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*
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 and
Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801
3To whom correspondence should be addressed. E-mail: nshay1{at}nd.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • soy • isoflavones • gene expression • cholesterol • mRNA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). This reduction has been observed in both human and animal studies. In humans, this reduction is more pronounced when hypercholesterolemic individuals consume soy. Animal studies often show profound reductions in cholesterol levels when replacing dietary animal protein, such as casein, with a vegetable protein, such as soy (3
). Cholesterol reductions in humans are somewhat less robust in nature in part due to the factors involved in human studies: use of soy as a supplement in studies rather than as 100% of dietary protein source, fewer experimental subjects, variations in age, body index, initial cholesterol levels, activity levels, and previous dietary patterns are all factors that may contribute to variations in the hypocholesterolemic effect of soy.
The cause of the cholesterol-lowering effect of soy is not known with any certainty. A variety of studies have been conducted to elucidate the factor(s) responsible. Recent reviews (4
–6
) discuss potential soy-regulated metabolic mechanisms that reduce cholesterol levels. Evidence suggests that the protein component and a nonprotein component of soy may both be contributing to the cholesterol-lowering effect of soy. The active protein component may relate to the bioactivity of specific soy proteins, peptides produced by partial digestion of soy proteins, or the specific amino acid composition of soy. Within the nonprotein fraction of soy, the isoflavones have been investigated in a number of studies as a likely agent that may be affecting cholesterol metabolism. Other components of this nonprotein fraction that may influence cholesterol metabolism include soy fiber, lignans, saponins and soy lipids.
In this study, we studied one aspect of the cholesterol-lowering effect of soy using a genetic discovery method. We previously hypothesized that soy intake affects gene expression in the liver (3
). Our own reports and those of others have confirmed this hypothesis (3
,7
,8
). The liver has a profound influence on blood cholesterol levels, being the source of circulating VLDL and HDL in the bloodstream. Additionally, the liver is responsible for the clearance of LDL from the bloodstream (9
) and it is a major source of secreted blood proteins, some of which may affect cholesterol levels or arterial function. Determining which genes are regulated by soy intake may provide a new research direction to determine how soy intake reduces cholesterol levels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All animal protocols were approved by the University of Illinois Laboratory Animal Care Advisory Committee. Adult Mongolian gerbils (Meriones unguiculatus) of mean weight 60 g were housed individually in plastic shoebox cages in an environmentally controlled room at 23°C with an alternating 12/12-h light/dark cycle. Gerbils were fed powdered rodent food (Purina, St. Louis, MO) for 1 wk to acclimate them to our facility and to powdered diets. Gerbils were randomly assigned to dietary treatment groups (n = 4) for three different studies. Study 1 lasted 28 d and there were three treatment groups. Gerbils were fed protein as casein (CAS), 4
reduced isoflavone-containing soy (ISP, Supro 670IF; Protein Technologies International, St. Louis, MO), or ISP supplemented with an isoflavone-containing soy extract. Study 2 lasted 14 d and gerbils were fed protein that was a mixture of casein and ISP to provide protein as 0% ISP, 33% ISP, 67% ISP, or 100% ISP. Studies 1 and 2 used intact protein sources. Although the ISP protein used had reduced isoflavone concentrations, there still were measurable levels of isoflavones in this source of protein (Table 3)
. Study 3 was designed to incorporate some protein sources that were isoflavone-free. To produce these isoflavone-free diets, purified amino acids were mixed in ratios resembling the amino acid profile of soy and casein (amino acid compositions are detailed in Table 3
). Study 3 lasted 21 d and there were eight diet treatment groups. Gerbils were provided one of four different protein sources: CAS, ISP, or purified amino acid mixtures that resembled the amino acid content of CAS and ISP. Each of the four protein sources was provided to gerbils with or without the isoflavone-containing soy extract to produce a total of eight diets. The three studies are outlined in Table 1
. In all studies, the diets were identical except for the source of protein and the amount of an isoflavone-containing alcohol extract of soy (Tables 2
and 3
). Due to a concern that purified amino acid mixtures would be inadequate, choline was supplemented to a final concentration of 3 g choline/kg diet in all study 3 diets. The concentration of isoflavones (algycone-form) in ISP was 0.05 mg/g (which is 
2–3% of the concentration of isoflavones in intact, unextracted soy) and was 11.4 mg/g in the soy protein extract. This extract (Table 3)
contains other organic compounds, including saponins, phospholipids, and phenolic acids. The quantitation of isoflavone concentrations was performed by HPLC by the manufacturer. At the end of each feeding trial, gerbils were killed by anesthetization by carbon dioxide inhalation followed by decapitation. Trunk blood was collected without anticoagulant to isolate serum. The abdominal cavity was opened and liver immediately removed for isolation of total RNA. Purified RNA and serum were stored at -80°C until samples were needed for subsequent use.
