed a high-fat diet Seung-Min Leea , Jiyoung Moona , Hyun Ju Doa , Ji Hyung Chungb , Kyung-Hea Leec , Yong-Jun Chac , Min-Jeong Shina,⁎ a

Department of Food and Nutrition, Institute of Health Sciences, Korea University, Seoul, Republic of Korea b Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea c Department of Food and Nutrition, Changwon National University, Changwon, Republic of Korea Received 25 August 2011; revised 9 January 2012; accepted 18 January 2012

Abstract In the present study, we hypothesized that onion peel extract (OPE) alters hepatic gene expression to improve blood cholesterol profiles. To investigate the effect of OPE to test our hypothesis, SpragueDawley rats were fed ad libitum for 8 weeks with the control, high-fat diet (HFD) or the high-fat diet with 0.2% OPE supplementations (HFD + OPE). Messenger RNA (mRNA) levels of genes in cholesterol metabolism and fatty acid metabolism were examined by semiquantitative reverse transcriptase polymerase chain reaction. The OPE in HFD reverted high fat–induced reduction in mRNA levels of sterol regulatory element-binding protein-2, low-density lipoprotein receptor, and hydroxyl-3-methylglutaryl coenzyme reductase genes in the liver comparable with the levels of the control group. Onion peel extract slightly increased stearoyl-coA desaturase 1 (SCD-1) expression compared with high-fat feeding. However, sterol regulatory element-binding protein-1c and fatty acid synthase were not affected by high-fat or OPE feeding. Onion peel extract also enhanced expression of ATP-binding cassette transporter A1, peroxisome proliferator–activated receptor γ2 and scavenger receptor class B type I genes when compared with high-fat feeding. However, OPE did not influence high fat–triggered changes in apolipoprotein A1 mRNA levels and liver X receptor α were not affected by either high-fat or OPE feeding. Our results suggest that OPE changes the expression of genes associated with cholesterol metabolism in favor of lowering blood low-density lipoprotein cholesterol and enhancing high-density lipoprotein cholesterol through increasing mRNA abundance of low-density lipoprotein receptor and ATP-binding cassette transporter A1 genes. © 2012 Elsevier Inc. All rights reserved. Keywords: Abbreviations:

Onion peel extract; LDL receptor; SREBP-2; HMG-CoA reductase; ABCA1, LXRα ABCA1, ATP-binding cassette transporter A1; ApoA1, apolipoprotein A1; CVD, cardiovascular disease; FAS, fatty acid synthase; HDL, high-density lipoprotein; HFD, high-fat diet; HMG-CoAR, hydroxyl-3-methylglutaryl coenzyme reductase; HPLC, high-performance liquid chromatography; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; LXRα, liver X receptor α; mRNA, messenger RNA; OPE, onion peel extract; PPARγ2, peroxisome proliferator–activated receptor γ2; SR-BI, scavenger receptor class B type I; SREBP-2, sterol regulatory element-binding protein-2.

1. Introduction ⁎ Corresponding author. Tel.: +82 2 940 2857; fax: +82 2 940 2850. E-mail address: [email protected] (M.-J. Shin). 0271-5317/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2012.01.004

Abnormal blood lipid profiles have been highly associated with an increased risk for metabolic disorders such as cardiovascular diseases (CVDs) [1]. It is well demonstrated

