Preventive Medicine 54 (2012) S57–S63

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Review

Isoflavones as a smart curer for non-alcoholic fatty liver disease and pathologicaladiposity via ChREBP and Wnt signaling

Mi-Hyun Kim, Kyung-Sun Kang ⁎Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, 599 Gwanakno, Sillim-dong, Gwanak-gu, Seoul 151-742, Republic of KoreaLaboratory of Stem Cell and Tumor Biology, Department of Veterinary Public Health, College of Veterinary Medicine, Seoul National University, 599 Gwanakno, Sillim-dong, Gwanak-gu,Seoul 151-742, 1001 Republic of Korea

⁎ Corresponding author at: Adult Stem Cell Research Cand Tumor Biology, Department of Veterinary PublicMedicine, Seoul National University, 599 Gwanangno, Gpublic of Korea. Fax: +82 2 876 7610.

E-mail address: kangpub@snu.ac.kr (K.-S. Kang).

0091-7435/$ – see front matter © 2012 Elsevier Inc. Alldoi:10.1016/j.ypmed.2011.12.018

a b s t r a c t

a r t i c l e i n f o

Available online 28 December 2011

Keywords:IsoflavonesNAFLDAdiposityChREBP signalingWnt signaling

Objective. Non-alcoholic fatty liver disease (NAFLD) and pathological adiposity has emerged as an impor-tant modern disease. Along with this, the requirement for alternative and natural medicine for preventingNAFLD and adiposity has been increasing rapidly and considerably. In this report, we will review the biolog-ical effect and mechanisms of soy isoflavones on NAFLD and pathologic adiposity mainly through the novelpathways, de novo lipogenic carbohydrate responsive element binding protein (ChREBP) and anti-adipogenic Wnt signaling.

Methods. This paper reviews in vitro and in vivo isoflavone studies published in 2002 to 2011 in North

America and East Asia.

Results. Collectively, the data support a beneficial relation of isoflavones and NAFLD and/or adiposity. Iso-flavones suppress ChREBP signaling via protein kinase A (PKA) and/or 5′-AMP activated protein kinase(AMPK)-dependent phosphorylation, which prevents ChREBP from binding to the promoter regions of lipo-genic enzyme. Furthermore, isoflavones directly stimulate Wnt signaling via estrogen receptors-dependentpathway, which inactivates glycogen synthase kinase-3 beta (GSK-3β), transactivate T-cell factor/lymphoid-enhancer factor (TCF/LEF), the effector of Wnt signaling, degrade adipogenic peroxisome proliferator-activatedreceptor γ (PPARγ), augment p300/CBP, the transcriptional co-activators of TCF/LEF.

Conclusions. Natural compound isoflavones may be useful alternative medicines in preventing NAFLD andpathological adiposity and this action may be partially associated with ChREBP and Wnt signaling.

© 2012 Elsevier Inc. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S57NAFLD and pathological adiposity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S58Phytoestrogen isoflavones as a curer for NAFLD and adiposity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S59ChREBP and Wnt signaling in NAFLD and adiposity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S59

De novo lipogenesis via ChREBP signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S59Anti-adipogenic Wnt signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S60

The anti-steatotic and anti-adipogenic mechanism of isoflavone via ChREBP and Wnt signaling . . . . . . . . . . . . . . . . . . . . . . . . . S60Isoflavones and ChREBP signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S60Isoflavones and Wnt signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S61

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S62Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S62Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S62References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S62

enter, Laboratory of Stem CellHealth, College of Veterinarywanak-gu Seoul 151-742, Re-

rights reserved.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a metabolic conditionwhich encompasses a wide spectrum of liver disease ranging from

Table 1Effects of genistein and daidzein on parameters of NAFLD and adiposity.