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Cholesterol and apolipoprotein concentrations in serum were measured using colorimetric assays essentially as described previously (3
).
RNA isolation.
Total liver RNA was isolated using an Ultraspec-II kit (Biotecx Laboratories, Houston, TX) following the manufacturer’s suggested protocol. Sample concentration and purity were analyzed spectrophotometrically. RNA was also visualized after size-separation by electrophoresis, to determine a size distribution of RNA consistent with intact RNA, i.e., 28S rRNA staining was estimated to be twofold greater than 18S rRNA staining.
RNA differential display.
Within each group of gerbils fed casein, ISP, or ISP+, hepatic RNA was pooled from four different gerbils to produce an RNA sample representing each experimental group. Differential display was performed as described (10
) using 4 unique 5'-end primers and 4 unique 3'-end primers, which produced 16 unique combinations for the polymerase chain reaction (PCR). After PCR was conducted in the presence of radiolabeled nucleotides, PCR products were size-separated by electrophoresis and PCR products were viewed by autoradiography after gels were dried onto a large sheet of filter paper. When PCR products were observed that appeared to be produced in a differential manner among the different dietary groups, the corresponding piece of the dried gel was excised and the PCR product was subcloned for further study.
Subcloning and sequencing.
Twenty radiolabeled PCR products exhibiting substantial differences in abundance on autoradiographs were recovered, amplified by an additional round of PCR, and subcloned into pCRII-TOPO (Invitrogen, Carlsbad, CA). The University of Illinois DNA Sequencing Core Center determined nucleotide sequences of cDNA. Sequences were searched using the National Center for Biotechnology Information (Bethesda, MD) server program BLAST (11
). Plasmid DNA was isolated using the Qiagen Plasmid Maxi kit (Qiagen, Valencia, CA). Plasmid DNA was digested with either EcoRI or BamHI/NotI, and the insert and vector were size-separated by electrophoresis on 2% agarose. The insert band was excised from the gel and purified from the agarose using the Qiaex II Gel Extraction Kit (Qiagen, Carlsbad, CA).
Northern blot analysis.
To verify that the mRNA selected from the initial display gels were differentially expressed, Northern blot analysis was performed essentially as described previously (12
). Each RNA sample (15 µg) was size-separated by MOPS/formaldehyde electrophoresis and capillary blotted to Hybond-N+ nylon membranes (Amersham, Arlington Heights, IL). Gel-purified cDNA (25 ng) were radiolabeled with 50 µCi 
-32P-dCTP (Amersham) using the Rediprime DNA labeling system (Amersham). Unincorporated dNTP were separated from radiolabeled DNA probes using NucTrap probe purification columns (Stratagene, LaJolla, CA). Probes were denatured at 100°C for 5 min immediately before hybridization. Blots were hybridized to cDNA probes at 60°C for 2 h. After washing blots, hybridization was visualized first by exposure to phosphorimagining screens (Molecular Dynamics, Sunnydale, CA), and then to Kodak X-AR film at -70°C. The Northern blots were then hybridized with 18S or 28S rRNA probes, which served as nonregulated internal standards. Exposed phosphorimaging screens were quantified by phosphorimaging, with normalization of mRNA values to the 18S or 28S rRNA.
Statistics.
All experiments were analyzed by analysis of variance (ANOVA). The level of significant difference was set at P < 0.05. Studies 1 and 2 were analyzed by one-way ANOVA. Study 3 used a three-way ANOVA, using amino acid profile (soy vs. casein), protein source (intact protein vs. amino acid mixture, and extract (supplemented vs. no supplement) as the three factors. When F tests were significant, Tukey’s post hoc test (13
) was used to identify pair-wise differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Gerbils fed soy-containing diets had lower serum cholesterol concentrations than those fed casein. Cholesterol concentrations in gerbils fed either ISP (2.2 ± 0.1 mmol/L) or ISP with soy extract (2.1 ± 0.1 mmol/L) were lower (P < 0.01) than those in gerbils fed casein (3.4 ± 0.2 mmol/L).
Identification of soy-regulated mRNA.
