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Nutrient-Gene Interactions

Hepatic Gene Expression Profiles Are Altered by Genistein Supplementation in Mice with Diet-Induced Obesity¹

Sujong Kim², Insuk Sohn^*, Yeon Sook Lee and Yong Sung Lee

Department of Biochemistry, College of Medicine, Hanyang University, Seoul 133–791, Korea; ^* Department of Statistics, Graduate School, Korea University, Seoul 136–701, Korea; and Department of Food and Nutrition, Seoul National University, Seoul 151–742, Korea

²To whom correspondence should be addressed. E-mail: sundance{at}amorepacific.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
We reported previously that genistein enhances the expression of genes involved in fatty acid catabolism through activation of peroxisome proliferator-activated receptor (PPAR) in HepG2 cells, suggesting that genistein holds great promise for therapeutic applications to lipid abnormalities such as obesity and hyperlipidemia in humans. In this study, we examined the changes in hepatic transcriptional profiles using cDNA microarrays in mice with high-fat diet (HFD)-induced obesity supplemented with genistein. C57BL/6J male mice (n = 10/group) were fed a low-fat diet (LFD), a HFD, or a HFD supplemented with 2 g/kg genistein (HFD+GEN) for 12 wk. Mice fed the HFD had abnormal lipid profiles and significantly greater body weight and visceral fat accumulation than the LFD-fed group. Genistein supplementation improved lipid profiles and hepatic steatosis and attenuated the increases in body weight and visceral fat in HFD-fed mice. The cDNA microarrays revealed marked alterations in the expression of 107 genes in the mice fed the HFD and/or the HFD+GEN. Of 97 transcripts altered in the HFD-fed group, 84 genes were normalized by genistein supplementation. However, several genes involved in fatty acid catabolism were not normalized but were still upregulated in the HFD+GEN-fed group, relative to the LFD-fed group. Furthermore, carnitine O-octanoyltransferase, which accelerates fatty acid oxidation, was not affected by the HFD, but was induced by genistein supplementation. These results are consistent with our previous study showing that genistein is an activator of PPAR in vitro. This study showed beneficial effects of genistein supplementation in preventing the development of obesity and metabolic abnormalities in mice with diet-induced obesity. Our results also provide interesting information about the genes associated with the beneficial effects of genistein as well as the mechanisms underlying the development and maintenance of the obesity phenotype in vivo.

KEY WORDS: • cDNA microarray • genistein • hepatic steatosis • high-fat diet • obesity

A high-fat diet (HFD)³ has been shown to adversely affect the health of humans and experimental animals (1,2), causing hyperinsulinemia and hyperglycemia; if continued for a longer period, it can lead to the development of obesity and diabetes (2). It has also been reported that a HFD increases fat-mediated oxidative stress, which can cause an increase in the incidence of cancer in experimental animals fed a HFD (3,4).

For many years, isoflavones attracted wide attention because of their potential beneficial effects in preventing menopausal symptoms, osteoporosis, cardiovascular diseases, and cancers (5). More recently, evidence has emerged that dietary isoflavones may play a beneficial role in lipid and carbohydrate metabolism (6,7). It was shown that higher daily isoflavone intake was associated with lower BMI in postmenopausal women (6), and isoflavone-containing soy protein diets significantly improved plasma lipid concentrations and insulin sensitivity in nonhuman primates (7).

