Nrf2 induces fibroblast growth factor 21 in diabetic mice Yuki Furusawa1,2, Akira Uruno1*, Yoko Yagishita1, Chika Higashi1,2 and Masayuki Yamamoto1* 1

Department of Medical Biochemistry, Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan 2 Pharmaceutical Research Center, Mochida Pharmaceutical Co. Ltd, Gotemba, Shizuoka 412-8524 Japan

Transcription factor Nrf2 (nuclear factor E2-related factor 2) is a master regulator of cellular defense system against oxidative and electrophilic stresses and is negatively regulated by an adaptor protein Keap1 (Kelch-like ECH-associated protein 1). Nrf2 also plays a pivotal role in metabolic homeostasis, such as lipid metabolism and energy expenditure as well as redox homeostasis. FGF21 (fibroblast growth factor 21) is known as a key mediator of glucose and lipid metabolism. Here, we found that Nrf2 is involved in FGF21 regulation in diabetic model mice. Nrf2 induction by genetic knockdown of Keap1 increased plasma FGF21 level and hepatic Fgf21 expression in diabetic db/db mice and high-calorie-diet-induced obesity model mice. Administration of CDDO-Im (oleanolic triterpenoid 1-[2-cyano-3,12-dioxooleane-1, 9(11)-dien-28-oyl] imidazole), a potent Nrf2 inducer, up-regulated plasma FGF21 level and hepatic Fgf21 expression in db/db mice, whereas CDDO-Im did not induce FGF21 in db/db mice with Nrf2 knockout background. Furthermore, in Keap1-knockdown db/db mice, Nrf2 enhanced expression of glucose- and lipid-metabolism-related genes in adipose tissues, which improved plasma lipid profiles. These results show that Nrf2 positively regulates FGF21 expression in diabetic mice. We propose that FGF21 is a potential efficacy biomarker that mediates metabolic regulation by the Keap1–Nrf2 system.

Introduction There are increasing demands for more effective therapies based on novel mechanism against metabolic disorders such as diabetes mellitus, hyperlipidemia and obesity. Fibroblast growth factor (FGF) 21 has been reported as a potential new therapy for metabolic disorders (Kharitonenkov et al. 2005). FGF21 is one of the FGF family molecules, which regulates glucose and lipid metabolism. Administration of FGF21 improves hyperglycemia, insulin resistance and hyperlipidemia and attenuates weight gain in diabetic model animals (Kharitonenkov et al. 2005, 2007; Wente et al. 2006; Inagaki et al. 2007; Coskun et al. 2008; Xu et al. 2009). Furthermore, treatment with FGF21 analogue in type 2 diabetes patients improves lipid profile and reduces fasting insulin level and body weight (Gaich et al. 2013). FGF21 is expressed in Communicated by: Kohei Miyazono *Correspondence: [email protected] or [email protected]

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liver, white adipose tissue (WAT) and pancreas (Nishimura et al. 2000; Wente et al. 2006; Muise et al. 2008). FGF21 exerts its functions by binding to FGF receptors with a cofactor b-Klotho (Kurosu et al. 2007; Ding et al. 2012). As b-Klotho is preferentially expressed in metabolic organs including liver, WAT and pancreas, FGF21 functions are directed to these specific tissues (Ito et al. 2000; Ding et al. 2012). Nrf2 (nuclear factor E2-related factor 2) is a basic region-leucine zipper-type transcription factor belonging to the Cap ‘n’ collar (CNC) family (Moi et al. 1994; Itoh et al. 1995). Nrf2 is negatively regulated by Keap1 (Kelch-like ECH-associated protein 1) through ubiqutination and proteasome-mediated degradation under unstressed condition. When Keap1 is exposed to oxidative and electrophilic stimuli (Yamamoto et al. 2008; Takaya et al. 2012), Nrf2 is avoided from the proteasome-mediated degradation (Itoh et al. 1999; Kobayashi et al. 2004; Motohashi & Yamamoto 2004; Zhang et al. 2004; Tong et al. 2007). Thereafter Nrf2 translocates and accumulates in the nucleus and forms heterodimer with small Maf

DOI: 10.1111/gtc.12186 © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Nrf2 induces FGF21