We produced >5000 radiolabeled PCR products using electrophoresis. Greater than 99.5% of the PCR products appeared identical in amount in the three dietary groups. Sixteen PCR products that appeared to be displayed at different levels of abundance were successfully subcloned and sequenced. From these clones, six corresponded to previously known mRNA, five are homologous to known cDNA, and four have sequences for which no known homologies were identified. The antithrombin III mRNA sequence was obtained from two independently selected PCR products; thus, the 16 cloned cDNA yielded only 15 different sequences. Three of the cDNA identified encode the serum proteins apolipoprotein B, transferrin, and antithrombin III. Other mRNA that had an identity or homology identified were phosphoribosylpyrophosphate synthetase-associated protein (PAP), cytochrome P450 II A2, mitochondrial NADH-dehydrogenase, proteasomal ATPase, ribosomal protein S10, phosphoenolpyruvate carboxykinase, melancortin-4 receptor, and an adenylate cyclase-related protein.
Each cDNA was then used as a probe, evaluating expression of the mRNA corresponding to the cloned cDNA. Because our major goal of this project was to identify abundant mRNA species that were robustly regulated, we established an arbitrary qualification of considering only mRNA whose expression was at least doubled. Also, for this initial project, we did not investigate further any mRNA that were not clearly detected by Northern blot analysis. Two mRNA confirmed to be differentially regulated by the dietary treatments to the greatest degree were a cytochrome P450 enzyme (CYP) and phosphoribosylpyrophosphate synthetase-associated protein (PAP) (14
). Although the gerbil genome is poorly characterized, the CYP mRNA identified is most highly homologous to the 3-methylcholantherene-inducible CYP450 of the hamster (15
) and in humans, to members of the phenobarbital-inducible CYP2A subfamily (16
). Phosphoribosylpyrophosphate synthetase-associated protein has been described as a repressive regulator of the phosphoribosyl pyrophosphate synthetase enzyme complex (14
). Using the cDNA for CYP and PAP, we performed Northern blot analysis on the individual gerbil RNA samples that were used previously to prepare pooled RNA used in the genetic screen. When gerbils consumed either ISP or ISP+ diet, the abundance of both the CYP and PAP mRNAs was 300% of the abundance when gerbils were fed casein (P < 0.05; Fig. 1
).
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Expression of both the CYP and PAP mRNA reflected the amount of ISP in the diet (Fig. 2
). Expression of CYP and PAP was significantly higher (P < 0.05) when gerbils were fed the 100% ISP diet (P < 0.05) compared with the 0% ISP diet, and expression levels when gerbils were fed 33% or 67% ISP were intermediate compared with 0% or 100% ISP. Because the ISP diets used in study 2 contained a low concentration of isoflavones and other phytochemicals, we conducted study 3. Cholesterol concentrations (data not shown) exhibited a step-wise pattern, with the lowest concentrations in the 100% ISP-fed gerbils, and highest in the 100% CAS-fed gerbils.
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Dietary treatments affected both cholesterol concentrations (Fig. 3
) and hepatic PAP and CYP mRNA levels (Fig. 4
). Cholesterol concentrations were lower (P < 0.05) in gerbils fed any ISP diet (intact or simulated, with or without extract supplementation) compared with those fed any CAS-based diet (intact or simulated, with or without extract supplementation). There was an interaction (P < 0.05) between the form of protein (intact vs. simulated) and the presence or absence of extract supplementation. For example, cholesterol concentrations were 3.9 ± 0.2 mmol/L in gerbils fed unsupplemented casein or ISP, compared with 3.2 ± 0.2 mmol/L in gerbils fed casein or ISP supplemented with extract (P < 0.05, n = 8). However, cholesterol concentrations were not affected by soy extract supplementation when purified amino acid mixtures were fed to gerbils. Gerbils fed casein- or soy-resembling amino acid mixtures without extract supplementation had a cholesterol concentration of 3.2 ± 0.2 mmol/L and a concentration of 3.3 ± 0.2 mmol/L when the mixtures were provided with soy extract (n = 8). Supplementation of the purified amino acids with soy extract increased CYP (P < 0.01) and PAP (P < 0.05) mRNA expression compared with mRNA levels when extract was not provided. Regarding mRNA expression, there were no interactions among amino acid profile, form of protein, and extract.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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5000 PCR products we screened do not represent the entire complement of mRNA estimated to exist in hepatocytes, we consider this study a partial step toward cataloging all mRNA induced by soy or soy isoflavones. Microarray technology rather than the differential display protocol may help complete this task.
