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* Department of Biological Sciences, University of Notre Dame, Notre Dame, IN and
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA
3To whom correspondence should be addressed. E-mail: nshay1{at}nd.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • soy isoflavones • cardiovascular disease • SREBP • HMG CoA synthase • LDL receptor
High total cholesterol and LDL cholesterol levels correlate with cardiovascular disease. Elevated cholesterol levels contribute to the formation of atherosclerotic plaques and eventually to thrombosis or myocardial infarction. Management of cholesterol levels is an essential part of treating cardiovascular disease. Soy intake has been shown to lower cholesterol levels and thereby reduce the risk of developing atherosclerosis. The beneficial effects of soy intake on plasma lipoprotein levels led to FDA approval of a health claim that "25 g of soy a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease." This health claim and other media publicity are promoting intake of both soy food products and isoflavone-containing soy supplements. However, the mechanism by which soy exerts its effects is unknown and still a matter of debate.
Soy contains protein, lipids, fiber, and phytochemicals including the isoflavones genistein, daidzein, and glycitein. It is unclear which of the components of soy is the major contributor to the hypocholesterolemic effect or whether different components have hypocholesterolemic effects (1–4). Prior work investigating isoflavone action focused on the estrogen-like activity of the isoflavones (5,6). However, there is much controversy as to whether this is the mechanism by which soy isoflavones act on lipid metabolism; for example, genistein has relatively low estrogen receptor binding activity (7). Studies also found that the isoflavone component of soy lowers LDL and total cholesterol levels in postmenopausal women (8) and C57BL/6 mice (9). Isoflavones were also shown to increase LDL receptor mRNA in postmenopausal women (8). However, other human diet trials showed no significant effects of isoflavones (1,2).
The LDL receptor is one of a number of genes regulated by the sterol regulatory element binding proteins (SREBPs)4 (10). Other genes regulated by the SREBPs include fatty acid synthase (11), 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase (12), and HMG CoA reductase (13). There are 3 main isoforms of SREBP encoded by 2 genes. SREBP-1a and SREBP-1c are encoded by a single gene but are produced by differential splicing (14,15). SREBP-1a and -1c are responsible primarily for the regulation of genes involved in fatty acid biosynthesis (16). SREBP-2 is encoded on by its own gene and is primarily responsible for the regulation of genes involved in cholesterol biosynthesis and metabolism (14,15). Each SREBP isoform is synthesized as a 
125-kDa precursor protein. Only when cellular sterol levels are low is SREBP escorted to the Golgi by the SREBP cleavage activating protein (SCAP) (17,18). In the Golgi 2 sequential cleavages occur, releasing the mature N-terminal portion (68 kDa) of SREBP that then translocates to the nucleus and activates transcription of sterol response element (SRE)–containing genes (19). If cellular sterol levels are high, SREBP remains anchored in the endoplasmic reticulum and there is no upregulation of SRE-containing genes (20,21).
Because SREBPs are critical for the regulation of intracellular sterol and lipid homoeostasis, we examined the effects of isoflavone-containing soy extract and individual isoflavones on SREBP levels and maturation as well as their effects on the SRE-regulated genes HMG CoA synthase, HMG CoA reductase, and the LDL receptor. Understanding the metabolic effects of the various components of soy is an important goal to help guide or design the modification of cholesterol-lowering soyfoods or dietary supplements.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture. HepG2 cells were obtained from American Tissue Culture Collection (HB-8065). They were maintained in minimum essential medium (MEM) containing 10% fetal bovine serum (FBS) (Invitrogen) and 1% antibiotic/antimycotic (Invitrogen) at 37°C and 5% CO2. For experiments on d 0, 8.5 x 105 cells/well were plated in 6-well dishes. On d 1 the medium was changed to serum-free medium with antibiotics. On d 2, cells were treated with vehicle, 1 µmol/L atorvastatin, 25 µmol/L cholesterol + 2.5 µmol/L 25-hydroxycholesterol, 10 g/L soy extract, 20 µmol/L genistein, and 20 µmol/L daidzein. Following a 24-h incubation cell protein was isolated using mammalian protein extraction reagent (M-PER, Pierce) according to the manufacturer’s protocol. Total RNA was isolated using a RNeasy kit (Qiagen) according to the manufacturer’s protocol.