Author(s), date Subjects Diet and treatment Effects Geographic regions

Demonty et al. (2002) Sprague–Dawley rats Casein-based diet supplemented withgenistein and daidzein (18.2 mg/g diet)for 21 days

Reduce blood and liver triglycerides Canada

Naaz et al. (2003) Ovariectomized mice Feeding genistein (0–1500 ppm) for21 days

Decrease adipose tissue weight throughthe reduction of lipoprotein lipase (LPL)gene

USA

Ascencio et al. (2004) Adult male rats Feeding soy protein (10 g/100 g diet) Inhibit the development of fatty liver viaregulation of sterol regulatory elementbinding protein (SREBP)-1 expression bymodulating serum insulin levels

Mexico

Lee et al. (2006) C57BL/6J mice (model of high fatdiet-induced obesity)

Genistein (1, 2, 4 g/kg body weight)-richdiet

Alleviate NAFLD by stimulating hepaticfatty acid β-oxidation and increasing anti-oxidative enzyme

Korea

Zheng et al. (2009) Rats (model of high fat diet-induced obesity)

Feeding puerarin for 4 weeks Reduce NAFLD via hepatic leptin signalingactivation (leptin receptor/JAK2/STAT3)

China

Mohamed Salih et al. (2009) Wister rat (model of insulinresistance)

Administration of genistein (1 mg/kgbody weight)

Reduce NAFLD via activation of theantioxidant profiles and decrease ininterleukine (IL)-6 and tumor necrosisfactor (TNF) α

India

Kim et al. (2010b) C57BL/6J mice (model of high fatdiet-induced obesity)

Genistein (1, 2, 4 g/kg body weight)-richdiet

Reduce NAFLD by regulating adipocytefatty acid β-oxidation and adipogenesis

Korea

Kim et al. (2011) C57BL/6J mice (model of high fatdiet-induced obesity)

Daidzein (0.1, 0.5, 1, 2 g/kg bodyweight)-rich diet

Prevent NAFLD through de novo hepaticlipogenesis and alternation of adipokines

Korea

Harmon et al. (2002) 3T3-L1 cell 100 μM of genistein and daidzein Block adipogenesis through the inhibitionof adipogenic transcription factor CCAT/enhancer-binding protein (C/EBP) βactivity

USA

Dang et al. (2003) Mouse bone marrow cell 0.01–50 μM of genistein Reduce adipogenesis through activation ofestrogen receptors (ERs) at lowconcentration (≤1 μM but augmentadipogenesis through peroxisomeproliferator-activated receptors (PPAR) γat high concentration (>1 μM)

USA

Hwang et al. (2005) 3 T3-L1 cell 100 μM of genistein and daidzein inhibit adipocyte differentiation processvia activating 5′-AMP-activated proteinkinase (AMPK) and evoke apoptosis ofmature adipocytes

Korea

Kim et al. (2006) 3T3-L1 cell/ovariectomized mice Genistein (0-400 mM) / genistein (0,150, 1500 mg/kg body weight) for 3 wks

Reduce body fat and mature adipocytes viaincrease in apoptosis

USA

Wu et al. (2007) Early postmenopausal women Intake isoflavone conjugates(75 mg/day) for 1 year

Prevent fat accumulation in the equolproducer

Japan

Cederroth et al. (2007) Outbred mice Feeding high soy-containing diet fromconception to adulthood

Reduce body weight and adiposity viaincrease in lipid oxidation and increase inlocomotor activity

Switzerland

Kim et al. (2010a) Adipose tissue-derived (AD) –mesenchymal stem cells (MSCs)

Genistein and daidzein (0.01–100 μM) Repress adipogenic differentiation of AD-MSCs via stimulation of Wnt signaling orlipolysis

Korea

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simple steatosis to hepatocellular carcinoma (Yoneda et al., 2007). Thehigh incidence of NAFLD in developed countries is related to the in-crease of risk factors including obesity, dyslipidemias, type 2 diabetes,and insulin resistance. Fat accumulation in the liver is a result of abnor-mal fatty acid metabolism including: (1) abnormal de novo synthesis oftriglyceride, (2) excessive delivery of free fatty acid (FFA) to liver, and(3) a surplus supply of proinflammatory and steatotic cytokines.Among these abnormal fatty acid metabolism, FFA and cytokines aremainly supplied from adipose tissue, an organ that stores surplus ener-gy. Indeed, a 1% increase in total adipose tissue induced a 22% increasein intrahepatic lipids (Thomas et al., 2005). Hence, appropriate mainte-nance of fat tissue can prevent NAFLD progress.

Current treatments for NAFLD typically include weight loss, exer-cise, improved diabetes control and the use of cholesterol-loweringmedications. Several possible treatments, such as ursodiol (Actigall)for lowering liver enzymes, metformin, pioglitazone (Actos), rosiglita-zone (Avandia) and betaine (Cystadane) for preventing diabetes, orlistat (Xenical) for blocking the absorption of dietary fat, and bariatricsurgery for rapid weight loss, are under investigation. However, todate, these treatments have side effects and do not prove efficient(Dowman et al., 2011). Thus, the requirement for complementary andalternative medicine has been growing. Isoflavones have demonstrated

to have favorable effects against menopausal symptoms, cardiovasculardisease, atherosclerosis, hyperlipidemia, cancer, and osteoporosis(Setchell and Cassidy, 1999; Tham et al., 1998). Isoflavones have beenreported to prevent NAFLD and adiposity in many studies through reg-ulation of peroxisome proliferator-activated receptors (PPARs), fattyacid β-oxidation, and oxidative stress (Table 1). However, there is littleinvestigation about the effect of isoflavones on de novohepatic lipogeniccarbohydrate responsive element binding protein (ChREBP) pathwayor anti-adipogenicWnt signaling pathway. Thus, the purpose of this re-port is to review the biological actions and mechanisms of soy isofla-vones on NAFLD and pathologic adiposity via ChREBP and Wntsignaling.

NAFLD and pathological adiposity

Non-alcoholic fatty liver disease (NAFLD) is defined as an excess offat in the liver in which at least 5% of hepatocytes display lipid drop-lets, as observed in patients who do not consume significant amountsof alcohol (Adams et al., 2005). In most industrialized countries, theprevalence of NFALD continues to rise. For example, an estimated30% of adults and 10% of children and adolescents in the United Statesexhibit NAFLD (Browning et al., 2004; Schwimmer et al., 2006).

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NAFLD was implicated as the primary liver disease that is associat-ed with obesity, because NAFLD can be developed by conditions thatare closely associated with obesity, including insulin resistance, oxi-dative stress and adiposity. Many research studies have suggestedthat more than 75% of obese adult subjects and 53% of obese childrendeveloped NAFLD, whereas the prevalence of NAFLD in the generalpopulation ranged from 5 to 33% (Franzese et al., 1997; Marchesiniet al., 2003).

The pathogenesis of NAFLD has a large spectrum of features thatinclude simple steatosis, non-alcoholic steatohepatitis (NASH), fibro-sis, cirrhosis and hepatocellular carcinoma (Yoneda et al., 2007). Anexcessive accumulation of triglyceride is the principal characteristicof hepatic steatosis. Steatosis is attributable to 1) excessive dietaryfat, 2) hepatic de novo synthesis via carbohydrate responsive elementbinding protein (ChREBP) pathway, and 3) a surplus supply of FFAand proinflammatory cytokines that are released from the adiposetissue.

The correlation between NAFLD and visceral fat is partially basedon the mechanism that obesity-induced macrophages infiltrationinto adipose tissue stimulates the release of proinflammatory cyto-kines, e.g., tumor necrosis factor (TNF) α, interleukin (IL)-6, and IL-1β, impairs insulin signaling, stimulating lipolysis, and eventually in-creasing FFA release from adipose tissue (Weisberg et al., 2003;Zhang et al., 2002). FFA is then taken up by the liver, activating nucle-ar factor (NF) χB, a master regulator of inflammation, and arising ox-idative stress through FFA oxidation (Cai et al., 2005). Moreover, theadipose tissue-secreted adipocytokines, leptin and adiponection, reg-ulate NAFLD. Leptin deficiency has been reported to suppress innateand acquired immune response, and in turn, favors proinflammatoryT-helper cell lymphocyte polarization, aggravate the pathology stageof NAFLD (Guebre-Xabier et al., 2000). Similarly, leptin receptor defi-cient condition showed simple steatosis and NASH via reduction ofhepatic fatty acid β-oxidation and hyperphagia (Cederroth et al.,2007). Adiponectin has been considered to stimulate hepatic fattyacid β-oxidation via activation of 5′ AMP-activated protein kinase(AMPK) and peroxisome proliferator-activated receptors (PPAR) α,which stimulate the expression of enzymes catalyzing fatty acid β-oxidation. Beside, adiponectin has been known to suppress NFχBand macrophage (Ouchi et al., 2000). This relationship betweenNAFLD and visceral fat evidenced one more from recent epidemiolog-ical researches. For example, Asians have a similar prevalence ofNAFLD (16–42% by imaging modalities and 15–39% by liver biopsies)with North Americans and Europeans due to a higher proportion ofcentral obesity than those of Europeans.

Phytoestrogen isoflavones as a curer for NAFLD and adiposity

Over many years, phytoestrogen isoflavones have received in-creasing amount of attention because of their favorable effects againstmenopausal symptoms, cardiovascular disease, atherosclerosis, hy-perlipidemia, cancer, and osteoporosis (Setchell and Cassidy, 1999;Tham et al., 1998). In raw foods and plants, isoflavones exist in thesugar-bound form (7-O-β-glycosides). However, during digestion,these glycosides undergo hydrolysis by the gastric acid, the brushborder membrane enzymes and the bacterial β-glycosides, and thenform absorbable aglycones (Shin et al., 2007).

The major isoflavones found in humans are genistein and daid-zein. Isoflavones have 2-phenylnaphthalene-type chemical structuresthat are similar to those of estrogens, and such a characteristic ofstructures contributes to estrogen receptor binding. Among the char-acteristics of the structures, the most important is the presence andposition of one aromatic ring with the hydroxyl group (OH). This hy-droxyl group participates in forming hydrogen bonds with twoα-helices of estrogen receptors (ERs) and is associated with the anti-oxidant potencies of isoflavones. Thus, genistein, which containsthree hydroxyl groups, has higher estrogen receptor binding affinity

to ERα than daidzein, which contains two hydroxyl groups (Kuiperet al., 1998).

After the release of aglycones from glycosides, genistein and daid-zein go through bacterial biotransformations including dehydroxyla-tion, reduction, C-ring cleavage and demethylation in the colon. Forexample, biochanin A and formononetin are biotransformed to thedemethylated intermediates, genistein and daidzein. Subsequently,genistein and daidzein are metabolized to p-ethyl phenol and equolindividually (Fig. 1). Equol has been highlighted due to its robust es-trogenic action and antioxidant activity. In vitro binding affinity toERs, equol showed higher value (0.4) than those (0.1) shown by daid-zein, when 17β-estradiol showed 1.0. Furthermore, equol had thegreatest antioxidant activity of all the isoflavones when tested usingFe (II) and Fe (III) metal ions and azo-derived peroxyl radicals-induced peroxidation in a liposomal system (Arora et al., 1998). How-ever, 30–50% of the adult human population could not convert daid-zein into equol (Rowland et al., 2000).

Most of the health benefits that are conferred by isoflavones areassociated with ERs-dependent or -independent mechanism basedon the concentration of endogenous estrogens and isoflavones, andindividual characteristics, such as gender and menopausal status(Penza et al., 2006). Genistein and daidzein exhibit weak estrogenicactivity approximately 10−2–10−3 of 17β-estradiol (Miksicek,1994), but are present in the body at concentrations that are 100-fold higher concentrations than endogenous estrogen (Adlercreutzet al., 1993). Thus, genistein and daidzein can exert an anti-estrogenicor estrogenic action by competing or replacing 17β-estradiol based onthe concentrations of 17β-estradiol in the body.

Dietary genistein and daidzein were reported to influence bodyweight gain, fat deposition, hyperlipidemia, insulin resistance and al-ternation of adipocytokines that are associated with NAFLD. Further-more, we reported previously that genistein decreased hepatic fataccumulation by up-regulation of genes involved in fatty acidβ-oxidation, such as uncoupling protein-2, PPARγ coactivator 1 andmitochondrial medium chain acyl-Co A dehyrogenase (Lee et al.,2006). These anti-steatotic and anti-adipogenic effects of isoflavoneare listed in Table 1.

ChREBP and Wnt signaling in NAFLD and adiposity

De novo lipogenesis via ChREBP signaling

The liver is the principal organ that is responsible for de novo lipo-genesis. The transcription factor sterol regulatory element bindingprotein (SREBP)-1c has previously emerged as a primary mediatorof insulin's action on lipogenic genes, such as acetyl Co carboxylase(ACC) and fatty acid synthase (FAS) (Foufelle and Ferre, 2002). How-ever, SREBP-1c activity alone is not sufficient to account for the stim-ulation of glycolytic and lipogenic gene expression, as deletingSRBEP-1c only results in a 50% reduction in fatty acid synthesis(Liang et al., 2002).

Basic/helix-loop-helix/leucine zipper (bHLH/LZ) transcription fac-tor ChREBP has shed light on the de novo lipogenic mechanism.ChREBP leads to the transcription of genes involved in glycolysis,resulting in the conversion of glucose excess to fatty acids. Glucoseactivates ChREBP by regulating the access of ChREBP from the cytosolinto the nucleus and then stimulates the binding of it to an E-boxmotif in the promoter of liver-type pyruvate kinase (L-PK), a key reg-ulatory enzyme in glycolysis (Iizuka et al., 2004). Interestingly, anoth-er previous study demonstrated the increase in expression ofmultiple liver lipogenic enzyme mRNAs but marked the reduction ofL-PK mRNA in mice lacking ChREBP gene expression in comparisonto WT mice (Ishii et al., 2004), which suggests that ChREBP can inde-pendently stimulate lipogenic enzymes, FAS, ACC, stearoyl-Co Adesaturase-9 (SCD)1, and glyceraldehyde 3-phosphate acyltransfer-ase (GPAT) (Uyeda and Repa, 2006), and is itself regulated at

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transcriptional level by its upstream genes SREBP1-c and liver X re-ceptors (LXRs) as well as glucose (Cha and Repa, 2007). In vivostudy, delivery of short hairpin ChREBP-RNA (shChREBP-RNA) effi-ciently knocked down ChREBP expression in liver of ob/ob miceunder both short- (2 days) and long-term (7 days) conditions, induc-ing a resultant decrease in lipogenic rates, leading to a 50% reductionin hepatic and circulating triglyceride concentrations via inhibition ofL-PK, ACC, FAS, and SCD1 as well as GPAT (Dentin et al., 2006).

Anti-adipogenic Wnt signaling

Most transcriptional regulators of adipogenesis seem to operatein a “feed-forward” fashion, in which they induce other pro-adipogenic factors and subsequently cooperate with those factorsto promote downstream gene expression. The first transcription fac-tors that participate in adipogenesis were members of the bZIP fam-ily CCAT/enhancer-binding protein (C/EBP)s, specifically C/EBPα,C/EBPβ and C/EBPδ (Darlington et al., 1998).

Upon committed adipogenesis, preadipocyte induces a rapid andtransient elevation of C/EBPβ and C/EBPδ. Next, these proteins theninduce the expression of C/EBPα and PPARγ. These transcription fac-tors induce or accelerate adipogenic differentiation by stimulatingadipogenic genes (C/EBP α and PPARγ) or insulin signaling (Bennettet al., 2002; Kennell and MacDougald, 2005; Wu et al., 1999).

Wnt/β-catenin signaling has been associatedwithmany human dis-eases, ranging from cancer to degenerative disease such as Alzheimer'sdisease (Luo et al., 2007), and it has been reported to maintain pre-adipocytes in an undifferentiated state by the inhibition of adipo-genesis (Bennett et al., 2002; Kennell and MacDougald, 2005).Wnts belong to a family of secreted proteins that act via paracrineand autocrine mechanisms to regulate many aspects of cell fateand development.

In the β-catenin-dependent (i.e., canonical) pathway,Wnts bind toFrizzled (Frz) receptors and low-density lipoprotein receptor-relatedprotein (LRP) 5/6 coreceptors. Subsequently, the Wnt signals are

Fig. 1. Genistein activates Wnt/β-catenin signaling through non-genomic and genomic estroShc4/ERK1/2/p90RS6KA3 pathway) phosphorylates GSK3β and subsequently escapes β-activators (P300/CBP, PACF, SMARCD1) of β-catenin and stimulates the transactivationreceptor-related protein Erk1/2: extracellular signal-regulated kinases 1/2; JNK3: c-Jun N-tGSK3β: glycogen synthase 3β; PACF: P300/CBP-associated factor; TCF/LEF: T cell factor/lym

transmitted by the association between Wnt receptors and Dishev-elled (Dsh). An event triggers the disruption of the complex contain-ing adenomatous polyposis coli (APC), axin, and glycogen synthasekinase (GSK)-3β, which saves β-catenin from GSK-3β-dependentdegradation. This leads to β-catenin translocation into the nucleusand combination of β-catenin with TCF, thereby stimulating the tran-scription of the Wnt responsive gene, cyclin D1 and PPARδ (Cadiganand Nusse, 1997). In vitro studies using preadipocyte lines demon-strated that ectopic expression of Wnt1, an activator of Wnt/β-catenin signaling, potently represses adipogenesis (Ross et al.,2000). Similarly, pharmacological agents that activate canonical Wntsignaling and stabilize free cytosolic β-catenin also block preadipocytedifferentiation (Bennett et al., 2002). Furthermore, overexpression ofWnt10b in mice causes a 50% reduction in adiposity under standardlaboratory conditions, and these mice resist expansion of adipose tis-sue under conditions of diet-induced and genetic obesity (Longoet al., 2004; Wright et al., 2007).

Expression of endogenous inhibitors of Wnt signaling modulatedadipogenesis. For example, expression of secreted frizzled-relatedprotein (SFRP) 2 is elevated in visceral adipose tissue compared tosubcutaneous depots (Gesta et al., 2006). Two additional inhibitorsof the Wnt pathway, SFRP5 and naked1, were found to be expressedin mature adipocytes and are positively correlated with increasingadiposity (Koza et al., 2006). Furthermore, male mice deficient inSFRP1 show a 22% decrease in percent body fat, while elevated levelsof SFRP1 are observed in individuals that display enhanced orbitaladipogenesis (Bodine et al., 2004).

The anti-steatotic and anti-adipogenic mechanism of isoflavonevia ChREBP and Wnt signaling

Isoflavones and ChREBP signaling

In our previous studies (Kim et al., 2010b, 2011; Lee et al., 2006),isoflavones reversed high fat-induced NAFLD features, including

gen receptors (ERs)-dependent pathway.Non-genomic ERs signaling (Shc4/JNK3/JUN orcatenin from GSK3β-mediated degradation. Genomic ERs interact directly with co-of β-catenin.ER: estrogen receptor; Gen: genistein; LRP5/6; low-density lipoproteinerminal kinases 3; Dvl: dishevelled, dsh homolog 1; APC: adenomatous polyposis coli;phoid-enhancer factor.

Fig. 2. Daidzein alleviates NAFLD by preventing ChREBP-dependent lipogenesis.Non-genomic (AMP/AMPK or cAMP/PKA) ER-dependent pathway phosphorylates both ChREBP andits upstream gene LXRs, which prevents ChREBP from being transported into the nucleus and binding to the carbohydrate responsive element (ChoRE) in the promoter regions oflipogenic enzyme genes, or decreases LXR binding activity for ChREBP by preventing LXR/RXR dimerization. This cascade inhibits the expression of lipogenic enzymes (ACC, FAS)and glycolysis enzyme (L-PK) and subsequently reduces free fatty acid (FFA) synthesis.ER: estrogen receptor; L-PK: liver-type pyruvate kinase; FAS: fatty acid synthase; ACC: acetylCo A carboxylase; SREBP-1c: sterol regulatory element binding protein; LXR: liver X receptor; ChREBP: carbohydrate responsive element binding protein; PKA: protein kinase A.

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much increased hepatic lipid droplets, elevation in the liver injurymarkers, alanine transaminase (ALT) or aspartate aminotransferase(AST), and increased hepatic lipid profiles. In particular, daidzein sup-plementation reduced de novo hepatic lipid synthesis by regulatingChREPB and its upstream LXR. The transcription factor SREBP-1cwas previously identified as a primary inter-mediator of insulin's ac-tion on lipogenic genes (Foufelle and Ferre, 2002). However, in ourstudy (Kim et al., 2011), despite the up-regulation of SREBP-1c bydaidzein supplementation, hepatic lipid profiles were lowered inmice that were fed on daidzein supplementation as compared withthose that were fed on the high fat diet. In addition, other studies re-veal that soy isoflavones either increase SREBP-2, which preferential-ly regulates genes involved in cholesterol synthesis (Mullen et al.,2004), or downregulates SREBP-1 by inhibiting the expression site-1protease, necessary for SREBP-1 maturation (Shin et al., 2007). Collec-tively, isoflavones probably inhibit hepatic lipid synthesis by directlyregulating ChREBP, irrespective with SREBP-1c. However, furthermechanistic studies are needed for elucidating the role of isoflavonein SREBP-1c regulation.

The transactivation of ChREBP can be modulated by LXRs and ser-ine/threonine kinase, such as protein kinase A (PKA) and AMPK aswell as SREBP-1c. LXRs have been reported to induce the expressionof ChREBP by directly binding to the ChREBP promoter, in order togenerate the fatty acids that are necessary for the formation of choles-terol esters (Cha and Repa, 2007). In contrast, PKA and AMPK havebeen shown to prevent ChREBP from being transported into the nucle-us or binding to the carbohydrate responsive element (ChoRE) that ispresent in the promoter regions of lipogenic enzyme genes via thephosphorylation of ChREBP (Kawaguchi et al., 2001, 2002), and indi-rectly decrease LXR binding activity for ChREBP by preventing LXR/retinoid X receptor (RXR) dimerization through the phosphorylationof LXR (Yamamoto et al., 2007; Yang et al., 2009). In our other study(Kim et al., 2010b), isoflavones increased the expression of AMPK viathe up-regulation of ERs-dependent PPARα. In addition, isoflavonestreatment increases the intracellular cAMP levels and the activity of

PKA by inhibiting phosphodiesterase in rat cancer cells (Lin et al.,2005). Our hepatic transcription profiles also evidenced that isofla-vones supplementation causes higher increased PKA and AMPK ex-pression and lower decreased LXRβ expression (Kim et al., 2011).

Isoflavones and Wnt signaling

The effects of isoflavones on Wnt signaling may vary based on thetarget tissues. A prospective study of 12,395 California Seventh-DayAdventist men who often drink soy milk showed that frequent con-sumption of soy milk was associated with a 70% reduction of prostatecancer (Jacobsen et al., 1998). Two cancer-related experimental stud-ies have showed that isoflavones inhibit the growth of cancer cells viaup-regulation of the expression of GSK-3β, enhancement of GSK-3βbinding to β-catenin, phosphorylation of β-catenin, or demethylationof Wnt5a promoter (Li et al., 2008; Wang and Chen, 2010), whichsuggest that isoflavone could inactivate Wnt signaling to inhibit thegrowth of prostate cancer cells.

However, in adipose tissue, isoflavones inhibited the expression ofWnt5a (Su et al., 2007), which is known as a suppressor of canonicalWnt signaling and increases triacylglycerol and the expressionof adipogenesis-related genes during adipocyte differentiation(Kanazawa et al., 2005). A study in our laboratory has also demon-strated that isoflavones genistein represses the adipogenic differenti-ation of human adipose tissue-derived mesenchymal stem cells (AD-MSCs) via Wnt signaling activation. In our microarray data, genisteinincreases positive canonical Wnt signaling regulators Wnt3a andβ-catenin, whereas it suppresses negative regulators of Wnt signalingsuch as SFRP1, Dickkopf-related protein (DKK)2 and Wnt 5a (Kimet al., 2010a). These results are consistent with a recent reportshown that treatment with genistein inhibits the cells migration ef-fect of DKK1 and increases the localization of β-catenin andE-cadherin (Kuang et al., 2009).