proteins (sMaf; Yamazaki et al. 2012), and binds to the antioxidant/electrophile responsive element (ARE/EpRE) in its target genes and induces their expressions (Hirotsu et al. 2012). This stress–response gene regulation system is called ‘the Keap1–Nrf2 system’ (Uruno & Motohashi 2011). Although the Keap1–Nrf2 system plays pivotal roles in antioxidant and detoxification functions (Itoh et al. 1997, 1999; Ishii et al. 2000; Uruno & Motohashi 2011), the Keap1–Nrf2 system is also involved in metabolic regulation (Mitsuishi et al. 2012). Nrf2 knockout (Nrf2
/
) mice are resistant to weight gain in high-fat diet (HFD)-induced obesity model (Pi et al. 2010) and show ameliorated insulin resistance (Meher et al. 2012). Meanwhile, Nrf2 deficiency impaired glucose tolerance and aggravated hyperglycemia in streptozotocin-induced diabetic model mice (Aleksunes et al. 2010) and obese ob/ob model mice (Xue et al. 2013). As the precise mechanisms of metabolic regulation by Nrf2 have not been fully elucidated, it is needed to identify metabolism-related Nrf2 target genes. In addition to Nrf2 knockout mice, effects of Nrf2 inducers on metabolic disorders have been reported. Repeated dose of synthetic oleanolic triterpenoid 1-[2cyano-3,12-dioxooleane-1,9(11)-dien-28-oyl] (CDDO) imidazole (CDDO-Im), an inducer of Nrf2 signaling pathway (Yates et al. 2007), prevents HFD-induced obesity by increasing energy expenditure (Shin et al. 2009). Furthermore, a CDDO derivative CDDO methyl ester (CDDO-Me) administration reduced blood glucose and plasma lipid levels in HFD-induced obesity model and diabetic db/db model mice (Saha et al. 2010). We have shown that Nrf2 induction by hypomorphic knockdown of Keap1 or administration of Nrf2 inducer CDDO-Im markedly suppresses onset of diabetes mellitus in mice (Uruno et al. 2013). It has been also shown that Nrf2 induction protects pancreatic b cells from oxidative and nitrosative stress in reactive species-mediated b-cell impairment model mice (Yagishita et al. 2014). These studies show significance of Nrf2 in metabolic regulation, as well as detoxification and cytoprotection, and propose that Nrf2 is a novel pharmacological target for treatment and prevention of metabolic disorders. In drug development process, target-engaged biomarkers are required to predict efficacy and safety of novel candidate of drugs (Frank & Hargreaves 2003; Lewin & Weiner 2004). Although biomarkers that reflect Nrf2 induction in vivo are required for development of Nrf2 inducers to treat metabolic disorders, metabolism-related markers directly linked to Nrf2 induction have not been determined. In this study, we identified that Nrf2 positively regulates Fgf21 in

liver. Furthermore, we showed that FGF21 is a biomarker of the Keap1–Nrf2 system activation in metabolically stressed mice and FGF21 seems to mediate metabolic regulations by Nrf2 signaling.

Results Microarray analysis shows secretory proteins induced by Nrf2 in db/db mouse liver

To identify target enzymes and proteins of Nrf2 related to metabolism, we examined expression of hepatic genes in Keap1 knockdown (Keap1flox/
) db/ db mice by means of microarray analysis (Table S1 in Supporting information). Keap1flox/
mice show significant genetic Nrf2 induction (Taguchi et al. 2010). The microarray analysis showed that expression levels of a number of genes encoding humoral factors, including growth factors and carrier proteins, were increased in db/db::Keap1flox/
mouse livers compared with those in littermate db/db::Keap1flox/+ control mice (Table S1 in Supporting information). Of these growth factors and carrier proteins, inductions of Fgf21 and Igfbp1 (Insulin-like growth factor binding protein-1) were particularly strong and significant. We furthermore examined the hepatic Fgf21 and Igfbp1 gene expression levels by means of qPCR. As our microarray analysis was carried out using 8-week-old mice, we first examined expression levels of these two genes in 8-week-old db/db mouse livers. In addition, we also examined Fgf21 and Igfbp1 gene expressions in 18-week-old db/db mice, as db/db mice overtly progresses toward severe diabetes mellitus during this period. Hepatic Fgf21 level was increased in both 8- and 18-week-old db/db::Keap1flox/
mice compared with db/db::Keap1flox/+ mice (Fig. S1A,B in Supporting information). In contrast, although the Igfbp1 expression level was higher in 8-week-old db/ db::Keap1flox/
mouse livers than that in control mouse livers (Fig. S1C in Supporting information), the expression level in 18-week-old db/db::Keap1flox/
mouse livers was comparable with that of db/db:: Keap1flox/+ mouse livers (Fig. S1D in Supporting information). Therefore, we decided to examine roles Nrf2 plays in the Fgf21 gene regulations in this study. Genetic induction of Nrf2 increases FGF21 level in db/db mice

As FGF21 is known as an important metabolic regulator, we examined how Nrf2 regulates Fgf21 gene expression. Notably, plasma FGF21 level was

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Yamazaki, H., Katsuoka, F., Motohashi, H., Engel, J.D. & Yamamoto, M. (2012) Embryonic lethality and fetal liver apoptosis in mice lacking all three small Maf proteins. Mol. Cell. Biol. 32, 808–816. Yates, M.S., Tauchi, M., Katsuoka, F., Flanders, K.C., Liby, K.T., Honda, T., Gribble, G.W., Johnson, D.A., Johnson, J.A., Burton, N.C., Guilarte, T.R., Yamamoto, M., Sporn, M.B. & Kensler, T.W. (2007) Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes. Mol. Cancer Ther. 6, 154–162. Ye, D., Wang, Y., Li, H., Jia, W., Man, K., Lo, C.M., Lam, K.S. & Xu, A. (2014) Fibroblast growth factor 21 protects against acetaminophen-induced hepatotoxicity by potentiating peroxisome proliferator-activated receptor coactivator protein-1a-mediated antioxidant capacity in mice. Hepatology 60, 977–989. Zhang, D.D., Lo, S.C., Cross, J.V., Templeton, D.J. & Hannink, M. (2004) Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol. Cell. Biol. 24, 10941–10953. Zhang, J., Kang, M.J., Kim, M.J., Kim, M.E., Song, J.H., Lee, Y.M. & Kim, J.I. (2008a) Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo. Nutr. Res. Pract. 2, 200–203. Zhang, X., Yeung, D.C., Karpisek, M., Stejskal, D., Zhou, Z.G., Liu, F., Wong, R.L., Chow, W.S., Tso, A.W., Lam, K.S. & Xu, A. (2008b) Serum FGF21 levels are increased in obesity and are independently associated with the metabolic syndrome in humans. Diabetes 57, 1246–1253. Received: 5 June 2014 Accepted: 28 August 2014

Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web site: Figure S1 Quantitative PCR analyses for hepatic Fgf21 and Igfbp1 gene expression levels. Table S1 Microarray expression analysis in Nrf2 induction model db/db mouse liver (humoral factors) Table S2 Primer design for qPCR

© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

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db/db mice. To our surprise, plasma FGF21 level was markedly increased 12 h after single dose of CDDOIm and the induction was maintained until 48-h postdose (Fig. 4A). We also determined expression level of Nqo1, a representative Nrf2 target gene. The administration of CDDO-Im enhanced hepatic Nqo1 expression with a peak at 24 h, and it is sustained by 48 h after the administration (Fig. 4B), indicating that the Nqo1 gene response to CDDO-Im is rather slower than the Fgf21 gene response. These results show that plasma FGF21 level closely reflects induction of Nrf2 in the liver of db/db mice.

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Figure 2 Nuclear factor E2-related factor 2 (Nrf2) decreases plasma triacylglycerol (TAG) and nonesterified fatty acid (NEFA) levels. Plasma TAG (A) and NEFA (B) concentrations in Keap1flox/
mutant and littermate Keap1flox/+ mice (n = 11– 13). *P < 0.05 and ***P < 0.001. †P < 0.01 vs Nrf2+/+::db/ db::Keap1flox/+ mice. n.s., not significant. Data represent mean  SEM.

increase the Fgf21 mRNA expression level in isolated pancreatic islets from wild-type mice (Fig. 3E). Consistent with the results of genetic Nrf2 induction by Keap1 knockdown, these results show that pharmacological Nrf2 induction increases hepatic Fgf21 expression level and plasma FGF21 level in diabetic db/db mice. Acute induction of FGF21 by CDDO-Im in db/db mice

To examine whether Nrf2 can induce acutely the FGF21 secretion, we next analyzed plasma FGF 21 level after single administration of CDDO-Im to the

Thus far, we have observed the increase in plasma FGF21 level in response to the Nrf2 induction in the db/db diabetic model mice. To examine whether this response of FGF21 can be seen in diet-induced obesity animals, we have extended our analysis to highcalorie-diet (HCD)-fed model mice. To this end, we have fed HCD to both Keap1flox/
and Keap1flox/+ control mice for 8 weeks (Uruno et al. 2013). Both plasma FGF21 level and hepatic Fgf21 mRNA expression level were markedly induced in Keap1flox/
mice compared with littermate Keap1flox/+ control mice, but these inductions were completely abolished by the Nrf2 deficiency (Fig. 5A,B). In contrast, in standarddiet (SD)-fed group, plasma FGF21 level and hepatic Fgf21 mRNA expression level were only slightly increased in the Keap1flox/
mice, but these increases were statistically not significant (Fig. 5C,D). These results suggest that Nrf2 enhances the Fgf21 gene expression preferentially in metabolically stressed conditions. FGF21 is a candidate for biomarker of Nrf2 induction and metabolic regulation

FGF21 has been reported to act as a metabolic regulator in adipose tissues by regulating carbohydrate metabolism and energy expenditure-related genes, such as Glut1 (glucose transporter 1), Ucp1 (uncoupling protein 1) and Cidea (cell death-inducing DNA fragmentation factor-a-like effector A; Kharitonenkov et al. 2005; Coskun et al. 2008; Fisher et al. 2012). In this study, we analyzed changes in the expression of these genes upon continuous FGF21 infusion using osmotic pump. After infusion of FGF21 for 3 days, plasma FGF21 level was significantly increased to 10.2 ng/mL compared with saline infusion (0.19 ng/

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Nrf2 +/+ Figure 3 Chemical nuclear factor E2-related factor 2 (Nrf2) inducer increases fibroblast growth factor 21 (FGF21). (A–D) Effects of repeated dose of Nrf2 inducer CDDO-Im on FGF21 secretion and expression in db/db mice. C57BL/KsJ background male db/ db mice were orally administered with CDDO-Im (10 mg/kg body weight) or vehicle (10% DMSO and 10% Cremophor-EL in PBS) once a day for 1 week (A). Plasma FGF21 concentration level of male db/db mice after vehicle and Nrf2 inducer CDDOIm administration (B) (n = 6–7). Fgf21 mRNA expression level in vehicle and CDDO-Im treated male db/db mouse liver (C), white adipose tissue (WAT) and brown adipose tissue (BAT) (D) (n = 6–7). (E) Effect of CDDO-Im on Fgf21 expression level in isolated islets. The isolated islets from ICR background wild-type mice were treated 10 nmol/L of CDDO-Im or 0.1% DMSO (as vehicle control) for 12 h (n = 3). Data are normalized with Hprt expression level, and data of vehicle-treated Nrf2+/+::db/db mice in each tissue (C and D) or vehicle-treated islets (E) are set as 1. *P < 0.05 and ***P < 0.001 vs vehicle-treated db/db mice. Data represent mean  SEM.

mL). Expressions of Ucp1 and Lipe (hormone sensitive lipase) genes were up-regulated in BAT of FGF21treated mice compared with those in BAT of salinetreated control mice (Fig. 6A). Expression of Glut1 mRNA was also increased in WAT of FGF21-treated mice (Fig. 6B). These increases were consistent with those reportedusing different FGF21 administration 868

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protocols (Kharitonenkov et al. 2005; Coskun et al. 2008; Fisher et al. 2012). We also found that FGF21 infusion induced Hk2 (hexokinase 2) gene expression in BAT (Fig. 6A). These results furthermore support the notion that FGF21 regulates expression of a set of glucose- and lipid-metabolism-related genes in adipose tissues.

© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

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Figure 4 Single administration of CDDO-Im acutely induces plasma fibroblast growth factor 21 (FGF21) level in db/db mice. (A) Plasma FGF21 level after CDDO-Im administration. CDDO-Im (10 mg/kg body weight) or vehicle was orally administrated to male db/db mice, and blood samples were taken after 12, 24 and 48 h CDDO-Im administration or 12 h vehicle administration. Data are plasma concentration (ng/mL) of FGF21 (n = 3–6). (B) Hepatic Nqo1 gene expression level after CDDO-Im administration. CDDO-Im or vehicle was orally administrated to male db/db mice, and hepatic Nqo1 expression level was evaluated after 12, 24 and 48 h CDDO-Im administration or 12 h vehicle administration. Data are normalized with Hprt expression level, and data of vehicle-treated db/db mice are set as 1. **P < 0.01, ***P < 0.001 vs vehicle-treated db/db mice. Data represent mean  SEM.

To explore how Nrf2 contributes to the FGF21mediated regulation of these genes, we next analyzed expression of these metabolism-related genes in BAT and WAT of genetically Nrf2-induced model mice. As was the cases for FGF21-treated wild-type mice, Glut1, Lipe and Cidea mRNA levels were significantly increased in BAT of db/db::Keap1flox/
mice and Hk2 mRNA level was also increased (Fig. 6C). Similarly, Glut1 and Hk2 mRNA levels were increased in WAT of db/db::Keap1flox/
mice (Fig. 6D). Showing very good agreement with the results for db/db::Keap1flox/
mice, Glut1 mRNA was signifi-

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Figure 5 Nuclear factor E2-related factor 2 (Nrf2) increases fibroblast growth factor 21 (FGF21) in diet-induced obesity model mice. Plasma FGF21 level (A and C) and hepatic Fgf21 mRNA expression level (B and D) in Keap1flox/
mutant and littermate control Keap1flox/+ mice. Mice were fed with highcalorie diet (A and B) or standard diet (C and D) for 8 weeks (male, n = 4–6). Data are normalized with Hprt expression level, and data of Nrf2+/+::Keap1flox/+ mice are set as 1 (B and D). *P < 0.05 vs Nrf2+/+::Keap1flox/+ mice. Data represent mean  SEM. n.s., not significant.

cantly increased in BAT and WAT of pharmacologically Nrf2-induced model mice using CDDO-Im (Fig. 6E,F). Ucp1 gene expression was also increased in WAT of CDDO-Im-treated mice (Fig. 6F). These results thus show that genetic and pharmacological induction of Nrf2 enhances expressions of a set of glucose- and lipid-metabolism-related genes in adipose tissues. The results furthermore suggest that FGF21 plays certain roles in the Nrf2-mediated regulation of these genes in adipose tissues. Transcriptional activation of Nrf2 in pharmacological and genetic Nrf2 induction model mice

We next examined whether Nrf2 activity was enhanced in liver and adipose tissues of pharmacolog-

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Figure 6 Nuclear factor E2-related factor 2 (Nrf2) regulates metabolism-related genes in adipose tissues. (A and B) Fibroblast growth factor 21 (FGF21) infusion increases metabolism-related gene expression levels in adipose tissues. Glucose- and lipidmetabolism-related gene expression levels in brown adipose tissue (BAT) (A) and white adipose tissue (WAT) (B) of wild-type male mice infused with FGF21 (18 lg/day) and saline for 3 days (n = 5 each). (C and D) Glucose- and lipid-metabolism-related gene expression in BAT (C) and WAT (D) of db/db::Keap1flox/
mutant and littermate db/db::Keap1flox/+ mice (n = 5–13). (E and F) Effects of repeated dose of Nrf2 inducer CDDO-Im (10 mg/kg bodyweight) on glucose- and lipid-metabolism-related gene expression in adipose tissues of db/db mice. CDDO-Im was orally administrated to db/db mice once a day for 1 week. Glucoseand lipid-metabolism-related gene expression in BAT (E) and WAT (F) after vehicle and CDDO-Im administration to db/db mice (n = 6–7). Data are normalized with Actb (A and B) or Hprt (C–F) expression level, and data of control saline-treated mice (A and B), db/db::Keap1flox/+ mice (C and D) or vehicle-treated db/db mice (E and F) are set as 1. *P < 0.05, **P < 0.01 and ***P < 0.001 vs saline-treated mice, db/db::Keap1flox/+ mice or vehicle-treated db/db mice. Data represent mean  SEM.

