Daidzein supplementation prevents non-alcoholic fatty liver disease through alternation of hepatic gene expression profiles and adipocyte metabolism

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Abstract

Introduction:

Globally, non-alcoholic fatty liver disease (NAFLD) continues to rise and isoflavones exert antisteatotic effects by the regulation of hepatic lipogenesis/insulin resistance or adiposity/a variety of adipocytokines are related to hepatic steatosis. However, there is very little information regarding the potential effects of daidzein, the secondary abundant isoflavone, on NAFLD. Here, we have assessed the hepatic global transcription profiles, adipocytokines and adiposity in mice with high fat-induced NAFLD and their alteration by daidzein supplementation.

Methods:

C57BL/6J mice were fed with normal fat (16% fat of total energy), high fat (HF; 36% fat of total energy) and HF supplemented with daidzein (0.1, 0.5, 1 and 2 g per kg diet) for 12 weeks.

Results:

Daidzein supplementation (0.5 g per kg diet) reduced hepatic lipid concentrations and alleviated hepatic steatosis. The hepatic microarray showed that daidzein supplementation (1 g per kg diet) downregulated carbohydrate responsive element binding protein, a determinant of de novo lipogenesis, its upstream gene liver X receptor β and its target genes encoding for lipogenic enzymes, thereby preventing hepatic steatosis and insulin resistance. These results were confirmed by lower insulin and blood glucose levels as well as homeostasis model assessment insulin resistance scores. In addition, daidzein supplementation inhibited adiposity by the upregulation of genes involved in fatty acid β-oxidation and the antiadipogeneis, and moreover augmented antisteatohepatitic leptin and adiponectin mRNA levels, whereas it reduced the mRNA or concentration of steatotic tumor necrosis factor α and ghrelin.

Conclusions:

These findings show that daidzein might alleviate NAFLD through the direct regulation of hepatic de novo lipogenesis and insulin signaling, and the indirect control of adiposity and adipocytokines by the alteration of adipocyte metabolism.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is defined as an excess of fat in the liver, in which at least 5% of hepatocytes evidence lipid droplets in patients who do not consume significant quantities of alcohol.1 In most industrialized countries, the prevalence of NAFLD continues to rise along with obesity,2 because it is developed as the result of conditions associated with obesity, including hepatic fat accumulation, oxidative stress, insulin resistance and adiposity.3, 4

NAFLD is frequently conceptualized as a four-step process, including simple steatosis, steatohepatitis accompanied with inflammation and fibrosis, cirrhosis and hepatocellular carcinoma. Thus, the most effective precaution against NAFLD might be to interrupt initial steps, simple steatosis, and its inflammatory form, steatohepatitis.1

The principle characteristic of hepatic steatosis is the excessive accumulation of triglycerides (TGs), which are available from lipids newly synthesized in the liver or a surplus supply of free fatty acid (FFA) released from adipose tissue. Steatohepatitis is aggravated by adipose tissue inflammation4 and the imbalance of adipocytokines (that is, leptin, adiponectin and tumor necrosis factor (TNF) α).5

Recently, a transcription factor carbohydrate responsive element binding protein (ChREBP) has emerged as a central determinant of hepatic lipogenesis through its transcriptional control of stearoyl CoA desaturase-1, acetyl CoA carboxylase and fatty acid synthase,6 and hepatic insulin resistance by the inhibition of thymoma viral proto-oncogene (Akt).7 On the other hand, the obesity-induced infiltration of macrophages into adipose tissue stimulates TNFα production, thereby impairing insulin signaling, stimulating lipolysis, and eventually increasing the FFA released from adipose tissue.8, 9 FFA is then taken up by the liver, accelerating statosis, and activating the nuclear factor (NF)κB, an inducer of steatohepatitis.10

In recent studies, the phytoestrogen genistein has been shown to attenuate NAFLD by the activation of hepatic fatty acid β-oxidation or the inhibition of adiposity through estrogen receptors -dependent or independent pathway.11, 12, 13

However, there is very little information regarding the antisteatotic effects of daidzein, the analogue of genistein, by the regulation of hepatic de novo lipogenesis, adiposity and adipocytokines, although its metabolite, equol, has higher binding affinity to estrogen receptors14 and anti-oxidative activity15 than genistein.