One of these mRNA species identified encodes a member of the cytochrome P450 IIA subfamily (CYP) and the second encodes phosphoribosylpyrophosphate synthetase-associated protein (PAP). Both were significantly induced in the liver when as little as 3% of the normal concentration of isoflavones was present in the diet (Fig. 5
). Addition of soy extract to purified amino acid mixtures, resembling either soy or casein, also increased CYP and PAP mRNA content (bars 5–8 in Fig. 4
). We hypothesize that even in the low-isoflavone containing soy protein, the low levels of soy isoflavones present are sufficient to increase CYP and PAP mRNA levels. This was observed in study 1 (Fig. 1)
and for CYP in study 3 (Fig. 4
, panel B, bars 1 vs. 2) but not for PAP mRNA. After evaluating PAP mRNA levels in study 3, we determined that there was one RNA sample in a CAS-fed rat in which an unusually high PAP mRNA level was measured. This unusually high value was reflected by the larger SEM observed for the CAS-fed gerbils in study 3 (Fig. 4
, panel A, bar 1). We chose not to arbitrarily remove this one unusual value of PAP mRNA expression. Supplementation with extract increased both PAP and CYP mRNA (Fig. 5)
. The PAP and CYP mRNA are similarly regulated, perhaps because the promoter regions of these two genes each contain aromatic hydrocarbon response element sequences (17
). The function of this regulatory sequence within the promoter region of cytochrome P450 genes is a part of the regulatory mechanism responding to xenobiotic challenge involved in toxin metabolism. However, the PAP protein is less well understood, and its function during xenobiotic challenge is not understood. If the PAP protein is indeed a negative regulator of PRPP-synthetase activity, xenobiotics may up-regulate PAP levels to help down-regulate PRPP synthesis, reducing nucleotide synthesis and DNA replication. This might slow DNA replication during a time when xenobiotics are a mutagenic threat to the cell. The mRNA for several CYP species are induced by consumption of soy (7
,8
). In rats fed soy or casein diets, CYP3A1, 3A9, and 3A18 were induced modestly by soy protein intake, while 3A2 was up-regulated threefold to fourfold. Cytochrome P450-2B1 was not induced by soy intake (7
). More recently, it was found that CYP1A1 and 1A2 are induced in soy-fed rats when isosafrole was included in the diet (8
). Isosafrole is a natural product found in some food oils and flavorings. We contend that the gerbil mRNA identified in this report corresponds to the CYP2A subfamily and may not correspond exactly to the CYP mRNA described previously (7
,8
).
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) plays a role in soy’s antiatherogenic effect. Very recently, evidence indicated that soy may act via LDL receptor- and plasma lipoprotein-independent pathways to inhibit atherosclerosis (19
), a scenario consistent with an action through a blood protein such as antithrombin. Another interesting candidate to consider is apolipoprotein B, the major protein characteristic of hepatic VLDL particles. Our screen initially indicated the ApoB cDNA was expressed least in gerbils fed ISP+ diet. This observation is consistent with reduced serum cholesterol concentrations, because the liver may be synthesizing less ApoB and secretion less VLDL when serum cholesterol concentrations are reduced. However, interpretation of this observation brings to mind a critical consideration that must be recognized when evaluating the results of feeding studies that may result in changed cholesterol levels. If blood cholesterol levels are reduced by soy intake, or any experimental treatment, a number of changes will be observed that are directly due to the reduction in cholesterol and not necessarily because soy intake produced those changes directly. An example would be any sterol-regulated gene, such as the LDL receptor. Although many investigators have measured LDL-receptor levels in soy intake studies, the conclusion that soy regulates LDL-receptor expression may in fact not be a direct effect of soy, but rather, a secondary effect caused by the reduction in cholesterol levels resulting from soy intake. Short-term studies will be required to determine whether gene expression changes are caused directly by soy intake, or by a longer-term reduction in serum cholesterol levels. In our own laboratory, we have shown that cholesterol levels are not significantly reduced by soy intake in gerbils until sometime between 7 and 14 d (data not shown). Along with the LDL-receptor, another important enzyme critical to cholesterol metabolism is cholesterol 7- hydroxylase, which commits hepatic cholesterol to the bile salt synthetic pathway. Although we expect 7- hydroxylase to be regulated coordinated with intracellular sterol concentrations, to our knowledge, no recent studies have measured 7- hydroxylase mRNA levels as a function of soy isoflavone content in the diet.