ical and genetic Nrf2 induction model mice. To this end, we first examined Nrf2 nuclear accumulation and Nqo1 expression level in SD-fed wild-type mice. Upon administration of CDDO-Im, Nrf2 was accumulated in the nuclei of hepatocytes, BAT and WAT (Fig. 7A–C). CDDO-Im increased the Nqo1 expression level in liver 3 h after administration and in liver, BAT and WAT 6 h after administration (Fig. 7D–F). We also examined whether CDDO-Im administration increases Nrf2 transcriptional activity 870

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in db/db mice. Repeated CDDO-Im administration to db/db mice induced Nqo1 expression level in liver, BAT and WAT compared with the vehicle administration, and the CDDO-Im-mediated Nqo1 induction was canceled by the Nrf2 knockout in all these tissues (Fig. 7G–I). We also determined Nqo1 expression level in genetic Nrf2 induction model mice (i.e., Keap1flox/
mice). We found that Nqo1 expression levels were induced in Keap1flox/
mouse liver and adipose tissues

© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

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Figure 7 Transcriptional activation of nuclear factor E2-related factor 2 (Nrf2) in pharmacological and genetic Nrf2 induction model mice. (A–F) Nrf2 accumulation in the nucleus and Nrf2 transcriptional activity monitoring by Nqo1 expression level. CDDO-Im (16.3 mg/kg body weight) or vehicle (10% DMSO and 10% Cremophor-EL in PBS) was orally administrated to C57BL/6J background male wild-type mice, and tissue samples were taken 3 and 6 h after CDDO-Im or vehicle administration. Immunoblot analyses for Nrf2 and lamin in nuclear extracts from liver (A), brown adipose tissue (BAT) (B) and white adipose tissue (WAT) (C) 3 h after vehicle (lanes 1–3) or CDDO-Im (lanes 4–6) administration. Nqo1 expression level in liver (D), BAT (E) and WAT (F) 3 and 6 h after vehicle or CDDO-Im administration (n = 3). (G–I) Effects of repeated dose of CDDO-Im on Nqo1 expression level in db/db mice. C57BL/KsJ background db/db mice were orally administered with CDDO-Im (10 mg/kg body weight) or vehicle once a day for 1 week. Nqo1 expression level in db/db mouse liver (G), BAT (H) and WAT (I) (n = 6–7). (J–L) Magnitude of Nqo1 inductions in Keap1flox/
mice. ICR background Keap1flox/+ and Keap1flox/
mice were fed with SD or high-calorie-diet (HCD; n = 5 each), or crossed with db/db mice (n = 11–13 for liver; n = 4–5 for BAT; n = 12–15 for WAT). Data are normalized with Hprt expression level. For experiments with CDDO-Im administration, data of control vehicle-treated wild-type mice (D–F) or vehicle-treated db/db (G–I) mice are set as 1. For genetic Nrf2 induction experiments (J–L), data of Keap1flox/+ mice in each SD-fed, HCD-fed or db/db mouse group are set as 1. ***P < 0.001 vs vehicle-treated or control Keap1flox/+ mice. Data represent mean  SEM. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

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under lean (SD-fed), obesity (HCD-fed) and diabetic (db/db) conditions (Fig. 7J–L). In liver (Fig. 7J) and BAT (Fig. 7K), magnitude of Nqo1 inductions in Keap1flox/
mice was mildly suppressed in HCD-fed and db/db mice, compared with SD-fed mice (Fig. 7L). These data indicate that both pharmacological and genetic inductions increase Nrf2 transcriptional activity in various metabolic conditions. Nrf2 is required for FGF21-mediated metabolic gene regulations

We next determined whether Nrf2 contributes to FGF21-mediated metabolic regulations in adipose tissues, as was the case for FGF21 secretion from liver. It has been reported that FGF21 signaling pathway and Keap1-Nrf2 signaling pathway mutually interact each other in the detoxification enzyme inductions in liver (Ye et al. 2014). Therefore, we postulated similar interactions of these pathways also in adipose tissues. We tested whether FGF21 administrationmediated metabolic change is affected by the Nrf2 knockout. For this purpose, we infused FGF21 to Nrf2 knockout and wild-type mice fed with HCD (Fig. 8A). We first examined plasma FGF21 level in HCDfed mice 7 days after the start of FGF21 infusion. In wild-type mice, FGF21 administration increased plasma FGF21 concentration compared with saline administration (18.4 and 6.5 ng/mL, respectively). These plasma FGF21 concentrations are similar to those in HCD-fed Keap1flox/
and Keap1flox/+ mice (24.0 and 5.2 ng/mL, respectively). FGF21 infusion also increased plasma FGF21 level in Nrf2 knockout mice compared with saline infusion (14.5 and 2.7 ng/mL, respectively). We next assessed roles that Nrf2 plays in FGF21stimulated Glut1 and Hk2 inductions. FGF21 administration increased Glut1 and Hk2 expression levels in HCD-fed wild-type (Nrf2+/+) mouse WAT (Fig. 8B, C). Interestingly, the FGF21-mediated Glut1 and Hk2 inductions were canceled by the Nrf2 depletion (Fig. 8B,C). FGF21 administration induced Nqo1 expression levels in wild-type mouse WAT, and the Nrf2 knockout completely canceled the FGF21-mediated Nqo1 induction (Fig. 8D). These data indicate that FGF21 signaling pathway and Keap1–Nrf2 signaling pathway mutually interact in gene regulations. We also evaluated whether Nrf2 knockout affects FGF21-mediated lipid metabolism. To avoid the variance of feeding conditions, corn oil was orally administrated to mice after 4 h of fasting. Three 872