In the current study, we have attempted to determine whether daidzein might alleviate NAFLD by the alteration of hepatic and adipose tissue metabolism, or adipocytokines and hormone alterations using global gene comparison, histological analysis and hormone assay.

Materials and methods

Experimental animals and diets

Mice studies were conducted with the approval and the guidelines of the Institutional Animal Care and Use Committee of the Seoul National University, which comply with national and international laws and policies (National Institutes of Health Guide for Care and Use of Laboratory Animals, 1996 (7th edition)). The 3-week-old C57BL/6J male mice (Seoul National University Animal Laboratories, Seoul, Korea), a good obesity model, were individually housed in animal facilities maintained at 22±2 °C with a 12-h light/dark cycle. The animals (n=18 per group) were assigned to six dietary groups; normal fat diet (NF), high-fat diet (HF) and the HF diet supplemented with daidzein (0.1, 0.5, 1 and 2 g per kg diet), and fed with an ad libitum experimental diet and water. The diets were on a modification of AIN 93-G.11, 16 The HF diet contained high levels of fat (36% fat of total energy), as compared with the fat (16% fat of total energy) in the NF diet. Daidzein (BioSpectrum Ltd., Seoul, Korea) was substituted for equivalent cornstarch in the HF diet (Supplementary Table 1).

Sample collection and biochemical analysis

After an overnight fasting, blood was collected through the orbital venous plexus following anesthetization with ketamine hydrochloride (60 mg per kg body weight), and then mice (n=18 per group) were immediately killed. The livers and three positions (visceral, perirenal and epididymal fat) of adipose tissues were rapidly dissected and frozen in liquid N2 and stored at −80 °C until used for RNA or lipid analysis. Specially, visceral fat were collected between small intestine and colon to be distinguished from perirenal fat. The other portion of livers and adipose tissues was stored in 10% formalin for histological analysis. Feces were weighed on 4 consecutive days for the analysis of lipid excretion. Serum was prepared by the centrifugation of blood at 956 × g for 20 min at 4 °C and stored at −80 °C until use. Serum total lipids were measured by the sulfo-phospho-vanillin reaction17 at the wavelength of 540 nm. Total cholesterol (TC), TG, alanine aminotransferase (ALT) and aspirate aminotransferase (AST) were assayed using the enzymatic and colormetric kit; TC (BioVision, Mountain View, NC, USA), TG (Sigma, St Louis, MO, USA), ALT (Sigma) and AST (Sigma). AST and ALT were used for determining hepatic dysfunction. Liver and fecal total lipids were extracted with a mixture of chloroform/methanol (2:1),18 and then TG and TC levels were determined with the extract of total lipids using enzymatic and colorimetric kits described above.

Histological analysis of liver and adipose tissue

Liver and epididymal fat were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sliced into 3 μm sections. The sections were stained with hematoxylin for negatively charged nucleic acid, counterstained with eosin for protein staining, then coverslipped with DPX (BDH, Poole, UK). The images were captured on a confocal microscope (Nikon, Eclipse TE 200, Tokyo, Japan). Adipocyte numbers were calculated by counting the nuclei in a fixed field area (0.9 mm2) at × 100 magnification. Adipocyte size was calculated by the following formula;19

where A, defined area; n, number of cell counted; m, final magnification of photograph. In all samples, at least five different fields of four different tissue sections were assessed.

Total RNA extraction and real-time PCR

Total RNA of liver and epididymal fat were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. For the relative quantification of gene expression, real-time PCR was conducted using a Biosystem 7500 real-time PCR system (ABI, Carlsbad, CA, USA). In all, 500 ng of complementary DNA was amplified with a 25 μl SYBR Green PCR master mix (ABI) and specific primer pairs. The primers are listed in the Supplementary Table 2. The relative expression levels of target gene were calculated as 2−ΔΔCt, where ΔΔCt=ΔCthigh fat or daidzein diet group−ΔCtnormal fat diet group and ΔCt=ΔCttarget gene−ΔCtβ−actin.