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that an elevated blood cholesterol level is known for a primary condition contributing to CVD [2,3]. Also, recent clinical trials using statins showed that a decrease in blood low-density lipoprotein (LDL) cholesterol reduces cardiovascular risk [4]. In addition, epidemiologic evidence has reported that low levels of serum high-density lipoprotein (HDL) cholesterol were associated with increased CVD morbidity and mortality [3]. In this regard, the strategy for ameliorating blood lipid profiles has been suggested to confer cardiovascular protective effects. Numerous data from epidemiologic and clinical studies have indicated a relationship between dietary intake of flavonoids and reduced risk of CVD [5,6]. Onion is among the most highly consumed vegetables worldwide, which contains many different forms of flavonoids, predominantly quercetin, and smaller quantities of kaempferol, isorhamnetin, anthocyanin, and taxifolin [7]. Besides quercetin, there are quercetin derivatives where glycosyl moieties are found in different positions of the molecule [8,9]. Many epidemiologic and animal studies suggest beneficial health effects of flavonoids and organosulfur compounds, which are abundant in onions [10], including antidiabetic, anticancer, and antifungal activity [11-14]. We previously reported the beneficial effects of onion supplementation in improving lipid metabolism in humans [15,16], suggesting it as an alternative and/or supplementary option for the prevention of CVD. For example, 12-week onion powder displayed significant reductions in circulating levels of total cholesterol and LDL cholesterol in hyperlipidemic patients [15]. Also, it was demonstrated that 10-week supplementation of quercetin-rich onion extract lowered serum levels of LDL cholesterol and increased serum levels of HDL cholesterol in male smokers [16]. Although accumulating data have reported positive effects of onion supplementation on lipid metabolism, the exact underlying mechanism explaining these effects is still unclear. In the current study, we hypothesized that onion peel extract (OPE) changes the expression of hepatic genes for cholesterol metabolism to modulate blood cholesterol levels. Therefore, we investigated whether the effects of OPE supplementation could modulate hepatic messenger RNA (mRNA) expression of genes regulating cholesterol homeostasis in rats with diet-induced hypercholesterolemia, which were extended from a previous study [17]. Our results may provide a mechanism to support the idea that the quercetin-rich OPE could modulate hepatic gene expression to ameliorate dysregulated blood lipid profiles.

2. Methods and materials 2.1. Preparation of OPEs Onion peel extracts were prepared with onion peels purchased from Nonghyup (Changnyeong, Korea). They were washed 3 times in tap water, extracted with 60% aqueous ethanol solution (50°C, 3 hours) in an extractor

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(1kL; Hansung F&C Co, Ltd, Incheon, Korea), and then filtered with filter press (Hankook Industry Co Ltd, Ansan, Korea). The filtrates were concentrated to 2.4° Brix as percent soluble solid, which was measured using a refractometer (Atago Co Ltd, Tokyo, Japan) in a vacuum concentrator (1kL; Hansung F&C Co). The concentrates were processed to a powder with a freeze dryer (SFDTS–200 kg; Samwon Industry Co, Ltd, Seoul, Korea) and passed through a #40 mesh to a fine powder, which contained 100 mg quercetin, along with 128 mg mixed flavonoids/g of OPEs (composition not known). 2.2. Quercetin content determination Quercetin measurements were done using high-performance liquid chromatography (HPLC) determination as previously described [17]. In brief, the hydrolysis of all glycosides to quercetin aglycone, followed by HPLC determination from samples, was analyzed. A 0.1-g sample (OPE) was mixed with 40 mL of 60% aqueous ethanol and 5 mL of 6 N HCl. After refluxing at 95°C for 2 hours, the hydrolyzed solution was filtered into a 100-mL flask with 60% aqueous ethanol. Approximately 10 mL of the solution filtered through a 0.45-μm filter before injection for HPLC analysis. Quercetin in OPE was quantified using a Hewlett-Packard 1100 series HPLC system (HewlettPackard, Palo Alto, CA, USA) with a ZORBAX C18 column (150 × 4.6 mm, 5 μm, XDB-C18; Hewlett-Packard). Elution was performed using a mobile phase made up of water–5% acetic acid–acetonitrile (40:30:30) at a flow rate of 1.0 mL/min. UV detector was measured at 370 nm, and the sample injection volume was 20 μL. Quantification was extrapolated from the pure quercetin (Sigma Chemical Co., St. Louis, MO) standard curve. 2.3. Animals Three-week-old Sprague-Dawley (SD) rats were used for an experiment of 0.2% OPE supplementation in high-fat diet (HFD) (33% fat for total energy consisting of 12.4% and 20.6% from corn oil and lard, respectively). After a 1-week adaptation period, the animals were randomly assigned to 1 of the 3 experimental groups (control, HFD, HFD + OPE) and fed for 8 weeks. Each group contained 8 animals. The HFD group was provided with a diet containing lard as a source of saturated fat. The HFD + OPE group was fed a diet containing 0.2% (wt/wt) of OPE. Diet composition is shown in Table 1. Test diets were stored at 4°C. Animals were fed the control diet, HFD, or HFD + OPE ad libitum. All experimental animals were purchased from Koatech (Pyungtek, Korea), which were grown in a pathogen-free environment and housed in a temperature-controlled (18°C– 24°C) and humidity-controlled (50%–60%) room. Animal body weight was measured every week. All the experimental procedures were approved by the Committee on Animal Experimentation and Ethics of Korea University.