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hours after corn oil administration, plasma TAG and NEFA levels were measured (Fig. 8A). FGF21 administration decreased plasma TAG level in wildtype mice (Fig. 8E). Plasma TAG concentration was also decreased in saline-treated Nrf2 knockout mice compared with saline-treated wild-type mice (Fig. 8E), but the reason for this decrease remains to be elucidated. Importantly, plasma TAG concentration was not furthermore decreased in FGF21-treated Nrf2 knockout mice compared with saline-treated Nrf2 knockout mice (Fig. 8E), indicating that the FGF21-mediated TAG suppression is canceled by the Nrf2 knockout. Plasma NEFA level was also decreased by FGF21 administration in wild-type mice (Fig. 8F). The Nrf2 depletion attenuated the FGF21mediated decline of plasma NEFA level (Fig. 8F). These data indicate that Nrf2 signaling is required for the FGF21-mediated lipid-lowering effects.

Discussion In this study, we found that Nrf2 increases hepatic Fgf21 expression and plasma FGF21 level in db/db and diet-induced obesity model mice. Furthermore, genetic induction of Nrf2 is found to lower plasma lipid levels in db/db diabetes model mice, and FGF21 is found to regulate expressions of glucose- and lipidmetabolism-related genes in adipose tissues in mice. As summarized in Fig. 9, these results support our contention that FGF21 is an efficacy biomarker that reflects activation of the Keap1–Nrf2 system in metabolically stressed conditions. It has been reported that FGF21 is mainly expressed in liver and its expression is regulated by peroxisome proliferator-activated receptor (PPAR) a (Badman et al. 2007; Inagaki et al. 2007; Lund
asen et al. 2007). It has also reported that FGF21 is expressed in WAT under the control of PPAR c (Muise et al. 2008; Dutchak et al. 2012). However, we have detected Nrf2-mediated up-regulation of Fgf21 expression only in liver, but not in the other tissues, including WAT, BAT and pancreatic islets. An intriguing observation is that whereas Nrf2 remarkably induces plasma FGF21 level and hepatic Fgf21 expression level in db/db and HCD-fed model mice, the inductions are largely attenuated in SD-fed mice, indicating that FGF21 is preferentially induced in the liver of mice under metabolically stressed conditions. Showing very good agreement with this observation, it has been reported that the Fgf21 expression level is enhanced in metabolically stressed conditions; for instance, high glucose stimulates Fgf21

© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

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Figure 8 Nuclear factor E2-related factor 2 (Nrf2) is required for fibroblast growth factor 21 (FGF-21)-mediated metabolic gene regulations. (A) C57BL/6J background male Nrf2 knockout and wild-type mice were fed with high-calorie-diet (HCD) for 6 weeks. At the end of HCD-feeding periods, mice were infused with FGF21 (6 lg/day) and saline for 7 days (n = 3 for wildtype mice; n = 5 for Nrf2 knockout mice). After 4 h fasting, corn oil emulsion was orally administrated (10 mL/kg body weight). Tissue and blood samples were taken 3 h after corn oil administration. (B–D) Role of Nrf2 on FGF21-mediated regulation of glucose-metabolism-related genes (B and C), and effect of FGF21 administration on Nqo1 expression level in white adipose tissue (WAT). Expression levels of Glut1 (B), Hk2 (C) and Nqo1 (D) in FGF21- or saline-treated wild-type and Nrf2 knockout mouse WAT. Data are normalized with Hprt expression level, and data of control saline-treated wild-type mice are set as 1. **P < 0.01 and ***P < 0.001. (E and F) Role of Nrf2 on FGF21-mediated plasma lipid-lowering effects. Plasma triacylglycerol (TAG) (E) and nonesterified fatty acid (NEFA) (F) levels in FGF21-treated wild-type and Nrf2 knockout mice. *P < 0.05 and **P < 0.01. †P < 0.01 vs saline-treated wild-type mice. n.s., not significant. Data represent mean  SEM.

expression by carbohydrate response element-binding protein (ChREBP) in the liver (Iizuka et al. 2009). Similarly, human studies have shown that plasma FGF21 level is increased in type 2 diabetes (Mashili et al. 2011), obesity, hyperlipidemia (Zhang et al. 2008b) and nonalcoholic fatty liver disease (NAFLD; Dushay et al. 2010). Exploiting genetic and pharmacological induction models of Nrf2, we have shown that Nrf2 positively

regulates FGF21 in severely stressed mouse livers. This conclusion is somewhat contradictory to the observation by Chartoumpekis et al. (2011) that Nrf2 negatively regulates the FGF21 expression. In their study, Nrf2 knockout (Nrf2
/
) mice were fed with HFD, but the diabetic model mice or pharmacological induction of Nrf2 has not been used. In this regard, we found that the plasma FGF21 level and liver Fgf21 mRNA level of Nrf2
/
mice were only

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Liver

Nrf2

FGF21 Plasma biomarker Adipose tissues Interaction with FGF21 signals

Nrf2

Direct regulations? Glucose and lipid metabolism-related genes

Figure 9 Model for the role of nuclear factor E2-related factor 2 (Nrf2) on regulation of glucose and lipid metabolisms by fibroblast growth factor 21 (FGF21) induction. Activation of Nrf2 signaling induces hepatic Fgf21 expression and secretion in metabolically stressed condition, and the plasma FGF21 elevation contributes to the increase in glucose- and lipidmetabolism-related gene expressions in adipose tissues. In addition, Nrf2 seems to directly regulate these gene expressions in adipose tissues, and Nrf2 is required for FGF21-mediated metabolic regulations in adipose tissues.