Microarray analysis

Gene expression profiles were assessed using an AB 1700 mouse chip, which contains 60-mer oligonucleotide (ABI) probes representing more than 34 000 mouse genes and harbor a set of 1000 controls for tracking system performance throughout the experiment. Each gene was derived from the Celera Genomics database and the Mouse Genome Sequencing consortium. Total RNA underwent complementary DNA synthesis and then dioxigenin-labeled cRNA synthesis using a chemiluminescent RT-IVT labeling kit v2.0 (ABI). The dioxigenin-labeled cRNA was hybridized to array that bound to antidioxigenin antibody, conducted the chemiluminescent (CL) reaction using a chemiluminescence detection kit (ABI), and then was detected using a chemiluminescent Microarray Analyzer (ABI). The scanned image was analyzed with AB 1700 chemiluminescent microarray analyzer software v 1.1.1 (ABI). In the present study, the target sequence signal, exclusively to the terms of S/N ratio 3 and Flag value 5000, was analyzed.20 We designated it as differentially expressed genes whose mRNA levels were changed 1.5-fold and considered to be significantly altered by Local-Pooled-Error test (P>0.05).21 Biological processes were analyzed by Protein Analysis Through Evolutionary Relationships (PANTHER, http://www.pantherdb.org).

Metabolic hormones assay

Serum insulin, leptin, adiponectin and ghrelin levels were assessed using immunoassay kits; insulin (Mercodia, Uppsala, Sweden), leptin (R&D Systems, Minneapolis, MN, USA), adiponectin (R&D Systems) and ghrelin (Phoenix Pharmaceuticals, Burlingame, CA, USA). Blood glucose levels were determined with an automatic dry chemistry analyzer (Daiichi Kagaku, Seoul, Korea). Insulin resistance was assessed through homeostasis model assessment:22 Insulin resistance=(insulin (μU ml−1) × blood glucose (mmol l−1))/22.5.

High performance liquid chromatography/mass spectrometry analysis

To quantify and identify free daidzein and equol in serum, the glucose conjugated forms of isoflavones were hydrolyzed with 1 ml of 0.2 M acetate buffer (pH 5.0) containing 50 μl of glucuronidase/sulfatase from Helix pomatia (Sigma) at 37 °C for 16 h. The hydrolyzed daidzein and equol were mixed with 6 ml of methanol for 1 h and subsequently centrifuged at 3000 × g for 20 min at 4 °C. The sample supernatants were dried using a vacuum centrifuge at 4 °C and then redissolved in 400 μl of methanol.23 The quantification analysis was conducted on an Hewlett Packard 1090 Series high performance liquid chromatography equipped with a diode-array detector, using a μBondapark C18 column (a 3.9 × 300 mm, 10 μm; Waters Co., Milford, MA, USA) connected with a RT-18 guard column (15 × 3 mm, 7 μm; Rainin Instrument Co., Oakland, CA, USA). The mobile phase was consisted of (A) water and (B) acetonitrile with gradient elution of 20% B at 0–5 min, 29% B at 5–10 min and 50% B at 10–20 min. The flow rate was 1.0 ml min−1, and the detection wavelength was set at 230 nm for equol and 254 nm for daidzein under ultraviolet spectra (190–600 nm). The identification analysis was conducted with triple quadrupole tandem mass spectrophometry (API 4000, ABI) capable of both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). All analyses were conducted in negative ion mode and unit mass resolution. Ionization interfaces temperature (ESI: 550 °C; APCI: 500 °C), needle voltage (ESI: 3500V; APCI: 3 V). Daidzein and equol were identified in terms of the masstocharge ratio (m/z).

Statistical analysis

Data were expressed as the means±s.e.m. Biochemical data were analyzed through one-way analysis of variance with Duncan’s test, and real-time reverse transcriptase-PCR data were analyzed through analysis of variance with Dunnett's test and t-test using SAS Proprietary Software Release 8.2 (SAS Institute, Inc., Cary, NC, USA). Microarray data were analyzed by Local-Pooled-Error tests suitable for a small replication21 using Avadis Prophetic version.3.3 (Strand Genomics, Bangalore, India) and R (version 2.5) program. P values of <0.05 were considered to be statistically significant.