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Table 1 Ingredient composition (in grams per kilogram) of the diets fed to rats Ingredients

Corn starch Casein Sucrose Corn oil Mineral mix a Vitamin mix b Cellulose DL-methionine Choline bitartrate Lard Cholesterol BHTc OPEs Total

Groups Control

HF

HF + 0.2% OPE

54 20 10 6 3.5 1.0 5 0.3 0.2 – – 0.001 – 100.001

43 20 10 6 3.5 1.0 5 0.3 0.2 10 1.0 0.001 – 100.001

43 20 10 6 3.5 1.0 5 0.3 0.2 10 1.0 0.001 0.2 100.201

n = 8 rats per group. BHT indicates butylated hydroxytoluene. a American Institute of Nutrition (AIN)-93 mineral mix. b AIN-93 vitamin mix, 3480 BHT.

2.4. Samples collection (liver) and storage At the end of the experimental feeding period, the rats fasted 12 hours before being euthanized with diethyl ether. The liver was then removed, weighed, rapidly frozen with liquid nitrogen, and stored in the freezer at −80°C.

sequences are presented in Table 2. Peroxisome proliferator– activated receptor γ2 (PPARγ) primers were taken from a published article [18], and the other primers were designed using primer 3. The polymerase chain reaction products were separated by electrophoresis on a 2% agarose gel. The separated bands were visualized using a UV transilluminator (Fluorchem FC2; Alpha Innotech, San Leandro, CA, USA). The images were captured using a Sigma EXDG camera (Nikon, Tokyo, Japan). The intensity of the bands was quantitated using Alphaview software (Alpha Innotech). Values were expressed in arbitrary unit. The mRNA levels were determined by relative values to that of an endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and expressed as fold change over the control. 2.6. Statistical analyses Statistical analysis was performed using the SPSS (SPSS Inc, Chicago, Ill). The variables were logarithmically transformed for statistical analysis, and for descriptive purposes, the mean values were presented using untransformed values. The results were presented as means ± SD, and the differences among the experimental groups were analyzed using 1-way analysis of variance (ANOVA) with Duncan multiple range at P b .05 at the criterion of significance. 3. Results

2.5. RNA extraction from animal liver and semiquantitative reverse transcriptase polymerase chain reaction

3.1. Body weight gain and food efficiency ratio in animals on control and test diets

Liver samples (0.05 g) were homogenized in 0.4 mL of lysis buffer (Qiagen, Valencia, CA, USA) using a Dounce homogenizer. Total RNA was extracted from liver tissue using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's protocol. Complementary DNA was synthesized from 1 μg of RNA using oligo-dT and Superscript II reverse transcriptase (Invitrogen, Grand Island, NY, USA). One microgram of complementary DNA was used for polymerase chain reaction. Rat primer

As described in the previous report [17], animals raised on 3 different diets, which were control diet (control group), HFD (high-fat group), and HFD + OPE (OPE group) (Table 1), were examined for their body weight gain and food intake. There were no significant differences in body weight gains in animals fed different diets ad libitum over the 8-week period (control group: 284.9 ± 12.6 g, HFD group: 284.6 ± 14.3 g, and HFD + OPE group: 292.4 ± 16.7 g). The HFD and HFD + OPE groups consumed a significantly

Table 2 Primers used in the experiment Gene (cycle)

Forward primer

Reverse primer

SCD-1 (30) SREBP-2 (30) SREBP-1c (30) LDLR (30) FAS (30) HMG-CoAR (30) ABCA1 (20) ABCG1 (32) SR-BI (24) ApoA1 (20) LXRα (24) PPARγ2 (32) GAPDH (30)

5′-TCCTGCTCATGTGCTTCATC-3′ 5′-AGACTTGGTCATGGGGACAG-3′ 5′-GGCATGAAAC CTGAAGTGGT-3′ 5′-CAGCTCTGTG TGAACCTGGA-3′ 5′-TCGAGACACA TCGTTTGAGC-3′ 5′-TGCTGCTTTG GCTGTATGTC-3′ 5′-GTACCCAGCGTCCTTTGTGT-3′ 5′-CTGCAAGAGAGGGATGAAGG-3′ 5′-TGCCCCAGGTTCTTCACTAC-3′ 5′-GCCACTGTGTATGTGGATGC-3′ 5′-TACAACCGGGAAGACTTTGC-3′ 5′-CTTGGCCATATTTATAGCTGTCATTATT-3′ 5′-TCTGACATGC CGCCTGGAGA A-3′

5′-GGATGTTCTCCCGAGATTGA-3′ 5′-GGGGAGACATCAGAAGGACA-3′ 5′-TGCAGGTCAG ACACAGGAAG-3′ 5′-TTCTTCAGGT TGGGGATCAG-3′ 5′-TCAAAAAGTGCATCCAGCAG-3′ 5′-TGAGCGTGAACAAGAACCAG-3′ 5′-CCCAAGAGAGTGGAGAGACG-3′ 5′-ACAGGAGGGTTGTTGACCAG-3′ 5′-CCCTACAGCTTGGCTTCTTG-3′ 5′-AACCCAGAGTGTCCCAGTTG-3′ 5′-TGCAGAGAAGATGCTGATGG-3′ 5′-TGTCCTCGATGGGCTTCAC-3′ 5′-TGGAGGCCAT GTAGGCCATG A-3′

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lower amount of food (17.2 ± 0.2 and 17.4 ± 0.6 g, respectively) than the control group (19.6 ± 0.8 g). Because the diet of the HFD and HFD + OPE groups was calorically denser than that of the control group (1825 vs 1632.8 joule/100 g), cumulative energy intake for 8 weeks was similar among the groups. Calculated food efficiency ratio represents weight gain efficiency based on the amount of total food intake. Food efficiency ratios significantly increased in animals in HFD and HFD + OPE group (0.31 ± 0.02 and 0.31 ± 0.02, respectively) compared with the control group (0.27 ± 0.01).

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3.2. The OPE feeding modulates hepatic expression of genes in LDL metabolism We examined whether high-fat composition affects the expression of genes in LDL metabolism. High-fat diet reduced hepatic mRNA levels of LDL receptor (LDLR), sterol regulatory element-binding protein-2 (SREBP-2), and hydroxyl-3-methylglutaryl coenzyme reductase (HMGCoAR) genes by 20% to 30% in comparison with the control diet (Fig. 1A, C). Both LDLR and SREBP-2 mRNA

Fig. 1. The effect of 8 weeks of 0.2% OPE supplementation on hepatic mRNA levels of LDLR (A), SREBP-2 (B), HMG-CoAR (C), SREBP-1 (D), FAS (E), and SCD-1 (F) genes in rat fed HFD. Control: a group on control diet, HF: a group on HFD, HF + 0.2% OPE: a group on HFD supplemented with 0.2% OPE. The results were expressed as means ± SD of 8 animal tissues. Tested by ANOVA with Duncan multiple range test. Values with the same superscript letter are not significantly different (P ≥ .05).

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expressions in the HFD group were significantly lower by about 20% compared with those of the control group (Fig. 1A, B). In the case of HMG-CoAR, there was an approximate 30% decrease in the transcript levels (Fig. 1C). These changes in transcript levels caused by HFD were reversed by supplementation of the OPE to that of the levels of the control diet (Fig. 1A-C). There were significant increases in mRNA abundance of LDLR, SREBP-2, and HMG-CoAR in the HFD + OPE group (Fig. 1A-C). In addition, it was examined whether genes involved in fatty acid metabolism were influenced by the OPE supplementation. Both SREBP-1c and fatty acid synthase (FAS) were not significantly affected in animals on HFD compared with those on the control diet (Fig. 1D-F). Interestingly, animals fed HFD + OPE displayed higher expression of staeroyl-coA desaturase 1 (SCD-1) than the HFD group (Fig. 1F). These results may indicate that the OPE is likely to affect transcription of genes in LDL metabolism but not fatty acid metabolism. 3.3. The OPE intake regulates hepatic expression of genes in HDL metabolism To investigate the effect of the OPE on HDL metabolism, genes associated with this regulation were examined in animal livers. In comparison with the control group, the HFD group displayed no significant changes in transcript levels of ATPbinding cassette transporter A1 (ABCA1), ATP-binding cassette subfamily G member 1 (ABDG1), scavenger receptor class B type I (SR-BI), and PPARγ2 genes (Fig. 2A-C, E, F) but showed a decrease in apolipoprotein A1 (ApoA1) mRNA levels (Fig. 2D). However, the HFD + OPE group significantly up-regulated expression of ABCA1 and SR-BI genes compared with the HFD group (Fig. 2A, C) and increased mRNA levels of ABCG1 in comparison with the control (Fig. 2B). In addition, OPE elevated PPARγ2 gene expression higher than both the control and the HFD groups (Fig. 2F). The high fat–induced change in ApoA1 gene, shown in the HFD group, was sustained in the OPE group as well, hence suggesting no influence by the OPE (Fig. 2D). There was no change in LXLα gene expression either by OPE or by high-fat feeding. These results suggest that OPE appears to modulate the expression of several genes involved in HDL metabolism.

4. Discussion In the present study, we examined the effects of OPE on the expression of hepatic genes involved in cholesterol metabolism in high fat–fed animals and examined possible cellular mechanisms conferring cholesterol-lowering effects of the OPE. To understand the metabolic effects of the OPE on blood cholesterol profiles, which were previously observed [15,16], we focused on the hepatic gene expressions that regulate hepatic cholesterol metabolism and, ultimately, affect blood cholesterol levels [19]. We hypothesized that OPE affects cellular cholesterol homeostasis by altering the

expression of hepatic genes for cellular cholesterol uptake and release. A major regulatory step for blood LDL cholesterol uptake to the liver is through LDLR-mediated transport [19], of which expression is regulated by SREBP2, a key transcription factor for cholesterol metabolism. When cellular cholesterol levels decline, SREBP processing in endoplasmic reticulum is initiated and the nuclear form of SREBP translocates to the nucleus to increase intracellular cholesterol levels. The nuclear SREBP up-regulates LDLR and HMGCoAR, a rate-limiting enzyme for cholesterol synthesis [20]. Besides LDLR and HMG-CoAR, SREBP-2 regulates the expression of its own SREBP gene, suggesting a positive feedback mechanism [21,22]. We confirmed that high fat– fed animals down-regulated the expression of SREBP-2 gene, which is likely due to the feedback mechanism induced by elevated levels of cellular cholesterol in the liver. Sterol regulatory element-binding protein target genes LDLR and HMG-CoA were subsequently down-regulated. Low mRNA levels of LDLR are well known contributors for an increase in plasma LDL cholesterol by reduced removal of circulating LDL cholesterol [23]. This reduced clearance of LDL cholesterol may build up cholesterol in the blood and lead to an increase in the risk of atherosclerosis. In contrast, the OPE supplementation reverted the decreased hepatic mRNA levels of SREBP-2 and LDLR in high fat– fed animals, which could ameliorate blood LDL cholesterol levels. However, the OPE also increased in the amount of transcripts of HMG-CoAR gene (Fig. 1C), which catalyzes the synthesis of cholesterol, thereby increasing the possibility of elevated blood cholesterol levels. However, its activity could be inhibited by intracellular cholesterol taken up by LDLR [24,25]. Taken together, we speculate that the mechanism underlying hypocholesterolemic effects of the OPE involves the regulations of SREBP in the induction of the SRE-regulated genes HMG-CoAR and LDLR, of which the net effect is to decrease circulating LDL cholesterol levels. Along with hepatic genes involved in LDL metabolism, our results showed that the OPE affected transcript levels of HDL regulatory genes. As evidenced by the experimental associations of plasma HDL cholesterol levels with either knockdown or overexpression of hepatic ABCA1 in mice [26,27], the liver has been demonstrated to be the major site for HDL formation. In the present study, the OPE enhanced the expression of cholesterol efflux gene, ABCA1 and ABCG1, which might allow the incorporation of cellular cholesterol to acceptor proteins such as ApoA1 to increase the production of HDL cholesterol [28,29]. The OPEinduced expression of ABCA1 and ABCG1genes may be mediated by directly up-regulating PPARγ2 and subsequently increasing levels of liver X receptor α (LXRα). This is a transcription factor for ABCA1 [30,31], even if there was no significant changes in LXRα mRNA levels in the present study. We also observed that SR-BI transcript levels, which were slightly decreased in the HFD group but not

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Fig. 2. The effect of 0.2% OPE supplementation on hepatic mRNA levels of ABCA1 (A), ABCG1 (B), SR-BI (C), ApoAI (D), LXRα (E), and PPARγ2 (F) genes in rat fed HFD for 8 weeks. Control: a group on control diet, HF: a group on HFD, HF + 0.2% OPE: a group on HFD supplemented with 0.2% OPE. The results were expressed as means ± SD of 8 animal tissues. Tested by ANOVA with Duncan multiple range test. Values with the same superscript letter are not significantly different (P ≥ .05).

significantly, were reversed by the OPE. SR-BI protein, a receptor for HDL cholesterol, is used not only to increase HDL cholesterol uptake to the liver but also to enhance biliary cholesterol secretion [32,33]. Therefore, SR-BI upregulation by the OPE may partly lead the clearance of cholesterol into the bile. Thus, OPE is likely to target genes including ABCA1, ABCG1, and PPARγ2 to affect HDL cholesterol metabolism.

There were no significant changes of SREBP-1c and FAS mRNA expressions in either the HFD group or the HFD + OPE group, suggesting that these genes are not likely disturbed by high-fat feeding and affected by OPE supplementation. Sterol regulatory element-binding protein-1c is responsible primarily for the regulation of genes involved in fatty acid biosynthesis, such as FAS [34]. Therefore, any lipid-modulating effects of OPE supplementation could be