Immunoblotting. Protein concentrations were determined using DC protein assay reagents (Bio-Rad) according to the manufacturer’s protocol in 96-well plates. Proteins were size-separated on 10% polyacrylamide:0.1% (w:v) SDS minigels together with prestained molecular weight markers (Pierce). Proteins were transferred to polyvinylidene fluoride membrane (Osmonics) at 495 mA for 1 h using a minitransblot system (Bio-Rad). Immunoblot analyses were performed using antibodies to SREBP-1 and SREBP-2 to detect the mature 68-kDa form. Immunoblot analysis was also performed for HMG CoA reductase. Antibodies were diluted in Tris-buffered saline/0.1% Tween 20 containing 5% nonfat dry milk. All appropriate secondary antibodies used were conjugated to horseradish peroxidase. Antibody complexes were visualized using ECL+ reagent (Amersham Biosciences) according to the manufacturer’s instructions. Immunoblots were exposed to Kodak BioMAX film and developed. The chemiluminescent signal was removed by treating membranes with 2% (w:v) SDS, 62.5 mmol/L Tris-Cl, pH 6.8, and 100 mmol/L 2-mercaptoethanol for 30 min at 55°C, prior to a second round of immunoblotting. Equal loading was confirmed using anti-tubulin antibody (Sigma, Cat. No. T-9026). Experiments were replicated at least 5 times and a representative blot is shown.
Northern blotting. RNA concentration was calculated by spectrophotometry (BIORAD, Smartspec 3000). Total RNA (15 µg) was size-separated on a 1% agarose-formaldehyde gel and transferred to a Hybond N+ nylon membrane (Amersham Biosciences). RNA was isolated from HepG2 cells and an RT-PCR reaction (Superscript RT-PCR, Invitrogen) was performed to generate a HMG CoA synthase cDNA. The forward primer used was 5'GAAGTTGGAACAGAGACAATCATCG-3' and the the reverse primer used was 5'-GGAAGTCATTCAGCAACATCCG-3'. The resulting PCR fragment generated was cloned into a TOPO TA sequencing vector (Invitrogen) and the DNA sequence was confirmed. The probe was labeled with 32P (Rediprime, Amersham) and purified (Nuc-trap probe purification columns, Stratagene) according to the manufacturer’s protocol. The probe was then hybridized overnight in 1–10 x 106 cpm/ml probe at 65°C. After hybridization, the blots were washed and exposed to Kodak BioMAX film. The blots were rehybridized with a probe to the 18S RNA to verify RNA content. Experiments were replicated at least 5 times and a typical blot is shown.
Plasmids and transient transfection assays. The pSynSRE and LDL-t-Luc plasmids were previously described (23,24). The pSV ß-galactosidase (ß-gal) vector was purchased from Promega. On d 0, HepG2 cells were plated in 24-well dishes at a density of 2 x 105 cells/well in MEM containing FBS without antibiotic/antimycotic. On d 1, cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Six hundred nanograms of either pSynSRE or LDL-t-luc and 0.4 µg of pSV ß-galactosidase were transfected into the cells. After 6–8 h the medium was removed and changed to serum-free MEM containing 1% antibiotic/antimycotic. On d 2 the cells are treated with vehicle, 1 µmol/L atorvastatin, 25 µmol/L cholesterol + 2.5 µmol/L 25-hydroxycholesterol, 10 g/L soy extract, 20 µmol/L genistein, 20 µmol/L daidzein, and 20 µmol/L glycitein. For mevalonate experiments cells were treated with 100 µmol/L to 1 mmol/L sodium mevalonate. After 24 h, cell lysates were obtained using 1x reporter lysis buffer (Promega) according to the manufacturer’s protocol. Cell lysates were analyzed for SRE-directed luciferase and ß-gal activities.