Results

Body weight, fat mass and lipid profiles

As shown in Figures 1a–c, C57BL/6J mice fed on the HF diet for 12 week gained body weight (6%, P<0.05), increased fat mass (25100%, P<0.05), elevated serum TG (32%, P<0.05) and TC (15%, P<0.05), and accumulated liver total lipids (43%, P<0.05), TG (23%, P<0.05) and TC (146%, P<0.05) as compared with mice fed on the NF diet. However, daidzein supplementation (0.5 g per kg diet) reduced body weight and fat mass in a dose-dependent manner, and lowered serum and hepatic lipids, as compared with those fed on the HF diet (P<0.05). In addition, the mice fed with the daidzein supplementation (1 g kg−1) excreted more total lipids (39%, P<0.05) and TG (47%, P<0.05) in feces than those fed on the HF diet (Figure 1c).

Figure 1
figure1

Effect of daidzein on body weight, fat mass and lipid profiles. Body weight and food intake (a), fat mass (b), and lipid profiles (c) of the C57BL/6J mice fed with normal fat diet (NF), high-fat diet (HF) and HF supplemented with daidzein (0.1, 0.5, 1 and 2 g per kg diet). All data are indicated as means±s.e.m. (n=6 per diet group). a,b,cMeans without a common letter differ, P<0.05 (Duncan’s test).

Histological analysis of liver and adipose tissue

As shown in Figure 2a, liver histology showed that the HF diet increased hepatocellular vacuolation as the result of lipid accumulation, expanded hepatocytes, shifted nuclei towards the plasma membrane and narrowed the sinusoidal channels. However, these features were alleviated by daidzein supplementation in a dose-dependent manner. The markers of liver injury or dysfunction, ALT (42%, P<0.05) and AST(46%, P<0.05), were elevated in the serum of mice fed on the HF diet as compared with that of mice fed on the NF diet, and this elevation was restored by daidzein supplementation (0.5 g per kg diet) (P<0.05) (Figure 2c).

Figure 2
figure2

Histological analysis of liver and adipose tissue. The microscopic picture (a) of liver (original magnification, × 200; Scale bar, 50 μm) and adipose tissue (original magnification; × 100; Scale bar; 100 μm) of C57BL/6J mice fed with the normal fat diet (NF), high-fat diet (HF) and HF supplemented with daidzein (0.1, 0.5, 1 and 2 g per kg diet) (n=4–5 per diet group); (b) adipocyte numbers and sizes on the basis of histological examination; (c) the concentrations of asparate aminotransferase (AST) and alanine aminotransferase (ALT) (n=6 per diet group). All data are indicated as means±s.e.m. a,b,cMeans without a common letter differ, P<0.05 (Duncan’s test).

The stereological examination of adipose tissue revealed that the hyperplasia of adipocytes was a primary contributor to the increase in fat mass induced by the HF diet, but prevented by daidzein supplementation in a dose-dependent manner (P<0.05) (Figures 2a and b).

Hepatic global gene expression profiles altered by HF diet or daidzein supplementation

A total of 692 genes and 230 genes were expressed differentially in the livers of mice fed on the HF diet or daidzein supplementation (1 g per kg diet) as compared with the livers of mice fed on the NF diet in chip analysis. Among the differentially expressed genes, we selected the genes associated with NAFLD, such as ChREBP signaling, insulin signaling, oxidative stress and fatty acid β-oxidation.

As shown in Table 1, the expressions of ChREBP, a central stimulator of lipogenesis, and its target genes encoding for lipogenic enzymes, such as ACACβ, fatty acid synthase, adenosine triphosphate citrate lyase and 1-acylglycerol-3-phosphate O-acyltransferase-2, were downregulated by 1.5- to 3.4-fold as the result of daidzein supplementation. Moreover, the expression of liver X receptor (LXR) β, a ChREBP activator,24 was reduced by fourfold level as the result of daidzein supplemenation, whereas the expression of protein kinase A (PKA), a ChREBP inhibitor,25 was augmented by 1.7-fold as the result of daidzein supplementation.