Moreover, our data revealed that these stimulatory effects of ge-nistein on Wnt signaling are exerted though non-genomic and/or

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genomic ERs-dependent pathways, that is, the genistein-activatedERs signaling, such as Shc 4/extracellular signal-regulated kinases(Erk) 2/p90RS6KA3 and Shc4/c-Jun N-terminal kinases (JNK) 3/JUN,were revealed to inactivate GSK-3β (Ding et al., 2005), transactivateT cell factor/lymphoid-enhancer factor (TCF/LEF) (Nateri et al.,2005), a final effector of Wnt signaling and degrade adipogenicPPARγ (Camp et al., 1999). Furthermore, genistein-augmentedp300/ CREB-binding protein (CBP), P300/CBP-associated factor(PACF), SMARCD1 both the binders of nuclear ERs and the transcrip-tional co-activators of TCF/LEF, accelerate the access of β-catenin tothe TCF/LEF promoter and link β-catenin to the basal transcriptionalmachinery or the RNA polymerase complex (Hecht et al., 2000). Bythe way, these effects are all offset by ICI 182,780, an estrogen antag-onist (Kim et al., 2010a).

Conclusions

Collectively, soy isoflavones appear to alleviate NAFLD in obesemice fed on a high fat diet, and this effect is the result from an alter-ation in hepatic and adipocyte metabolism as follows: 1) by reducingde novo hepatic lipogenesis via ChREBP signaling, and 2) by reducingfat mass via the activation of anti-adipogenic Wnt signaling, which,in turn, can lead to inhibit the release of steatotic or steatohepatiticadipocytokines, such as TNFα and ghrelin. The proposed mecha-nisms of isoflavones on ChREBP and Wnt signaling are illustrated inFigs. 1 and 2.

However, there is a different set of pathways in anti-steatotic andanti-adipogenic effects of individual isoflavone, e.g., daidzein sup-presses the adipogenic differentiation of AD-MSCs via PKA-dependent hormone sensitive lipase, whereas genistein inhibits theadipogenic differentiation of AD-MSCs via ERs-dependent Wnt sig-naling activation (Kim et al., 2010a). This difference between genis-tein and daidzein might be related to the estrogenic activitychanged based on ER subtypes. The ranking of the estrogenic potency(as the percent activity relative to E2) is E2 (100)>genistein (0.56)>daidzein (0.11) for the ERα subtype, and estrogen (100)>daidzein(5.1)>genistein (2.2) for the ERβ subtype. Interestingly, the respon-siveness of Wnt signaling has been reported to be increased inERα-positive cells (Miyakoshi et al., 2009; Ray et al., 2008). Second,a metabolite of daidzein, O-desmethylangolensin, has been shownto inhibit the transcriptional activity of 5α-dihydrotestosterone(DHT) and alpha2-adrenoceptor antilipolytic signaling (Arner,2005). Third, the function of genistein as a tyrosine kinase inhibitormight induce a different set of pathways between genistein and daid-zein. Glucose is a primary energy source for the adipogenesis and itstransport is controlled by an insulin-dependent pathway. Interesting-ly, genistein inhibits expression of the genes encoding insulin signal-ing including insulin receptor substrate (IRS)-1, PKB and glucosetransporter (Glut) 4 as a tyrosine kinase inhibitor (Szkudelski et al.,2005). This point will make genistein more powerful in anti-adipogenic action than daidzein.

Thus, the intake of natural compound isoflavones may be useful inpreventing NAFLD and pathological adiposity and recognition aboutthe difference between them will help to facilitate the design ofnovel strategies to treat NAFDL and pathological adiposity using iso-flavones. Furthermore, this action of isoflavones may be partially as-sociated with ChREBP and Wnt signaling.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgment

This work was supported by the Bio & Medical Technology Devel-opment Program of the National Research Foundation (NRF) fundedby the Korean government (MEST) (No. 2010-0020265).

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