Statistics. Results of transfection experiments are calculated as luciferase/ß-gal and are expressed as means ± SEM, n = 3 per experiment. Vehicle is defined as control (100%) and results are expressed as a percentage of control. A one-way ANOVA was carried out to test for statistical significance. Tukey’s test was used for pairwise analysis of treatment groups. Means without a common letter differ (P < 0.05).
LDL uptake.
HepG2 cells were plated on glass coverslips in 6-well dishes (7 x 105 cells/well) and grown for 48 h in complete MEM. Cells were then washed in PBS and incubated with serum-free medium for 24 h. Cells were incubated for 20 h with vehicle, 25 µmol/L cholesterol + 2.5 µmol/L 25-hydroxycholesterol, or 6 mg/L (20 µmol/L) genestein. Fluorescently labeled DiI-LDL (10 mg/L, Biomedical Technologies) was then added to the treatment mixture for 4 h. Cells were subsequently washed twice with PBS for 5 min and fixed to coverslips with fixative solution (11% formaldehyde, ddH2O, 10x PBS, Triton X) for 10 min. Coverslips were air dried and mounted on slides with Vectashield (Vector Laboratories). Analysis was carried out using confocal microscopy (Nikon, Diaphot 200). This experiment was carried out 3 separate times.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To examine the effects of soy extract and the individual isoflavones on the expression of mRNAs regulated by the SREBPs, total RNA was isolated from HepG2 cells following cell treatments identical to those used in Fig. 1a and b, and a Northern blot analysis was performed. In concert with the results in Fig. 1a and b sterol-free, atorvastatin, soy extract, and individual isoflavone treatments increased HMG CoA synthase mRNA levels (Fig. 1c).
Because the levels of SREBP-2- and SREBP-regulated genes increase after exposure to isoflavone-containing soy extract or isoflavones, we wished to examine whether these effects were mediated by SRE control. To this end, HepG2 cells were transfected with SRE-regulated luciferase reporter constructs derived from the HMG CoA synthase (pSynSRE) or the LDL receptor promoters (LDL-t-Luc). When transfected cells were treated with increasing concentrations of soy extract, we observed a dose-dependent increase in SRE-regulated luciferase activity. Consistently, when treated with 5 or 10 mg/L soy extract (total isoflavone concentration 12–24 µmol/L), luciferase activity was well above that of 1 µmol/L atorvastatin and roughly equivalent effects were produced by 1 µmol/L atorvastatin and 1 mg/L soy extract (Fig. 2). This relationship was consistently observed throughout our studies. We examined the effects of the individual isoflavones on SRE-regulated expression from both the HMG CoA synthase (Fig. 3a) and the LDL receptor (Fig. 3b) promoters. Both genistein and daidzein robustly induced luciferase expression from both promoters whereas glycitein did not induce to any appreciable level. These data prompted us to discontinue evaluation of glycitein in our studies. Taken together, the data in Figs. 1 through 3 provide a compelling case for isoflavone-enhanced SREBP-2 maturation and SREBP-2-regulated gene expression via sterol regulatory elements. Although they are provided as one set of representative experiments (Figs. 123), these data are entirely consistent with over 30 independent experiments we completed using HepG2 cells.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Soy extract and isoflavones consistently induced SREBP-2 regulated gene expression over that of atorvastatin, although on a molar basis the concentration of isoflavones used was well above that of atorvastatin. Glycitein did not significantly induce SREBPs and SRE-regulated gene expression in our studies. This may be due to the presence of a methoxy-group on glycitein that is not present on the other isoflavones or reduced cellular uptake of glycitein. The negative effect of glycitein prompted us to discontinue further evaluation of glycitein.