Table 1 Hepatic genes expression profiles altered by the HF Diet or daidzein supplementation

The suppression of ChREBP signaling can result in improvements in insulin signaling through Akt activation,26 or the inhibition of hepatic fat accumulation. Indeed, daidzein supplementation improved insulin signaling related intermediates, such as phosphatidylinositol 3-kinase, Akt2 and glucose transporter-2. Insulin signaling can also be controlled by oxidative stress or inflammation. As shown in Table 1, daidzein supplementation normalized the suppression of the HF diet on superoxide dismutase-2 and glutathione S-transferaseα3, which detoxifies xenobiotics or removes reactive oxygen species,27 and the augmentation of the HF diet on c-Jun N-terminal kinase-1, TNFα and inhibitor of NFκB kinase-ɛ, which induce insulin resistance through hepatic inflammation.

PPARα, a central activator of fatty acid β-oxidation, was not altered by the HF diet or daidzein supplementation. However, its target genes, ACAD10 and long chain acyl CoA synthetase (ACSL)4, were reduced in mice fed on the HF diet, and reversed again as the result of daidzein supplementation. PPARγ, known to improve insulin sensitivity in the liver, also increased by 1.5-fold the levels of expression in mice fed on the daidzein supplementation diet, but not the HF diet.

These findings suggest that soy daidzein might alleviate NAFLD directly through the transcriptional control of hepatic lipogenesis, insulin signaling, oxidative stress, inflammation and fatty acid β-oxidation.

Confirmation of microarray data

Changes in gene expression were confirmed by a small set of genes using real-time reverse transcriptase-PCR. The genes were selected from Table 1. When we compared the gene expression profiles obtained from microarray analysis and real-time reverse transcriptase-PCR results, their gene expression patterns were quite similar in terms of the direction (up- or downregulation) and the degree of expressional changes (Table 2).

Table 2 Confirmation of the expression of genes selected among hepatic gene expression profiles using real-time RT-PCR

Changes of insulin, blood glucose and adipocytokines associated with NAFLD

Fasting insulin and blood glucose concentrations were elevated in mice fed on the HF diet as compared with mice fed on the NF diet, but this elevation was reversed again by daidzein supplementation in a dose-dependent manner (P<0.05) (Figure 3a). These values resulted in lower homeostasis model assessment insulin resistance scores in daidzein supplementation (0.5 g per kg diet) than those in the HF diet (P<0.05) (Figure 3a).

Figure 3
figure3

Insulin resistance and metabolic hormone. Blood glucose, insulin and homeostasis model assessment (HOMA) insulin resistance index (a), leptin, adiponectin and ghrelin (b) were determined in serum of C57BL/6J mice fed with normal fat diet (NF), high-fat diet (HF) and HF supplemented with daidzein (0.1, 0.5, 1 and 2 g per kg diet). All data are indicated as means±s.e.m. (n=6 per diet group). a,b,c,d,eMeans without a common letter differ, P<0.05 (Duncan’s test).

As shown in Figure 3b, antisteatotic and antifibrogenic adiponectin levels in serum were not altered by the HF diet, and were rather reduced as the result of daidzein supplementation (0.5 g per kg diet) (P<0.05). This result may be attributable to the reduction of fat mass, considering the fact that adiponectin is one of the hormones secreted from adipose tissue. However, despite the reduction of fat mass, antisteatotic leptin concentrations in serum were elevated in mice fed on the daidzien supplementation diet, as well as the HF diet (P<0.05).

Thus, we determined the mRNA level of leptin and adiponectin for excluding the influence of differential fat mass. As might be expected, adiponectin and leptin had higher levels of expression in mice fed on the daidzein supplementation diet (1 g per kg diet), as compared with those fed on the HF diet (Figure 4a). The increase in adiponectin resulted in the downregulation of TNFα expression, which involves hepatic insulin resistance and inflammation (Figure 4a).28 Serum ghrelin levels, adipogenic and orexigenic hormone, were not altered in mice fed on the HF diet, but declined in mice fed on the daidzein supplementation diet (0.5 g per kg diet) (P<0.05).

Figure 4
figure4

Alteration of gene involved in adipocytokines, fatty acid β-oxidation and adipogenesis. mRNA was extracted from adipose tissue of the C57BL/6J mice fed with normal fat diet (NF), high-fat diet (HF) HF supplemented with daidzein (1 g per kg diet). mRNA levels of adiponectin, leptin and TNFα (a), mRNA levels of genes involved in fatty acid β-oxidation; AMPK, ACAS and ACAD (b), (c), and mRNA levels of genes involved in adipogenesis; PPARγ and β-catenin (c). Data were expressed as Means±s.e.m., (n=3–5 per diet group). mRNA level was determined with real-time RT-PCR, and normalized to β-actin mRNA expression, where the values for NF diet were set 1.00. *Different from NF diet group, P<0.05 (Dunett’s multiple range test) and Different between HF diet group and daidzein supplementation group, P<0.05 (t-test).

These findings show that daidzein might attenuate NAFLD by improving insulin resistance and altering adipocytokines toward antisteatohepatitis.

Adipocyte metabolism altered by HF diet or daidzein supplementation

The daidzein-limited adipocyte hyperplasia may result from the alteration of a variety of genes involved in adipogenesis or fatty acid β-oxidation. In Figure 4b, the genes encoding for enzymes that catalyze fatty acid β-oxidation, including ACAS and ACAD, were augmented by 4.5- to 6.8-fold higher levels of expression in mice fed on the daidzein supplementation diet (1 g per kg diet) as compared with mice fed on the NF diet (P<0.05). This activation of fatty acid β-oxidation was associated with the upregulation of expression of their upstream genes, including adiponectin and 5′-adenosine monophosphate-activated protein kinase (AMPK) (P<0.05). Furthermore, β-catenin, an antiadipogenic transcription factor, was augmented by daidzein supplementation (1 g per kg diet) (P<0.05) (Figure 4c), even if β-catenin stability or nuclear translocation, which predicts to be enhanced by the increase in AMPK, was not measured.

Collectively, daidzein appears to inhibit the hyperplasia of adipocytes, a primary risk factor of NAFLD, by stimulating fatty acid β-oxidation and suppressing adipogenesis.

Serum daidzein and its metabolite, equol, concentrations

As shown in Table 3, liquid chromatography-ultraviolet/mass spectrometry demonstrated that serum daidzein levels in mice fed with daidzein 0.1, 0.5, 1 and 2 g per kg diets for 12 week were 0.13, 0.7, 2.2 and 3.9 μmol l−1, and its metabolite equol levels were 0.04, 0.3, 0.7 and 1.3 μmol l−1. These levels are within the range encountered in a variety of human population groups consuming a diet containing soy products;29, 30, 31 human who consume single meals based on soybean flour evidence serum daidzein levels of 3.14 μmol l−1. Similarly, human infants fed on soy-based infant formula have plasma daidzein levels of 1.16 μmol l−1 and plasma equol levels of 1.23 μmol l−1.

Table 3 Serum daidzein and its metabolite equol concentration

Discussion

In the current study, the consumption of a high-fat diet increased body weight, the amount of fat stored, and lipids in the serum and liver (Figure 1) (P<0.05). These unfortunate features led to NAFLD along with a significant elevation of the liver injury markers, ALT and AST (Figure 2), as was also the case in obese fa/fa rats fed on a high-fat diet.32 However, these unfortunate features were reversed as the result of daidzein supplementation.

First, daidzein supplementation reduced the hepatic de novo lipid synthesis by regulation of SREBP-1c, LXR and ChREPB. The transcription factor SREBP-1c has been previously identified as a major intermediator of insulin action on lipogenic genes, including acetyl CoA carboxylase and fatty acid synthase.33 However, SREBP-1c activity alone is not sufficient to account for the stimulation of lipogenesis, as SRBEP-1c gene deletion in mice results in only a 50% reduction in fatty acid synthesis.34 Indeed, despite the upregulation of SREBP-1c by daidzein supplementation in our hepatic transcript profile (Table 1), hepatic lipid profiles were lowered in mice fed on daidzein supplementation as compared with those fed on the HF diet (Figure 1c).