Genistein, the principal soy isoflavone, has been investigated extensively for its hypolipidemic, antilipogenic, antioxidant, and antitumor activities as well as estrogenic activity in diverse biological systems (5,8). The majority of the pleiotropic effects of genistein are mediated by changes in the expression of the genes involved; however, definitive molecular mechanisms have not yet been clearly defined. Several genes that encode enzymes or signal mediators involved in metabolism and tumorigenesis were shown to respond to genistein (8). For example, we reported previously that genistein is an activator of PPAR , and subsequently induces expression of PPAR -target genes involved in fatty acid ß-oxidation, such as carnitine palmitoyltransferase1-liver (Cpt1l), in HepG2 human hepatoma cells (9). Although these investigations revealed the molecular mechanisms of genistein, the results in most cases have been obtained in a "gene by gene" manner in diverse systems. Because a global analysis of gene expression in response to changes in physiologic status appears to be essential for understanding the molecular mechanisms underlying the alterations, we investigated the hepatic transcriptional profiles of HFD-fed obese mice and their alteration by genistein supplementation using cDNA microarrays.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Mice and diets. C57BL/6J male mice (3 wk old) were obtained from the SNU Animal laboratories (Seoul, Korea) and housed individually in stainless steel wire-mesh cages in a room kept at 23 ± 1°C with a 12-h light:dark cycle (light period: 0800–2000h). After familiarization with the facility for 1 wk, the body weights were measured (20.21 ± 0.2 g) and then 30 mice were randomly assigned to 1 of 3 dietary groups⁴ (n = 10) for 12 wk; a low-fat diet (LFD), a HFD, and the HFD supplemented with genistein (2 g/kg diet) (HFD+GEN). The diets were based on a modification of the recommendations of the AIN (10). The HFD contained 18% (wt/wt) fat (36% of total energy), compared with 7% (wt/wt) fat (16% of total energy) in the LFD. For the HFD+GEN diet, commercially available genistein (BioSpectrum) was substituted for equivalent cornstarch in the HFD at 2 g/kg diet. The dosage utilized in this study was selected on the basis of our preliminary quantification of plasma genistein using LC-MS detection. This dosage produced a plasma genistein level of 4.1 µmol/L, similar to that in humans consuming a diet containing soy products (11–17); it is a level that was shown to have biological activities in vivo and in vitro (6,7,9). Mice had free access to food and distilled water, which were provided fresh every day. Food intake was recorded every other day, and body weight was measured weekly throughout the study. This study was conducted in conformity with the policies and procedures of the Institutional Animal Care and Use Committee of the Seoul National University.

    Sample collection and analytical methods. On completion of the experiment, all mice were weighed, and blood was collected into EDTA-treated blood collection tubes from mice anesthetized with ketamine hydrochloride (60 mg/kg body weight) after overnight food deprivation. Plasma was prepared by centrifugation of blood at 1000 x g for 15 min at 4°C and stored at –80°C until analysis. Immediately after mice were killed by exsanguination, livers were perfused in situ with ice-cold saline. Livers and adipose tissues were then collected and weighed. Three portions (1 g each) from each liver were frozen immediately in liquid N₂ and stored at –80°C until used for preparation of RNA and lipid. Plasma total cholesterol (TC), glutamic-oxaloacetic transaminase (GOT), and -glutamic-pyruvic transaminase (-GPT) were measured with an automatic dry chemistry analyzer, Spotchem (Daiichi Kagaku). Plasma FFA and triglyceride (TG) levels were measured by enzymatic and colorimetric methods, using an FFA assay kit (Roche Diagnostics) and a Sigma Triglyceride (GPO-Trinder) kit, respectively. Plasma glucose was determined with a glucose assay kit (Sigma). For liver lipid analysis, total lipids were extracted with a mixture of chloroform:methanol (18), and liver TG and TC were measured enzymatically as described above after the total lipids were dissolved in Triton X-100.

    Microarray analysis. The mouse 10K cDNA microarray used in this study consisted of 10,336 spots, as previously described (19,20). It included 6531 transcripts from the National Institute of Aging, 1243 transcripts from Brain Molecular Anatomy Project, 2060 transcripts from InCyte Pharmaceuticals (Fremont), and yeast DNA and housekeeping genes as negative controls.

Total RNA was prepared from livers using Trizol (Invitrogen). Fluorescence-labeled cDNA probes were prepared from 20 µg of total RNA using an amino-allyl cDNA labeling kit (Ambion). Equal amounts of the RNA from 6 mice of each group were mixed, and each sample was equally divided; one half was used to generate Cy3-labeled cDNA, the other half was used to generate Cy5-labeled cDNA for dye swapping. The Cy5 and Cy3 probes were mixed, and hybridization was performed at 55°C for 16 h, as previously described (19,20).

The 2 fluorescent images (Cy3 and Cy5) were scanned separately by a GMS 418 Array Scanner (Affymetrix), and the image data were analyzed using ImaGene 4.2 (Biodiscovery) and MAAS (Gaiagene) software (21). For each hybridization, the emission signal data were normalized by multiplying the Cy3 signal values by the ratio of the means of the Cy3 and Cy5 signal intensities for all spots on the array. To eliminate the unreliable data, the following criteria were adopted: 1) PCR amplification of the sequence spotted on the array was deemed acceptable only if the amplification was confirmed and a single size product was obtained; 2) accurate printing of each spot was required, as shown by the emission signal from >40% of the spot area; 3) the signal from the fluorophore labels had to be higher than 256 (2⁸).