mildly increased compared with those in Nrf2+/+ mice even in the db/db background (db/db::Keap1flox/+ ::Nrf2
/
vs. db/db::Keap1flox/+::Nrf2+/+ and vehicletreated db/db::Nrf2
/
vs. vehicle-treated db/db:: Nrf2+/+). Therefore, we surmise that the difference is derived from the experimental conditions, and their results and our results are consistent with each other. One important remaining question in this study is the mechanism how Nrf2 induces Fgf21 gene expression level. Our preliminary transactivation assays show mild induction of luciferase reporter activity by CDDO-Im treatment. We found several candidates of AREs/EpREs in the regulatory regions of mouse Fgf21 gene. However, our examination of Nrf2 binding to these regulatory elements by chromatin immunoprecipitation (ChIP) assay, so far and by our hand, did not give rise to any Nrf2 bindings to the ele874

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ments. We surmise a number of possibilities that explain this situation, for instance presence of distal enhancer for Fgf21 gene or presence of important transcriptional co-activators. It has been known that administration of FGF21 induces glucose uptake by the adipose tissues and fatty acid oxidation through inductions of glucoseand lipid-metabolism-related genes, such as Glut1 and Ucp1 (Kharitonenkov et al. 2005, 2007; Coskun et al. 2008; Xu et al. 2009). We have newly added that FGF21 regulates Hk2 gene in the adipose tissues. These results show that FGF21 regulates carbohydrates catabolism by increasing glucose transport and expressions of glycolysis-related genes. One of the salient observations of this study is that we have identified the interaction between FGF21 signaling pathway and Keap1–Nrf2 signaling pathway in adipose tissues. Metabolic regulations by FGF21 administration were significantly canceled by the Nrf2 knockout, indicating that Nrf2 is involved in FGF21 sensitivity in adipose tissue as well as FGF21 secretion from liver. Mechanisms how Nrf2 regulates adipose FGF21 sensitivity remain to be elucidated. As for the Glut1 gene induction, FGF21 positively regulates Glut1 expression level through FGF21 response element located 2.9 kb upstream from transcription start site (Ge et al. 2011). As ARE-like sequence is 3.7 kb upstream from transcription start site, it seems likely that FGF21 augments Glut1 expression level through coordinated regulation of FGF21 response element and ARE by FGF21 and Nrf2. In addition, FGF21 accelerates nuclear Nrf2 accumulation in liver (Ye et al. 2014). We also found that FGF21 administration increases Nqo1 expression level in adipose tissues. These findings suggest that the positive loop between FGF21 signaling pathway and Keap1–Nrf2 signaling pathway may be involved in metabolic regulations. The FGF pump infusions used in this study have attained much higher plasma FGF21 level than those in SD-fed lean mice and in db/db mice. In this regard, plasma FGF21 concentration in HCD-fed mice is much higher than SD-fed mice. Plasma FGF21 concentration reaches approximately 5 ng/mL in HCDfed control mice, but Nrf2 induction dramatically enhances plasma FGF21 concentration, and the level reaches above 20 ng/mL in HCD-fed Keap1flox/
mice. These observations support our contention that FGF21 is a biomarker of HCD-induced obesity. In conclusion, Nrf2 positively regulates the expression of Fgf21 gene in liver under metabolically stressed conditions. Our results indicate that FGF21 is a potential efficacy biomarker of the Keap1–Nrf2 system

© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Nrf2 induces FGF21

enhancement. FGF21 is a pivotal target for metabolic regulations by the Keap1–Nrf2 system.

Experimental procedures Mice Keap1flox/
mice and Nrf2 knockout mice were described previously (Itoh et al. 1997; Taguchi et al. 2010). ICR background db/+::Keap1flox/flox mice were crossed with db/+:: Keap1+/
mice to generate littermate db/db::Keap1flox/+ (db/ db::Keap1F/+) and db/db::Keap1flox/
(db/db::Keap1F/–) mice. The db/db::Keap1flox/
mice were backcrossed with ICR mice at least five generations. C57BL/Ksj background db/m and db/ db mice were purchased from CLEA Japan. Nrf2 knockout db/db mice were backcrossed with C57BL/Ksj mice at least eight generations. The db allele were genotyped by the end point analysis using TaqMan Genotyping Master Mix and ABI Custom SNP (forward primer, 50 -CCAACTTCCCAACA GTCCATACAAT-30 ; reverse primer 50 -CTCATCAAATGT TATTTCTTAGTCATTCAAACCA-30 : probe for wild allele 50 -VIC-TGATGGAGGGAAACAA-30 ; probe for db mutant allele, 50 -FAM-TTGATGGAGGTAAACAA-30 ) using 7300 Real-Time PCR System (95 °C, 10 min for 1 cycle; 92 °C, 15 s, 60 °C, 90 s for 50 cycles). In HCD-fed experiment, SD group was fed with standard chow (MF, 3.59 kcal/g, Oriental Yeast) and HCD group was fed high-fat diet (HFD-60, 5.06 kcal/g, 62.2% fat of calorie, Oriental Yeast) alone for 3 weeks followed by concomitant feeding with 20% sucrose in drinking water for 5 weeks (Uruno et al. 2013). All mice were handled according to the regulations of the Standards for Human Care and Use of Laboratory Animals of Tohoku University and Guidelines for Proper Conduct of Animal Experiments of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