Thus, daidzein is likely to inhibit hepatic lipid synthesis through the regulation of another lipogenic stimulator. Recently, the transcription factor ChREBP has emerged as a central and novel determinant of lipid synthesis in the liver through its transcriptional control of the lipogenic genes35 because of its binding to the carbohydrate response element in the promoter of lipogenic genes. In the ChREBP−/− mice, liver TG content is decreased compared with wild-type mice. Furthermore, complete inhibition of ChREBP in ob/ob mice reduces the effects of the metabolic syndrome, such as obesity, fatty liver and glucose intolerance.35 Indeed, in our hepatic transcript profiles (Table 1), ChREBP and its target genes, including ACACβ, 1-acylglycerol-3-phosphate O-acyltransferase-2, adenosine triphosphate citrate lyase and fatty acid synthase, were also downregulated as the result of daidzein supplementation.

The transactivation of ChREBP can be modulated by LXRs, SREBP-1c and serine/threonine kinase, including PKA and AMPK. LXRs have been reported to induce the expression of ChREBP through its target gene SREBP-1c or binding to the ChREBP promoter, to generate the fatty acids necessary for the formation of cholesterol esters.24, 36 In contrast, PKA and AMPK have been shown to prevent ChREBP from being transported into the nucleus or binding to the carbohydrate response element present in the promoter regions of lipogenic enzyme genes through the phosphorylation of ChREBP.25, 37 Recently, cyclic adenosine monophosphate, an activator of PKA, has also been reported to block recruitment of hepatic nuclear factor-4α and CREB binding protein to L-type pyruvate kinase promoter, a major target gene of ChREBP.38 Interestingly, daidzein supplementation reduced the expression of LXRβ, which might result from an increase in PKA expression (Table 1).

This suppression of the ChREBP may improve hepatic insulin resistance, a major contributor of NAFLD progress, through less hepatic fat accumulation or Akt activation. Recent study showed that ChREBP knock-down mice restored insulin sensitivity through the activation of Akt.26 Our hepatic transcript profiles (Table 1) also demonstrated that daidzein supplementation upregulated the intermediate genes involved in insulin signaling, including phosphatidylinositol 3-kinase, Akt and glucose transporter-2.39 This improvement of hepatic insulin signaling might block the hepatic uptake of FFA released from adipose tissue for using energy fuel. In addition, daidzein supplementation lowered serum insulin and blood glucose levels as well as a lower homeostasis model assessment insulin resistance index score (Figure 3a), which seems to be as a result of the increase in glucose uptake by the improvement of hepatic insulin signaling.

The improvement of hepatic insulin signaling can also be induced by the removal of hepatic reactive oxygen species and proinflammatory cytokines. The depression of hepatic defense systems (glutathione S-transferase-α3 and superoxide dismutase 2) and subsequently the activation of the NFκB pathways induce hepatic insulin resistance as a result of the phosphorylation of serine residues of the insulin receptor substrate.40, 41 Interestingly, daidzein supplementation normalized the depression of glutathione S-transferase-α3 and superoxide dismutase 2 by the HF diet, and further downregulated the expression of c-Jun N-terminal kinase and IκB kinase that is augmented by the HF diet (Table 1), which might be associated with the higher antioxidative capacity of daidzein metabolite, equol, rather than other isoflavones.15

Besides these results, our hepatic transcript profiles showed that daidzein supplementation reduced hepatic lipid levels through the activation of ACAS and ACAD, which are involved in fatty acid β-oxidation,42, 43, 44, 45, 46, 47 and this activation was due to the upregulation of PPARα, their upstream gene.48