    Real-time RT-PCR. Total RNA (4 µg) was reverse-transcribed in 25 µL of reaction mixture, containing MuLV reverse transcriptase (2.5 U), RNAse inhibitor (1 U), 5 mmol/L MgCl₂, 50 mmol/L KCl, 10 mmol/L Tris-HCl, (pH 8.3), 2.5 µmol/L oligo (dT) primer, and 1 mmol/L dNTPs. The reaction mixture was heated to 42°C for 60 min and then denatured at 85°C for 5 min. cDNA was amplified with ICycler (BioRad) in 50 µL of reaction mixture containing AmpliTaq DNA polymerase (1 U, Perkin Elmer), 50 mmol/L Tris (pH 8.3), 0.25 g/L bovine serum albumin, 3 mmol/L MgCl₂, 0.25 mmol/L dNTPs, 1/50,000 dilution of SYBR green I (Molecular Probes), and 0.25 µmol/L appropriate forward and reverse PCR primers (Supplemental Table 1).¹ The following cycling conditions were used: one denaturing cycle at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 60°C for 45 s, and 72°C for 1 min. Relative RNA levels were determined by analyzing the changes in SYBR green I fluorescence during PCR according to the manufacturer’s instructions. ß-Actin was amplified in parallel and the results were used for normalization. The correct size of the PCR products was confirmed by electrophoresis on a 2% agarose gel stained with ethidium bromide. Purity of the PCR products was determined by melting point analysis, using the ICycler software.

    Statistical analysis. Mice data were expressed as means ± SEM. Data were analyzed by 1-way ANOVA with Tukey’s post-hoc test using the SPSS version 10.0 statistical package for Windows. Differences were considered significant at P < 0.05.

In the microarray analysis, we calculated the median gene expression ratios from 6 independently repeated microarray experiments. We used the significant analysis of microarray (SAM) method for multiclass response data to test whether differences in gene expression were significant (22).

The data were calculated as follows. M_ij = log₂ (R_ij/G_ij), i = 1, 2, ..., p genes, j = 1, 2, ..., n samples. Let C_k = indices of observations in class k, n_k = slide numbers in C_k, _ik = _jCkM_ij/n_k, _j = _j M_ij/n. For a gene i, a statistic is d_i = r_i/(s_i + s_o); i = 1, 2, ..., p. Where r_i = [{ n_k/n_{k k=1}^K n_k (_ik – _i)²]^1/2, s_o is fudge factor, and s_i = [1/ (n_k – 1) ( 1/n_k) _k=1^K _jCk (M_ij – _ik)²]^1/2. We took the genome-wide significance level at the SAM () = 0.66, and adopted a cutoff of 2.0-fold change based on our experience. Genes showing significant differences in expression were classified into different functional categories, based on Gene Ontology with modifications (23,24).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Body and tissue weights and plasma and liver biochemistry. Food consumption did not differ among the groups (Table 1). Feeding C57/6J mice the HFD for 12 wk increased final body weight (23%, P < 0.05), and relative weights of epididymal (60%, P < 0.05) and perirenal adipose tissues (39%, P < 0.05), compared with the LFD-fed mice (Table 1). However, the HFD+GEN-fed group gained 55% less body weight than the HFD-fed group (P < 0.05). The final adipose tissue relative weights were also lower in the HFD+GEN-fed group than in the HFD-fed group (65 and 46% of HFD group, P < 0.05; Table 1). Liver TG and TC were increased by 1.4-fold and 3.7-fold in the HFD-fed group, respectively, compared with the LFD-fed group (P < 0.05) (Table 1). The accumulation of liver TG due to the HFD was lower in the HFD+GEN-fed group (20%, P < 0.05); however, liver TC was not affected by genistein (Table 1). Plasma TC was markedly increased in the HFD-fed mice (48%, P < 0.05), whereas plasma TG was decreased (25%, P < 0.05); FFA and glucose levels did not differ between the HFD- and LFD-fed groups (Table 1). The genistein treatment attenuated the increase in plasma TC due to the HFD (33%, P < 0.05) and reduced FFA and glucose levels relative to both other groups (Table 1). Plasma GOT and GPT activities did not differ among the 3 groups (data not shown).