The results of this study are not the first to demonstrate the effect of soy or soy isoflavones on gene expression. As mentioned previously, we and others have measured levels of apolipoprotein and cytochrome P450 mRNA in rodent liver (3
,7
,8
). Other investigations have shown that soy isoflavone supplements regulate estrogen-dependent gene expression in the rat brain (20
,21
). In a long-term human study, LDL-receptor mRNA levels were found to be reduced in monocytes of soy-fed postmenopausal women (22
).
Although the primary focus of our study was to examine gene expression, it may be that some new information was determined regarding the cholesterol-lowering effect of soy. In studies 1 and 2, little additional effect on cholesterol lowering was observed when low-isoflavone soy was supplemented with isoflavone-containing soy extract. However, a concern regarding purified amino acid diets prompted us to supplement study 3 diets with choline chloride. Not only were the purified amino acid diets supplemented with choline, but also the ISP- and CAS-containing diets. Thus, in study 3, when choline chloride was added to the diet at 3 g/kg, there was a partial lowering of cholesterol levels when low-isoflavone soy was fed, but then also a further reduction of cholesterol levels when soy was further supplemented with the isoflavone-containing supplement. This may help explain some of the inconsistency observed in a variety of different isoflavone supplementation studies. When choline is present at adequate or above adequate levels, there may be a cholesterol-lowering effect of soy isoflavones that is not observed when choline is available at a lower level. It is possible that our amino acid mixtures were hypocholesterolemic by themselves, and a further reduction in cholesterol by isoflavone supplementation was masked by this effect (e.g., bars 1 and 2 vs. bars 5 and 6; and bars 5 and 6 vs. bars 7 and 8, Fig. 3
). This effect may warrant further investigation. Certainly, there are a wide range of results obtained from the many different diets and experimental protocols used to examine cholesterol lowering. The identification of a bioactive component within soy is a complex task and any many factors to consider when examining food components for specific cholesterol-lowering activity. The extraction or separation method must be carefully considered, because there is a possibility that isolation or extraction of a particular fraction may chemically alter or oxidize certain bioactive compounds, reducing their effect when studied as an extract. Another possibility is that there may be two or more bioactive components within soy that affect cholesterol metabolism and a synergy is lost when factors are studied independently. In any event, we suggest these possibilities must be considered when confronted with a negative or less robust response when studying soy isolates or fractions for cholesterol-lowering activity. These concerns may lead to the interpretation advocated in a recent review (6
). It was concluded that isoflavones might play a role in the cholesterol lowering effect: "studies with ethanol-extracted soy (devoid of isoflavones) indicated a loss of effect, but the extract itself (isoflavone rich) has no hypocholesterolemic activity."
In summary, we have successfully identified a number of mRNA that are putatively regulated by soy intake. Two mRNA, CYP and PAP, were up-regulated by the presence of soy extract in the diet (P < 0.05). Although some have indicated that the cholesterol-lowering effect resides mainly within the protein fraction of soy and not in the isoflavone fraction, we suggest that all of the biological effects of soy isoflavones are still only partially understood. Because the xenobiotic response seems to be a potent mechanism inducing some mRNA species, we speculate that the cholesterol-lowering effects of soy will be ultimately caused by other cellular mechanisms in addition to the estrogenic or anti-estrogenic effects of isoflavones.
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FOOTNOTES |
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2 Supported in part by funding provided by University of Illinois at Urbana-Champaign Value-Added Program, University of Notre Dame and Protein Technologies International, St Louis, MO.
4 Abbreviations used: ANOVA, analysis of variance; CAS, casein; CYP, cytochrome-P450; ISP, isolated alcohol-washed reduced-isoflavone soy protein; PAP, phosphoribosylpyrophosphate synthetase-associated protein; PCR, polymerase chain reaction; PPRP, phosphoribosylpyrophosphate.
Manuscript received 24 January 2002. Initial review completed 12 February 2002. Revision accepted 16 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Anderson, J. W., Johnstone, B. M. & Cook-Mewell, M. E. (1995) Meta-analysis of the effects of soy protein intake on serum lipids. N. Engl. J. Med. 333:276-282.
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21. Pan, Y., Anthony, M. & Clarkson, T. B. (1999) Effect of estradiol and soy phytoestrogens on choline acetyltransferase and nerve growth factor mRNAs in the frontal cortex and hippocampus of female rats. Proc. Soc. Exp. Biol. Med. 221:118-125.[Medline]
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