The effects of isoflavones on SRE-directed gene expression have not been well characterized. Recently, hepatic SREBP-1 expression in rats provided with soy protein was evaluated (26). The LDL receptor has been measured in several studies (27–29) but it is unclear whether changes in LDL receptor levels are caused by a direct action of soy isoflavones or affected indirectly by the lipid-lowering effect of soy intake. Short-term in vivo studies may help clarify this question. In our cell culture model in order for isoflavones to exert their effect on SREBP maturation and signaling sterols must be present at low levels. We showed that increased levels of cholesterol decrease the effectiveness of isoflavones in inducing the expression of SREBP-regulated genes. Because sterols decrease the expression of SREBP and SRE-regulated genes, increasing the amount of sterols should decrease the levels of expression of these genes. Therefore, if intake of sterols is low we hypothesize that isoflavones will increase SREBP and SRE-regulated genes and thereby decrease already high cholesterol levels. However, it remains to be determined in vivo whether high dietary cholesterol will lead to an increase in SREBP and SRE-regulated genes.
We hypothesize that there are at least two different mechanisms by which isoflavone-containing soy extract and individual isoflavones may induce SREBP-regulated gene expression in HepG2 cells. Isoflavones may inhibit HMG CoA reductase enzyme activity with a statin-like effect. The S-alkenyl cysteines of garlic exert a hypocholesterolemic effect by inhibiting HMG CoA reductase activity (30). It was also shown that phosphorylation of HMG CoA reductase at serine 871 by AMP-activated protein kinase decreases enzyme activity (31). Recently it was shown that isoflavones are weak inhibitors of HMG CoA reductase but with a much higher Ki value than the statins (32). However, the manner by which HMG CoA reductase is inhibited was not proposed and it is possible that isoflavones may decrease HMG CoA reductase levels by altering its phosphorylation. Alternatively, isoflavones could exert their effects directly on the maturation of SREBP, perhaps by acting as ligands of the SCAP. The steroid-like analogue GW707 and the nonsteroidal molecules GW300, GW532, and GW575 have been shown to be ligands of SCAP that upregulate the expression of the LDL receptor and the mature forms of both SREBP-1 and -2 in a similar fashion as we showed for the isoflavones (33). Our subsequent investigations did not rule out either of these mechanisms (Figs. 6and 7). Although we did observe a decrease in SRE-regulated gene expression when cells were cotreated with mevalonate and soy extract, the reduction was not to the same degree as that observed when cells were treated with mevalonate alone or cotreated with mevalonate and atorvastatin (Fig. 7). Mevalonate alone and mevalonate plus atorvastatin decreased SRE-directed gene expression 80% whereas mevalonate plus soy extract reduced expression by only 50%. These data suggest that both mechanisms may be involved.
We also recognize that isoflavones may affect other pathways involved in lipid and sterol metabolism. It was recently shown that soy isoflavones induce peroxisome proliferator activator receptor (PPAR)–regulated gene expression (34,35). PPARs are involved in fatty acid catabolism, adipocyte differentiation, and insulin sensitization. Because sterols are now understood to regulate via several different mechanisms, including action as both a SCAP ligand and agonist of the liver-X-receptor (LXR), it is entirely possible that isoflavones act on multiple pathways as well. It remains to be determined whether in vivo effects of soy intake are mediated mainly by isoflavones or another soy component. In vivo evaluation of isoflavones as agonists of SRE- and PPAR-regulated gene expression will help determine whether isoflavones contribute to the hypolipidemic effect of soy intake and which pathway or pathways are most important.
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FOOTNOTES |
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2 Supported by NIH AT000862 and The Solae Company.
4 Abbreviations used: ß-gal, ß-galactosidase; daid, daidzein; gen, genistein; fetal bovine serum, FBS; HMG CoA, 3-hydroxy-3-methylglutaryl coenzyme A; MEM, minimum essential medium; mev, mevalonate; M-PER, mammalian protein extraction reagent; PPAR, peroxisome proliferators activator receptor; SCAP, sterol regulatory element binding protein cleavage activating protein; SE, soy extract; SRE, sterol regulatory element; SREBP, sterol regulatory element binding protein; stat, statin; sterol, 25-hydroxycholesterol; veh, vehicle.
Manuscript received 28 June 2004. Initial review completed 20 July 2004. Revision accepted 20 August 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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