    Genes that were affected by the HFD and normalized by genistein. Of the 97 gene transcripts altered in the HFD-fed mice, 80 were completely normalized, 1 was partially normalized, and 3 were oppositely regulated by genistein treatment (Table 2). These included genes encoding lipogenic enzymes such as acetyl-CoA synthetase 2, glycerol-3-phosphate acyltransferase, and malic enzyme, which were 88, 60, and 62% lower in mice fed the HFD than in those fed the LFD, respectively, and were normalized in mice fed the HFD+GEN. The genes encoding enzymes of cholesterol biosynthesis, such as squalene epoxidase (Sqle), NAD(P)-dependent steroid dehydrogenase-like, farnesyl diphosphate farnesyl transferase 1 (Fdft1), and sterol-C4-methyl oxidase-like, were also decreased by at least 50% in the HFD-fed mice, and normalized by genistein supplementation. In contrast, genes related to fatty acid ß-oxidation and ketogenesis such as acyl-CoA oxidase 1 (Acox1) and HMG-CoA lyase were augmented in the HFD-fed mice compared with those fed the LFD. These upregulated genes in the HFD-fed mice were also normalized by genistein. Expression of a class of genes associated with carbohydrate metabolism, such as sorbitol dehydrogenase 1, fructose bisphosphatase 2 (Fbp2), and aldolase1 A isoform (Aldo1), showed a similar pattern.

The genes that play roles in signal transduction/apoptosis/cell cycle and transcription regulation also showed expression patterns similar to those described above (Table 2): growth arrest specific 8 (Gas8), DNA primase, p58 subunit (Prim2), small protein effect 1 of Cdc42, Wint6, Proliferin related protein (Plfr), serine/threonine kinase 6, 11 and 36, insulin-induced gene1 (Insig1), nuclear factor, erythroid derived 2 like 2 (Nrf 2), Fos, myelocytomatosis oncogene (c-Myc), and Max dimerization protein 5. The genes encoding transporters (lactotransferrin, intestinal calcium binding protein), cell-adhesion protein (catenin src), cytoskeleton organizers (tubulin 2 and ß5, -aminobutyric acid receptor-associated protein-like 1, destrin), and trafficking-regulators (kinesin family member 23, lectin, mannose-binding 2) were also upregulated in HFD-fed mice and normalized by genistein. CD36/FAT, which is responsible for the transport of long-chain fatty acids into muscle and adipose tissue, was also upregulated by the HFD, as shown in several obese animal models, and normalized by genistein (25).

    Genes that were affected by the HFD and not normalized by genistein. Several genes were increased in the HFD-fed mice, but were not normalized by genistein supplementation (Table 3). Acetyl-CoA acyltransferase 1 (Acaa1), involved in peroxisomal fatty acid ß-oxidation, is an example. Hydroxysteroid (17-ß) dehydrogenase 4 (Hsd17b4), which is a multifunctional protein to catalyze the oxidation of estradiol with high preference as well as peroxisomal fatty acid ß-oxidation (26), also increased 2.5- and 1.5-fold in the HFD-fed and HFD+GEN-fed groups, respectively. Other genes showing a similar expression pattern included 2 genes of the cytochrome P₄₅₀ families (CYP3a11, CYP4a10), aldehyde dehydrogenase family 3, subfamily A2, AMP deaminase 3, glutamate-cysteine ligase, catalytic subunit, and heat shock response genes (Hspca, Hspcb, Hspa8).

    Genes that were not affected by the HFD but were regulated by genistein. Genistein supplementation increased the transcription levels of 3 additional genes that were not changed in the HFD group [carnitine O-octanoyltransferase (Crot), eukaryotic translation initiation factor 3 subunit 4 , and ATP-binding cassette subfamily C member 3; Table 4]. Crot, which is implicated in peroxisomal fatty acid ß-oxidation, had an expression pattern that differed from that of other genes involved in lipid catabolism. Five additional genes [7-dehydrocholesterol reductase (Dhcr7), enolase 3 ß, insulin-like growth factor binding protein 1, endoglin, and calpain 6] and 2 expressed sequence tags (ESTs) that were not altered in the HFD-fed mice, were lower in the HFD+GEN group than in the LFD-fed mice. Downregulation of Dhcr7, the ultimate enzyme of cholesterol de novo synthesis, could explain in part the hypocholesterolemic effect of genistein.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

As expected, the largest numbers of genes expressed differentially in the HFD- and/or HFD+GEN-fed groups were those involved in metabolism (Tables 2, 3, 4). Because cholesterol homeostasis is maintained by a feedback mechanism involving the cholesterol itself as an end-product repressor (27), the downregulation of genes involved in cholesterol synthesis was in good accord with the hypercholesterolemia present in the HFD-fed mice. The downregulation of lipogenic genes and the upregulation of genes implicated in the uptake and catabolism of fatty acid might be associated with the physiologic status of the HFD-fed mice such as fatty liver and increased fat accumulation (Table 1). The gene expression profiles of mice in this study led us to hypothesize that in HFD-fed mice, the uptake of fatty acids into liver was augmented by increased CD36/FAT, resulting in subsequent accumulation of TG in the liver, and that hepatic TG accumulation might have driven the upregulation of genes involved in lipid catabolism and the downregulation of lipogenic genes by a feedback mechanism. Of the 29 metabolism-related genes altered by the HFD, the expressions of 21 genes were normalized or reversed by genistein supplementation. These alterations might be closely related to the improvement in weight-related and biochemical variables by genistein. On the other hand, CYP4a10, which is involved in peroxisomal lipid -oxidation, and Acaa1 and Hsd17b4, which are implicated in peroxisomal fatty acid ß-oxidation, were not normalized by genistein in HFD-fed mice. Furthermore, Crot, which accelerates fatty acid oxidation by transferring chain-shortened fatty acyl groups from the peroxisomes to the mitochondria, was not changed by the HFD, but was induced by genistein supplementation (28). These results suggest that the hypolipidemic effect of genistein could be ascribed in part to the upregulation of genes involved in fatty acid catabolism in liver. It is important to identify possible mechanism(s) by which genistein simultaneously regulated several genes of fatty acid metabolism. One possible mechanism is via transcription factors such as PPARs. PPARs were reported earlier to regulate the expression of genes involved in the balance among uptake, oxidation, and synthesis of fatty acids (29). Recently, genistein was shown to act as an activator of PPAR and PPAR and to elevate PPAR-directed gene expression in vitro and in vivo (9,30,31). Therefore, any change in mRNA levels of PPARs is of particular interest. Because PPAR transcripts were rejected according to technical criteria, we examined mRNA levels of PPARs by real-time RT-PCR. The expression of PPARs increased >1.0-fold in HFD-fed mice, and their increases were augmented by genistein supplementation (Table 5). Cpts, which are PPAR-responsive genes, also showed similar expression patterns. The observed changes in the expression of genes involved in fatty acid catabolism, including Cpts, might be ascribed in part to the increased expression of PPARs. These results agree with our previously reported study, which showed that genistein enhances the expression of genes involved in fatty acid catabolism through activation of PPAR in HepG2 human hepatoma cells (9).

Another possible mechanism by which genistein exerts antilipogenic effects in mice with diet-induced obesity is via estrogen receptors (ERs) in adipose tissue. In fact, genistein is a phytoestrogen that mimics estradiol in the activation of ER in vitro (32,33). It was also shown to be estrogenic in vivo and cause uterine growth, both in intact and ovariectomized animals (34). Similarly, Naaz et al. (35) suggested that the antilipogenic effects of genistein on adipose tissue are mediated through ER, based on the results of experiments in ER knockout mice given injections of genistein. Because our microarray approach was performed on liver tissue of mice given dietary genistein, however, the molecular events in adipose tissue cannot be determined. The gene expression profiles in adipose tissue of our mice and the biochemical parameters and hepatic transcription of ER knockout mice fed genistein should be examined in the future.