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mediated through the regulation of cholesterol homeostasis rather than that of lipogenesis. Meanwhile, the OPE-fed animals had a significantly higher SCD-1 expression compared with the HFD group. SCD-1 is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids, which are used for synthesis of triglycerides, phospholipid, and cholesterol esters [35]. The esterification of cholesterols with long-chain fatty acids can enhance their packaging in the cores of lipoproteins in the blood, thereby sequestering cholesterol within this core until it is removed by controlled mechanisms [36]. Through this change, the OPE might lead to an increased sequestration of cholesterol in the cell and decrease free cholesterol levels. Although the potential mechanism of a lipid-modulating effect of OPE is proposed in this study, the identification of the bioactive components in onion that are responsible for the observed effects still remains to be determined. Onion is a source of polynutrients such as flavonoids, thiosulfinates, and other sulfur compounds that exerts antioxidative, antidiabetic, and anticarcinogenic effects [37]. Among the possible components contained in onion, one could guess that the effects of the OPE might be mainly derived from the quercetin in it. Indeed, the OPE used in our study contained quercetin constituting 10% of total dry weight. However, one cannot exclude the possibility that the hypolipidemic effects were derived from other compounds rather than quercetin. Indeed, it was demonstrated that S-propyl cysteine isolated from onion reduced the secretion of apolipoprotein B100 and TG by HepG2 cells [38]. Other constituents in the OPEs combined with quercetin may contribute to enhancing the expression of LDLR and ABCA1 genes and, in turn, lowering LDL cholesterol and increasing HDL cholesterol levels. Therefore, the identification of the bioactive component in onion, in terms of hypolipidemic effects, needs further study. In summary, we have, for the first time, demonstrated that OPE alters hepatic gene expressions involved in cholesterol metabolism and appears to ameliorate dysregulated plasma cholesterol profiles induced by an HFD in the rat. A limitation of this study is that blood samples failed to confirm whether regulation of hepatic gene expression by OPE ultimately affects blood cholesterol levels in this animal model. Thus, further study is necessary. In addition, the measurement of tissue cholesterol could provide additional information whether OPE could lower the amount of tissue cholesterol. In light of this, it would be of benefit to confirm tissue protein levels by Western blot analysis of the gene proteins of interest. However, our in vitro data using human hepatoma cells demonstrated that OPE could significantly increase both mRNA and protein levels of LDLR and SREBP-2 gene, correspondingly, supporting our results in rats (see supplementary figures). Taken together, our findings suggest that the ameliorating effect of OPE supplementation on dyslipidemia is likely to be achieved by induced expression of LDLR, ABCA1, and ABCG1 genes with increasing mRNA abundance of regulatory transcrip-

tion factors SREBP-2 and PPARγ2, respectively. Further human studies would confirm our findings. Considering that most of the onion peels end up as waste with limited use, this study raises the possibility of a supplementary use of onion peels in the management of hypercholesterolemia, an important risk factor for CVD. Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0004530). Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.nutres.2012.01.004. References [1] Keys A, Taylor HL, Blackburn H, Brozek J, Anderson JT, Simonson E. Mortality and coronary heart disease among men studied for 23 years. Arch Intern Med 1971;128:201-14. [2] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915-24. [3] Gordon DJ, Probstfield JL, Garrison RJ, Neaton JD, Castelli WP, Knoke JD, et al. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies. Circulation 1989;79: 8-15. [4] National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation 2002;106:3143-421. [5] Geleijnse JM, Launer LJ, Van der Kuip DA, Hofman A, Witteman JC. Inverse association of tea and flavonoid intakes with incident myocardial infarction: the Rotterdam Study. Am J Clin Nutr 2002;75: 880-6. [6] Arts IC, Hollman PC. Polyphenols and disease risk in epidemiologic studies. Am J Clin Nutr 2005;81:317S-25S. [7] Slimestad R, Fossen T, Vagen IM. Onions: a source of unique dietary flavonoids. J Agric Food Chem 2007;55:10067-80. [8] Lee J, Mitchell AE. Quercetin and isorhamnetin glycosides in onion (Allium cepa L.): varietal comparison, physical distribution, coproduct evaluation, and long-term storage stability. J Agric Food Chem 2011; 59:857-63. [9] Benitez V, Molla E, Martin-Cabrejas MA, Aguilera Y, Lopez-Andreu FJ, Cools K, et al. Characterization of industrial onion wastes (Allium cepa L.): dietary fibre and bioactive compounds. Plant Foods Hum Nutr 2011;66:48-57. [10] Hertog MG, Hollman PC. Potential health effects of the dietary flavonol quercetin. Eur J Clin Nutr 1996;50:63-71. [11] Powolny AA, Singh SV. Multitargeted prevention and therapy of cancer by diallyl trisulfide and related Allium vegetable-derived organosulfur compounds. Cancer Lett 2008;269:305-14. [12] Jung JY, Lim Y, Moon MS, Kim JY, Kwon O. Onion peel extracts ameliorate hyperglycemia and insulin resistance in high fat diet/ streptozotocin-induced diabetic rats. Nutr Metab (Lond) 2011;8:18. [13] Campos KE, Diniz YS, Cataneo AC, Faine LA, Alves MJ, Novelli EL. Hypoglycaemic and antioxidant effects of onion, Allium cepa: dietary

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