Daidzein supplementation also functions as hepatic antisteatotic materials by the regulation of adipocyte metabolism. Recently, adiponectin has been shown to exert protective effects against NAFLD in obese ob/ob mice,49 hepatic injury or fibrogenesis in mice to whom lipopolysaccharide or CCl4 were administered.28, 50 The central mechanism is the improvement of hepatic insulin signaling and the activation of fatty acid β-oxidation,51 and the inhibition of inflammatory NFκB and TNFα signaling.52 Leptin has also been shown to attenuate liver TG secretion and fatty acid synthesis, although increasing hepatic fatty acid β-oxidation.53, 54 Leptin deficiency suppressed both the innate and acquired immune response, thereby favoring T-helper cell lymphocyte polarization and making the liver steatosis.55

In the present study, daidzein supplementation augmented leptin and adiponectin mRNA levels, and reduced the levels of TNFα mRNA in adipose tissue. The activation of adiponectin resulted in the upregulation of its downstream genes, PPARα and AMPK (Figure 4),51 which encode for many of the enzymes involved in fatty acid β-oxidation, such as ACAS, ACAD and carnitine palmitoyltransferase I.42, 45, 46 In our current study, ACAS and ACAD levels were increased by dadizein supplementation (Figure 4b). Daidzein supplementation also increased the expression of β-catenin, an antiadipogenic transcription factor56 (Figure 4c). In human and murine preadipocytes, a downregulation of canonical Wnt-signaling was a prerequisite to initiate adipogenesis; the overexpression of Wnt 10b in mouse preadipocytes inhibited the expression of CCAT/enhancer binding protein (C/EBP)α, a major adipogenic transcription factor, and subsequently kept preadipocyte in an undifferentiated state in vitro and in vivo.56 In addition, AMPK, which was increased by daidzein supplementation, has been demonstrated to phosphorylate β-catenin at Ser 552 and consequently enhance β-catenin stability and nuclear translocation.57

The activation of these genes might lead to inhibit the hyperplasia of adipocyte (Figure 2b), thereby lowering the concentration of ghrelin correlated positively to fat mass (Figure 3b). Ghrelin has been reported to induce hyperglycemia and inhibits the release of insulin from β-cells in mice.58, 59

The hypolipidemic action of daidzein might be partially because of reduction of lipid absorption. Recent data showed that the fermented soygerm isoflavones (daidzein, genistein and glycitein) significantly inhibited pancreatic lipase activity in dose-dependent manner, thereby suppressing absorption of excessive lipid into a body.60

These finding were in consistent with our data that is, increase in fecal TG excretion by daidzein supplementation, even if we did not measure the pancreas lipase activity. The inhibition of pancreas lipase activity can reduce the conversion of TG to monoglyceride or FFA, a final lipid form for absorption, which subsequently can inhibit the lipid absorption.

Thus, we thought that this daidzein-induced lipid malabsorption led to decrease the energy intake, thereby being able to prevent NAFLD.

Collectively, daidzein supplementation appears to alleviate NAFLD in obese mice fed on a high-fat diet, which results from the alteration of hepatic and adipocyte metabolism; (1) the reduction of hepatic de novo lipogenesis, (2) the improvement of hepatic insulin signaling, (3) the diminution of fat mass by the activation of catabolism and antiadipogenesis and (4) the upregulation of antisteatotic or antisteatohepatitic adipocytokines. Our findings can possibly be interpreted as relevant for humans, as our study-used daidzein and equol concentrations that are within the range encountered in various populations that consume foods rich in isoflavones, and C57BL/6J mice have phenotype similar to that of human disease; the induction of obesity and insulin resistance under ad libitum access to a high-fat diet, and the maintenance of normal conditions under restriction to a low-fat chow diet, as in humans.61, 62, 63 In addition, it will be the epoch-making discovery that daidzein affects ChREBP signaling, adiponectin and ghrelin as their relationships have yet to be assessed.

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Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST, No. 2010-0020265) and by the Research Fund from the Research Institute of Human Ecology of Seoul National University.

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Correspondence to K-S Kang or Y-S Lee.

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Supplementary Information accompanies the paper on International Journal of Obesity website

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Keywords

  • non-alcoholic fatty liver disease
  • daidzein
  • hepatic de novo lipogenesis
  • insulin signaling
  • adipocyte metabolism

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