Genes involved in APR or inflammatory processes had increased expression in HFD-fed mice and their increased expression was modified by genistein treatment (Table 2). Evidence has suggested that there is an association between a chronic, low-grade inflammation state characterized by elevated acute-phase reactants and the progression of obesity-associated deteriorations such as atherosclerosis and metabolic syndrome X (36). Therefore, the downregulation of APR genes by genistein could be a possible explanation for the previously reported beneficial effects of genistein such as its antiatherosclerotic and antidiabetic effects (5). One of the most interesting results was that several hepatic genes related to detoxification or defense responses were expressed an average 3-fold higher in the HFD-fed group, and their increased expressions were generally normalized or reversed by genistein treatment (Table 2). In particular, expression of metallothionein 1 and Gst genes was markedly increased in the HFD-fed group. Ishii et al. (37) showed that the expression of metallothionein and Gst is regulated by a common "antioxidant response element (ARE)," and suggested that their gene products play a protective role against oxidative damage in various tissues by neutralizing reactive oxygen species. In the present study, Nrf 2, a transcription factor that was shown to play an important role in ARE-mediated expression of metallothionein and Gst (37,38), was also increased 2-fold in the HFD-fed group and was normalized in the HFD+GEN-fed group. Ferritin heavy chain, another Nrf2-responsive gene, was also slightly increased in HFD-fed mice [70%, SAM () = 1.18], and normalized by genistein (not presented, because fold change <2.0) (39). Aldehyde dehydrogenase isotypes, classified into the metabolism category in this report, were shown previously to play pivotal roles in the detoxification of lipid peroxidation products such as 4-hydroxynonenal (40,41). Therefore, the >1-fold increase in the expression of these gene family members in HFD-fed mice can be explained as a defense response against a lipotoxic environment, such as fat accumulation, in liver tissue. A simultaneous increase in the expression of a number of antioxidative stress genes together with their major transcription regulator in HFD-fed mice suggests that global transcriptional regulation of biological defense responses to oxidative stress elevated by a long-term HFD may exist. The HFD-induced transcriptional alterations of genes in detoxification and defense responses were normalized or reversed by genistein supplementation. However, the question whether these normalizations induced by genistein were mediated by its hypolipidemic activity or antioxidant activity remains unanswered. Related to "defense and stress responses" is the elevated expression of Hsp in the HFD- and HFD+GEN-fed mice: the molecular chaperones, which are postulated to be responsible for repairs of misfolded or damaged proteins in a highly oxidative and lipotoxic environment, were not normalized by genistein supplementation (Table 3).

The changes in mRNAs for genes likely to influence the overall cell cycle, growth, apoptosis, and signal transduction did not follow a consistent trend. Some positive regulators of the cell cycle (Gas8, Prim2, Wnt6, and serine/threonine kinase 6), several negative regulators of cell cycle, and putative proapoptotic signal transducers (Plfr, and serine/threonine kinase 11), were highly expressed in HFD-fed mice and normalized by genistein, with almost identical expression patterns (Table 2). On the other hand, mitogenic epidermal growth factor receptor was decreased in HFD-fed mice and normalized in those fed HFD+GEN. One possible explanation for these seemingly conflicting changes in the expression of genes related to growth, the cell cycle, and apoptosis, is that different cells or nuclei within the liver responded differently (e.g., nuclei in hepatocytes and endothelial cells). One of the most interesting findings in this category was the decrease in the expression of Insig1 in HFD-fed mice and its normalization by genistein supplementation. Insig1 is a critical component of the sterol-sensing system that regulates processing of sterol regulatory element binding proteins (SREBPs), the major transcription factors regulating the expression of lipogenic and cholesterogenic genes (42). Therefore, the changes in the expression of genes involved in lipid and cholesterol metabolism described above (Table 2) might be ascribed in part to the different processing of the SREBPs due to changes in expression of Insig1 because gene expression of SREBPs was not significantly altered in the HFD and HFD+GEN groups.

In the interpretation of experiments with animals, whether these results may have relevance for humans is one of the most important questions. Our preliminary analysis using LC-MS showed that the peak plasma genistein level in mice fed genistein at 2 g/kg diet for 3 wk was 4.1 ± 0.2 µmol/L (mean ± SEM, n = 5), which is well within the range encountered in various population groups consuming foods rich in isoflavones (11–17). For example, humans consuming 3 meals containing soymilk on a daily basis have serum genistein levels up to 4.6 µmol/L (13). Similarly, consumption of 1 soy-based meal resulted in peak serum genistein concentrations of 2.4 µmol/L (14). Finally, human infants fed soy-based infant formula have plasma genistein levels ranging from 1.5 to 4.4 µmol/L (16). Therefore, there are numerous situations in which natural soy product consumption produces plasma genistein levels in humans equaling or exceeding those shown to have an antiobesity effect in mice.

Genistein has multiple biological effects; it can act as an estrogen at low concentrations (<1 µmol/L) and as a PPAR ligand at high concentrations (1 µmol/L) (30). Because dietary isoflavones are subjected to extensive first-pass clearance by liver, hepatocytes may be exposed to a genistein concentration higher than the plasma genistein level (>4.1 µmol/L). Furthermore, ER activation by estradiol was shown to limit lipid oxidation via suppression of Cpt1l gene expression and to promote triglyceride synthesis in experimental animals (43,44), which is contrary to our real-time PCR and biochemical data. Recent microarray analysis also showed that ER activation-induced hepatic gene expression changes in mice shared a very small portion with our microarray data, although it might be improper to compare 2 studies because of differences in age, sex, and diet between them (45). Other data from experiments using DNA microarray analysis for examining the effects of genistein in the developing rat uterus showed that genistein alters the expression of 6–8 times as many genes as does a physiological estrogen such as 17 ß-estradiol, suggesting that genistein might regulate ER-independent signal pathway(s) (46). Altogether, the effects of genistein on hepatic transcriptional profiles and lipid profiles might be through PPARs rather than ERs. Although it is difficult to explain how the effective use of a nuclear receptor pathway by genistein is established under different physiologic conditions, tissue distribution and the presence of downstream effectors of nuclear receptors may determine a predominant pathway in the cells/tissues.

In summary, the microarray data indicated that the transcriptional regulation networks associated with long-term HFD intake and genistein supplementation are very complex. These wide ranges of effects were expected because multiple cellular processes are regulated by nutritional factors as well as hormones. The present unbiased approach toward characterization of gene expression suggests that a number of previously unappreciated regulatory molecules play roles in the development and maintenance of the obesity phenotype in vivo and depicts the diverse transcriptional regulations through which genistein exerts its beneficial effects in mice with diet-induced obesity.

	ACKNOWLEDGMENTS

 
We thank Professor Woon Ki Paik for critical review of this manuscript.

	FOOTNOTES

 
¹ Primers used for real-time RT-PCR are available with the online version of this paper at www.nutrition.org.

³ Abbreviations used: Acaa1, acetyl-CoA acyltransferase 1; Acox1, acyl-CoA oxidase 1; Aldo1, aldolase1 A isoform; APR, acute-phase response; ARE, antioxidant response element; Cpt, carnitine palmitoyltransferase; Crot, carnitine O-octanoyltransferase; CYP, cytochrome P₄₅₀; Dhcr7, 7-dehydrocholesterol reductase; ER, estrogen receptor; EST, expressed sequence tag; Fbp2, fructose bisphosphatase 2; Fdft1, farnesyl diphosphate farnesyl transferase 1; Gas8, growth arrest specific 8; GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase; Gst, glutathione S-transferases; HFD, high-fat diet; HFD+GEN, high-fat diet supplemented with genistein (2 g genistein/kg diet); Hsd17b4, hydroxysteroid (17-ß) dehydrogenase 4; Hsp, heat shock proteins; Insig1, insulin-induced gene1; LFD, low-fat diet; Nrf 2, nuclear factor, erythroid derived 2 like 2; Plfr, proliferin related protein; PPAR, peroxisome proliferator-activated receptor; Prim2, DNA primase, p58 subunit; SAM, significant analysis of microarray; Sqle, squalene epoxidase; SREBP, sterol regulatory element binding protein; TC, total cholesterol; TG, triglyceride.

⁴ Containing (g/kg diet) LFD: cornstarch, 529.4; sucrose, 100; soybean oil, 70; HFD: cornstarch, 419.5; sucrose, 100; soybean oil, 180; HFD+GEN: cornstarch, 417.5; sucrose, 100; soybean oil, 180; genistein, 2 (10).

Manuscript received 23 June 2004. Initial review completed 26 July 2004. Revision accepted 8 October 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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