CDDO-Im administration CDDO-Im was obtained from Mochida Pharmaceuticals Co., Ltd. (Gotemba, Japan). Mice were orally dosed with 10% DMSO, 10% Cremophor-EL and phosphate-buffered saline as vehicle or CDDO-Im [10 mg/kg (18.1 lmol/kg) or 16.3 mg/kg (30 lmol/kg) body weight] once a day for 1 week or single dose. Blood and tissue samples were collected 20 h after the last dose under anesthesia unless otherwise mentioned.

FGF21 infusion Murine recombinant FGF21 (BioVendor) infusion was delivered using Alzet miniosmotic pumps (Durect Corporation). Pumps were filled with either FGF21 solution [0.75 lg/h (18 lg/day) for 3 days, or 0.25 lg/h (6 lg/day) for 7 days] or saline and inserted into the interscapular region under anesthesia.

Corn oil administration Corn oil was administrated as emulsion, mixture of 80 mg cholic acid (Sigma-Aldrich), 6 mL saline and 6 mL corn oil (Wako; Zhang et al. 2008a). After 4-h fasting, mice were orally administrated corn oil emulsion (10 mL/kg body weight). Three hours after administration of corn oil emulsion, blood and tissue samples were taken from mice.

Isolation of pancreatic islets Pancreatic islets were isolated from ICR background wild-type mice by ductal collagenase injection. Collagenase V (Wako) in Hanks’ solution was injected into common bile duct, and pancreata were digested by 30-min digestion at 37 °C (Uruno et al. 2013). Islets were hand-picked, and the isolated islets were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin and 100 lg/ mL streptomycin for 12 h, then treated with 10 nmol/L CDDO-Im or 0.1% DMSO (as vehicle control) for 12 h.

Microarray analysis Three independent RNA samples from 8-week-old male db/ db::Keap1flox/+ and db/db::Keap1flox/
mouse livers, respectively, were used for the microarray analyses. The Agilent 4 9 44 K Whole-Mouse Genome Oligo Microarray slides were hybridized, washed and scanned on an Agilent Microarray Scanner according to the manufacture’s protocol. The expression data were analyzed by utilizing GENESPRING software (Silicon Genetics).

Measurements of plasma FGF21, TAG and NEFA Blood samples were taken from inferior vena cava of mice after anesthetized with isoflurane. Plasma concentrations of FGF21, TAG and NEFA were measured using Mouse/Rat FGF21 Quantikine ELISA Kit (R&D Systems), DRI-CHEM7000 (Fuji Film) and Free Fatty Acid Quantification Kit (BioVision), respectively, according to the manufacturer’s instructions.

Quantitative real-time PCR Total RNA was extracted using Isogen RNA extract kit (Nippon Gene) and reverse-transcribed to cDNA using SuperScript III (Invitrogen) or PrimeScript RT Master Mix (Takara Bio; Uruno et al. 2011a). Quantitative real-time PCR was carried out using specific primers and probes (see Table S2 in Supporting information) using an ABI 7300 system. Relative expression of genes was normalized against Hprt or Actb mRNA.

Immunoblot analyses For immunoblot analysis for Nrf2, nuclear fraction was prepared from liver and adipose tissues using NE-PER nuclear

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Y Furusawa et al. and cytoplasmic extraction reagents (Thermo Scientific) according to the manufacturer’s instructions. Immunoblot analysis was carried out as previously described (Uruno et al. 2011a,b; Hirotsu et al. 2014). Immunoblot analyses were carried out using following antibodies: rat monoclonal anti-Nrf2 antibody (clone #103, 1 : 200; Hiramoto et al. 2014) and goat polyclonal anti-Lamin B antibody (Santa Cruz M-20, 1 : 1000).

Statistical analysis All data are represented as mean  SEM. Statistical significances are evaluated using Student’s t-test and one-way ANOVA (analysis of variance) followed by Bonferroni or Fisher’s least significant difference post hoc test for multiple comparisons unless otherwise mentioned.

Acknowledgements We thank Ms Sayoi Inomata (Tohoku University) for technical assistance and Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support. This work was supported by the Grants-in-Aids for Scientific Research on Innovative Areas and Scientific Research from the Ministry of Education, Science, Sports and Culture (MEXT) (M.Y.), create revolutionary technological seeds for science and technology innovation (CREST) from JST (M.Y.), Grants-in-Aids for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (Number 24249015 (M.Y.) and 90396474 (A.U.)), the Takeda Foundation (M.Y.), the Naito Foundation (M.Y.) and the Japanese Foundation for Applied Enzymology (A.U.). Y.F. and C.H. are employees of Mochida Pharmaceutical Co. Ltd. M.Y. has received grant support from Mochida Pharmaceutical Co., Ltd. No other potential conflicts of interest relevant to this article were reported.

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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web site: Figure S1 Quantitative PCR analyses for hepatic Fgf21 and Igfbp1 gene expression levels. Table S1 Microarray expression analysis in Nrf2 induction model db/db mouse liver (humoral factors) Table S2 Primer design for qPCR

© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd