Exendin-4 regulates lipid metabolism and fibroblast growth factor 21 in hepatic steastosis Jinmi Lee, Seok-Woo Hong, Se Eun Park, Eun-Jung Rhee, Cheol-Young Park, Ki-Won Oh, Sung-Woo Park, Won-Young Lee PII: DOI: Reference:
S0026-0495(14)00135-8 doi: 10.1016/j.metabol.2014.04.011 YMETA 53017
To appear in:
Metabolism
Received date: Revised date: Accepted date:
13 January 2014 17 April 2014 29 April 2014
Please cite this article as: Lee Jinmi, Hong Seok-Woo, Park Se Eun, Rhee Eun-Jung, Park Cheol-Young, Oh Ki-Won, Park Sung-Woo, Lee Won-Young, Exendin-4 regulates lipid metabolism and fibroblast growth factor 21 in hepatic steastosis, Metabolism (2014), doi: 10.1016/j.metabol.2014.04.011
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ACCEPTED MANUSCRIPT Exendin-4 regulates lipid metabolism and fibroblast growth factor 21 in hepatic steastosis
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Jinmi Leea, Seok-Woo Honga, Se Eun Parkb, Eun-Jung Rheeb, Cheol-Young Parkb, Ki-Won Ohb,
Institute of Medical Research, Kangbuk Samsung Hospital, Sungkyunkwan University School of
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Sung-Woo Parkb, Won-Young Leeb
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Medicine, Seoul 110-746, Republic of Korea
Department of Endocrinology and Metabolism, Kangbuk Samsung Hospital, Sungkyunkwan
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Correspondence:
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University School of Medicine, Seoul 110-746, Republic of Korea
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Won-Young Lee, MD, PhD
Division of Endocrinology and Metabolism Department of Internal Medicine, Kangbuk Samsung Hospital Sungkyunkwan University School of Medicine # 108 Pyung-Dong, Jongro-Ku, Seoul 110-746, Republic of Korea Tel: 82-2-2001-2579 Fax: 82-2-2001-2049 E-mail: [email protected]
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ACCEPTED MANUSCRIPT Word count of text: 4970, abstract: 265, number of references: 44, and number of tables: 1
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and figures: 4
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Funding
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This study was supported by a grant from Daewoong Pharmaceutical (# MRI-110907-004/5)
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and by the National research Foundation (NRF) grant funded by the Korea government (NRF2013R1A1A2063069). The funders had no role in the study design, data collection and analysis,
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decision to publish, or preparation of the manuscript.
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Conflicts of interest
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All authors declare that there are no conflicts of interest.
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ACCEPTED MANUSCRIPT Abstract
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OBJECTIVE: Hepatokine fibroblast growth factor (FGF) 21 takes part in the regulation of
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lipid metabolism in the liver and adipose tissue. We investigated whether exendin-4 regulates the expression of FGF21 in the liver, and whether the effects of exendin-4 on the regulation of
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FGF21 expression are mediated via silent mating type information regulation 2 homolog (SIRT)1
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or SIRT6.
MATERALS/METHODS: The C57BL/6J mice were fed a low fat diet, high fat diet, or high
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fat diet with 1 nmol/kg/day exendin-4 intraperitoneal injection for 10 weeks. HepG2 used in vitro study was treated with palmitic aicd (0.4 mM) with or without exendin-4 (100 nM) and FGF21
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(50 nM) for 24 hours. The change of FGF21 and its receptors expression by exendin-4 were
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measured using quantitative real-time RT-PCR and Western blot. The intracellular lipid content in HepG2 cells was evaluated by Oil Red O staining. Inhibition of FGF21, SIRT1 and SIRT6, by
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lipid metabolism.
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10 nM siRNA was performed to establish the signaling pathway of exendin-4 action in hepatic
RESULTS: Exendin-4 increased the expression of FGF21 and its receptors in high fat dietinduced obese mice. In addition, recombinant FGF21 treatment reduced lipid content in palmitic acid-treated HepG2 cells. We also observed significantly decreased expression of peroxisomal proliferator-activated receptor (PPAR) α and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in hepatocytes transfected with FGF21 siRNA. In cells treated with exendin-4, inhibition of SIRT1, but not SIRT6, by siRNA significantly repressed the expression of FGF21 mRNA, whereas decreased SIRT1 expression by inhibition of FGF21 was not observed.
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ACCEPTED MANUSCRIPT CONCLUSIONS: These data suggest that exendin-4 could improve fatty liver by increasing
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Keywords: Exendin-4; SIRT1; FGF21; FGFRs; fatty acid oxidation
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SIRT1-mediated FGF21.
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Abbreviation: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; SIRT1,
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silent mating type information regulation 2 homolog 1; NAD, nicotinamide adenine dinucleotide; HF, high-fat; PA, palmitic acid; Ex-4, exendin-4; GLP-1, glucagon-like peptide; NAFLD, non-
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alcoholic fatty liver disease; TG, triglycerides; PPARα, peroxisomal proliferator-activated receptor α; MCAD, medium-chain acyl-coenzyme A dehydrogenase; GLUT1, glucose
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transporter 1; AMPK, AMP-activated protein kinase; PGC-1α, PPAR γ coactivator protein-1α;
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PKA, protein kinase A.
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ACCEPTED MANUSCRIPT 1. Introduction
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The fibroblast growth factor (FGF) family consists of 22 members that have important roles in
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several cell types to regulate diverse physiologic functions. In particular, some members of the FGF19-family, including FGF19, FGF21, and FGF23, function as regulatory hormones of
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glucose, lipid, bile acid, phosphate, and vitamin D metabolism [1, 2]. Moreover, hepatokine
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FGF21 was found as a novel therapeutic agent for diabetes. Hepatic FGF21 expression is regulated by cyclic-AMP-responsive-element-binding protein H (CREBH) and peroxisomal
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proliferator-activated receptor (PPAR) α, and regulates lipid homeostasis [3, 4]. FGF21 is secreted from the liver, functions as a potent activator of glucose uptake in adipocytes via glucose
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transporter (GLUT)-1 [5]. In both ob/ob and db/db mice, administration of FGF21 was shown to
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decrease plasma glucose and triglycerides (TG) [6], whereas liver-specific knockdown of FGF21 led to hepatic insulin resistance by increase of gluconeogenesis and glycogenolysis [7]. In
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mice [8].
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addition, FGF21 treatment improved hepatic steatosis and insulin resistance in diet-induced obese
The silent mating type information regulation 2 homolog (sirtuin, SIRT) family is nicotinamide adenine dinucleotide (NAD)+-dependent enzymes that modify multiple target proteins by deacetylation. Until now, seven members of the sirtuin family have been identified in humans, [9, 10]. In particular, SIRT1 and SIRT6 of seven sirtuins are crucial in metabolic homeostasis. SIRT1 regulates hepatic fatty acid oxidation through positive regulation of AMPactivated protein kinase (AMPK) and PPARα, which are involved in fatty acid β-oxidation in the liver and skeletal muscle [11]. In contrast, the SIRT1 deacetylase-deficient mice on a high-fat diet have excess hepatic lipid accumulation and insulin resistance [12]. In our previous study, we 5
ACCEPTED MANUSCRIPT observed that increased SIRT1 by exendin-4 protected mice against diet-induced obesity and steatohepatitis by increased fatty acid oxidation [13]. Kim et al. [14] reported that SIRT6
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expression is positively regulated by SIRT1 in the liver, and liver-specific knockout of SIRT6
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causes fatty liver via an increase of glycolysis and reduction of β-oxidation. Also, increased SIRT6 expression was shown in rosiglitazone-mediated amelioration of hepatic steatosis [15]. In
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this study, we investigated whether exendin-4 regulates the expression of FGF21 in hepatocytes,
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and whether this mechanism is mediated via SIRT1 or SIRT6.
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ACCEPTED MANUSCRIPT 2. Materials and methods
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2.1. Animals
Male C57BL/6J mice of 6 weeks of age were obtained from Central Laboratory (Shizuoka
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Laboratory Animal Center, Shizuoka, Japan) and bred in standard conditions under a 12 hour
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light/dark cycle. All animal experiments were conducted in accordance with the Ethics Committee for Animal Experiments of the Sungkyunkwan University Kangbuk Samsung
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Hospital. Mice were randomly assigned to a low-fat diet (control, 10 kcal % fat consist of 5.6 kcal % lard and 4.4 kcal % soy bean oil, 20 kcal % protein, and 70 kcal % carbohydrate), high-fat
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(HF) diet (HF, 45 kcal % fat consist of 39.4 kcal % lard and 4.4 kcal % soy bean oil, 20 kcal %
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protein, and 35 kcal % carbohydrate), or a HF diet plus 1 nmol/kg/day exendin-4 (Sigma-Aldrich Corp., St. Louis, MO, USA) via intraperitoneal (IP) injection. During the 10-week treatment
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period, mice of each group were injected every other day with exendin-4 or saline (control) and
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allowed to access to their specific diet and water ad libitum. Body weight was checked twice per week. At the end of treatment, animals were sacrificed; liver tissues were collected and stored at 80 °C until required for this study.
2.2. Cell culture and transfection
A hepatocellular carcinoma cell line, HepG2, was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and was grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum (Gibco, 7
ACCEPTED MANUSCRIPT Grand Island, NY, USA), 1 % penicillin/streptomycin (Gibco, Grand Island, NY, USA) at 37 °C in 5 % CO2. To examine the effects of exendin-4 and FGF21 on hepatic steatosis, HepG2 cells
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were treated with palmitic acid (PA) (0.4 mM) with or without Ex-4 (100 nM) and FGF21 (50
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nM) (Sigma-Aldrich Corp., St. Louis, MO, USA) for 24 hours. Specific siRNAs of SIRT1 [1137490], SIRT6 [1137529], FGF21 [1052655], FGFR1 [100382] (Bioneer, Daejeon, Korea),
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and negative control (Invitrogen, Carlsbad, CA, USA) were purchased and transiently transfected
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into HepG2 cells using Lipofectamine RNAiMAX reagent, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). After incubation for 24 hours, cells were harvested
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to assay for gene knockdown.
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2.3. Total RNA isolation and real-time RT-PCR
Total RNA was isolated from the liver tissue and cultured hepatocytes using Trizol reagent
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(Invitrogen, Carlsbad, CA, USA). Complementary DNA was generated from 2 μg total RNA
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using moloney murine leukemia virus reverse transcriptase (MMLV-RT) and oligo (dT)12-18 primer (Invitrogen, Carlsbad, CA, USA) as described previously [13]. Analysis of mRNA was conducted by real-time PCR (Light-Cycler 480; Roche, Lewis, UK) using SYBR green (Roche, Lewis, UK) and specific primers (Bioneer, Daejeon, Korea) (Supplementary Table 1.) according to the manufacturer’s instructions. After pre-incubation at 95 °C for 10 minutes, thermal cycling conditions consisted of 40 cycles of 94 °C for 15 seconds, 55 °C for 10 seconds, and 72 °C for 20 seconds. Data were normalized using β-actin as an internal control and calculated using the comparative Ct method (2-delta delta Ct).
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ACCEPTED MANUSCRIPT 2.4. Western blot analysis
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Cultured hepatocytes were homogenized at 4 °C in RIPA buffer (Cell Signaling Technology,
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Danvers, MA, USA) supplemented with a cocktail of protease and phosphatase inhibitors, and protein was obtained by centrifugation at 13,000 rpm for 20 minutes at 4 °C. The concentration
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of the sample was determined using the Bradford protein assay (Bio-Rad Protein Assay, BioRad,
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Hercules, CA) with bovine serum albumin standard (Thermo Scientific, Rockford, IL, USA). After the assay, 20 μg of each sample was separated on 4-12 % bis-Tris Nupage gels (Invitrogen,
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Carlsbad, CA, USA) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Chalfont St. Giles, UK). The membranes were blocked and incubated overnight at 4°C with the
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following primary antibodies: FGFR1 (#9740; Cell Signaling Technology, Danvers, MA, USA)
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and β-actin (#4967; Cell Signaling Technology, Danvers, MA, USA). After being washed, the membranes were reacted with horseradish peroxidase conjugated secondary antibodies.
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Antibody–antigen complexes were visualized with enhanced chemiluminescence Western
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blotting detection reagents (Invitrogen, Carlsbad, CA, USA).
2.5. Lipid detection
For analysis of hepatic triglycerides, total hepatic lipids were extracted from frozen tissue. Total TG content was determined by enzymatic assays (TR0100, Sigma-Aldrich Corp., St. Louis, MO, USA) and normalized to protein concentration. The intracellular lipid content in HepG2 cells was evaluated by Oil Red O staining. Briefly, cells were washed 3 times with PBS, fixed for 30 minutes with 4 % paraformaldehyde in PBS, stained with Oil-Red O for 1 hour at room 9
ACCEPTED MANUSCRIPT temperature, and then rinsed with distilled water. Finally, cell images were captured under a microscope (magnification, x400). For quantitative analysis of lipid accumulation, 1 mL of
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isopropanol was added to the stained culture plate, and the absorbance of extracted dye was
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measured at 540 nm.
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2.6. Statistical analysis
All statistical analyses were performed using PASW Statistics 17 (SPSS Inc., Chicago, IL,
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USA). The data are presented as the mean ± standard error. For the evaluation of the significant differences between the mean values in the experimental groups, the one-way analysis of
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variance (ANOVA) test was used. Multiple comparisons between the experimental groups were
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adjusted with the Bonferroni correction. Null hypotheses that showed no differences between the
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groups were rejected if the p-values were less than 0.05.
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ACCEPTED MANUSCRIPT 3. Results
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3.1. Effects of exendin-4 on the expression of FGF21 and its receptors
To investigate whether exendin-4 regulates FGF21 and its receptors in the liver tissue of mice,
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the expression levels of hepatic FGF21 and its receptors such as FGFR1, 2, and 4, and co-
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receptor β-klotho were measured by real-time PCR and Western blot. In the exendin-4-treated group, the expression levels of FGF21, FGFR1, FGFR2, FGFR4, and β-klotho mRNA and
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FGFR1 protein were significantly increased compared with those in the HF group (Fig. 1A). Regulation of FGF21 by exendin-4 in liver tissues was consistently shown in HepG2 hepatocytes.
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Exendin-4 increased the expression of FGF21 mRNA in a dose-dependent manner, and markedly
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increased the expression of FGFR1 mRNA and protein in hepatocytes (Fig. 1B). Therefore, these
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results suggest that exendin-4 regulates the expression of FGF21 and FGFRs in hepatocytes.
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3.2. Relationship between FGF21 and hepatic lipid accumulation
The body weight gain, liver weight and hepatic TG of the HF group were higher than those of the control group; those of the exendin-4-treated group were significantly lower compared with those of the HF group (Table 1). No differences in food intake were observed between the exendin-4-treated group and HF group (data not shown). We investigated whether FGF21 as well as exendin-4 were associated with inhibition of hepatic lipid accumulation by applying in vitro models. Palmitic acid (PA) was treated in HepG2 cells for 24 hours to induce lipid accumulation. Exendin-4 and recombinant hepatic FGF21 were then added to cells pretreated with or without 11
ACCEPTED MANUSCRIPT PA for 24 hours, and intracellular lipids were detected by Oil red O staining (Fig. 2A). As shown in Fig. 2B, PA induced an increase of intracellular lipid content, and exendin-4 and FGF21
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significantly decreased PA-increased lipid accumulation. In addition, FGF21 inhibition by siRNA
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induced a decrease in the expression levels of PPARα and medium-chain acyl-coenzyme A dehydrogenase (MCAD) mRNA, which are key regulators of fatty acid oxidation, in HepG2 cells
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treated with exendin-4 (Fig. 2C). Therefore, these data suggest that increased expression of
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hepatic FGF21 may be associated with the protective effects of exendin-4 on hepatic lipid accumulation. In addition, to investigate whether exendin-4 regulates an autocrine effect of
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FGF21 via FGFR1, HepG2 cells were transfected with siRNA for FGFR1. FGFR1 inhibition induced decreased expression levels of FGF21 and PPARα in cells treated with exendin-4 (Fig.
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2D).
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3.3. Regulation of FGF21 by SIRT1 in HepG2 cells
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In the previous study, we found that SIRT1 is regulated by exendin-4 in hepatocytes [13]. Moreover it was observed significant increase of SIRT6 expression by exendin-4 in the liver of high fat diet-fed mice (Supplementary Fig. 1). Therefore, we investigated the relationship between FGF21 and sirtuin in hepatocytes treated with exendin-4. The expression levels of SIRT1, SIRT6, and FGF21 mRNA were monitored in HepG2 cells treated with exendin-4, after pretransfection with siRNA targeting SIRT1, SIRT6, and FGF21 for 24 hours. Inhibition of SIRT1, but not SIRT6, decreased the expression of exendin-4-mediated FGF21 mRNA (Fig. 3AB). Conversely, we examined whether the expression of hepatic SIRT1 or SIRT6 could be affected by FGF21. No change in the expression of SIRT1 and SIRT6 was observed in FGF21 12
ACCEPTED MANUSCRIPT siRNA–transfected cells (Fig. 3C). These data suggest that the increase of FGF21 by exendin-4 is
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mediated via SIRT1 in hepatocytes.
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ACCEPTED MANUSCRIPT 4. Discussion
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In this study, hepatic FGF21 and it receptors were increased by pharmacological dose of
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exendin-4 in diet-induced obese mice. In addition, recombinant FGF21 treatment resulted in improved lipid accumulation in HepG2 cells treated with PA due to increased fatty acid oxidation.
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Furthermore, our findings reveal that the expression of FGF21 is positively regulated by
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increasing SIRT1, but not SIRT6, which has beneficial effects on lipid and glucose homeostasis in the liver.
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Disruption of energy balance causes various liver diseases such as non-alcoholic fatty liver disease (NAFLD) which is strongly associated with obesity and type 2 diabetes [16, 17]. In this
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study, we presented in vivo and in vitro models of hepatic steatosis induced by high fat diet and
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PA, respectively, and this was confirmed through a comparison of lipid contents. The liver plays a central role in lipid and glucose homeostasis and is a major organ for the
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production of secretory proteins. Similar to adipokines, hepatokines act as a biomarker in insulin
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resistance and type 2 diabetes [18, 19]. Hepatokine FGF21 is a novel therapeutic agent known to attenuate obesity and diabetes [20, 21]. In the present study, we found that pharmacological treatment of exendin-4 increased the expression of hepatic FGF21, FGFR1, FGFR2, FGFR4 and β-Klotho mRNA and FGFR1 protein in mice as well as FGF21 mRNA and FGFR1 mRNA and protein in vitro models. The affinity between FGF21 and its receptors appear different in each tissue, and upon the presence of the co-receptor β-Klotho, FGF21 binds to FGFR1 with higher affinity compared to others [22, 23]. Interestingly, during inhibition of FGFR1 by siRNA, an increase of FGF21 and PPARα expression by exendin-4 was not shown (Fig. 2D), suggesting that an autocrine effect of FGF21 stimulated by exendin-4 treatment in hepatocytes is mediated by 14
ACCEPTED MANUSCRIPT FGFR1. We confirmed that recombinant FGF21 in hepatocytes increases the expression of FGF21 mRNA in a dose-dependent manner (data not shown) and decreases TG accumulation
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(Fig. 2A-B). Previous studies reported that liver-derived FGF21 may act in an endocrine or
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antucrine/ paracrine manner to regulate ketogenesis, gluconeogenesis and fatty acid oxidation [24], and that FGF signaling is inhibited with the FGFR inhibitor [25]. However, we consider that
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more detailed further research is needed to clarify the possible mechanism for the down-
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regulation of the expression of FGF21 and PPARα mRNA by inhibition of FGFR1. FGF21 regulates fatty acid metabolism through induction of PPAR γ coactivator protein-1α
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(PGC-1α) in the liver [26]. Also, the expression of FGF21 was increased in primary hepatocytes treated with metformin which is known to have a protective effect against NAFLD and insulin
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resistance through activation of AMPK [27]. In accordance with this finding, we observed that
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inhibition of FGF21 in HepG2 cells treated with exendin-4 induces decreased expression of PPARα and MCAD, which are regulators of fatty acid β-oxidation. Thus, these results suggest
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that the decrease in body weight gain and hepatic TG levels in exendin-4 treated mice may be due
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to increased FGF21-mediated fatty acid oxidation. Meanwhile, modulation of FGF21 by exendin-4 has been previously reported. Samson et al. [28] found that exendin-4 decreases serum- and hepatic FGF21 levels in NAFLD in a diet-induced obese mouse model, which is different from our present results. Although, FGF21 acts importantly on improvement of insulin sensitivity, glucose tolerance and hepatic steatosis, they reported that since metabolic syndromes such as obesity and type 2 diabetes are state of FGF21 resistance, plasma FGF21 levels increased under these disorders and FGF21 resistance in the liver was improved by exendin-4 treatment. We believe that these conflicting results may be attributable to the differences in the fat content of the diet, treatment period and concentration of 15
ACCEPTED MANUSCRIPT exendin-4, and in the age and number of animal models. In our study, the duration of exendin-4 administration was much longer than that in the previous report, and the age of the animal model
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used in this study was younger. Further studies are needed to assess this point.
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In addition, upon metabolic dysfunction, expression patterns of FGF21 are controversial. Zhang X et al. [29] reported that serum FGF21 levels are increased in individuals with metabolic
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syndrome. They suggested that their models of metabolic syndromes are in FGF21 resistant state-
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[28, 30, 31], whereas, in clinical and animal study, multiple studies reported no correlation between FGF21 and weight loss [32], metabolic syndrome, NALFD [33] and adiponectin, which
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is a biomarker of the metabolic syndrome [34]. In addition, glucagon-like peptide (GLP)-1 analogue liraglutide leads to an improvement of FGF21 and insulin resistance induced by a
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combination of HFD, ApoE deficiency and adiponectin knockdown via increasing FGF21
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activity [35] and FGF21 treatment in skeletal muscle cells treated with palmitic acid and in MSGIR mice improved insulin resistance [36, 37]. Therefore, to clarify the effect of exendin-4 on the
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regulation of hepatic FGF21, additional studies on diet-induced obesity conditions in human and
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animal models should be undertaken. SIRT1 and SIRT6, members of the sirtuin family, are NAD+-dependent deacetylases that act in the modification of lipid and glucose metabolism in the liver [14, 38-40]. In our previous study, we demonstrated that exendin-4 increases the expression of SIRT1 in the liver of mice [13], and that the protein kinase A (PKA) pathway which mediates the GLP-1-dependent action activates SIRT1 to maintain energy homeostasis [41]. Knockdown of hepatic SIRT1 and SIRT6 leads to insulin resistance and fatty liver [14, 42, 43], whereas resveratrol, a possible activator of SIRT1, improves insulin resistance, hyperglycemia and hepatic steatosis [44]. In this study, the expression of SIRT1 and FGF21 was increased by exendin-4. In addition, SIRT1 inhibition 16
ACCEPTED MANUSCRIPT decreased the expression of FGF21 in cells treated with exendin-4, whereas the expression of SIRT1 was not affected by knockdown of FGF21. Thus, these data suggest that the effect of
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exendin-4 on the regulation of hepatic FGF21 may be mediated by SIRT1, indicating that SIRT1
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is an upstream of FGF21 in hepatocytes.
In other words, the strength of this study is the observation that the expression of FGF21 and
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PPAR-alpha, which are associated with fatty acid catabolism, was down-regulated by FGFR1
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inhibition, and that SIRT1 was identified as an upstream regulator of FGF21. On the other hand, there are a few limitations in our study. First, in addition to SIRT1, a variety of factors may
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contribute to the regulation of FGF21. Since this study was only investigated using specific siRNA in vitro to clarify the relationship between SRIT1 and FGF21, we have not identified the
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effects of other regulators in the regulation of FGF21. Second, after inhibition of FGFR1 by
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siRNA transfection, change in interaction between FGF21 and FGFR1 was not directly tested. Since a decrease of FGFR1 expression was observed, we expected that the binding between two
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proteins may be reduced. These are needed to confirm through further experiments.
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In conclusion, the present study demonstrated that exendin-4 increases FGF21-mediated fatty acid oxidation by increasing SIRT1 in hepatocytes (Fig. 4). Therefore, the protective effect of exendin-4 in an in vivo hepatic steatosis model may also be mediated through the SIRT1-FGF21 pathway, and hepatic FGF21 may serve as a therapeutic target for the treatment of fatty liver disease associated with metabolic diseases, such as obesity and type 2 diabetes mellitus.
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ACCEPTED MANUSCRIPT Author contributions
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Conceived and designed the experiments: JL SWH WYL. Performed the experiments: JL SWH.
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Analyzed the data: JL SWH SEP EJR CYP KWO SWP WYL. Contributed
reagents/materials/analysis tools: JL SWH SEP EJR CYP KWO SWP WYL. Wrote the paper: JL
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EJR WYL.
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ACCEPTED MANUSCRIPT References
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1 Kharitonenkov A. FGFs and metabolism. Current opinion in pharmacology 2009;9(6): 805-10.
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2 Kurosu H, Kuro-o M. Endocrine fibroblast growth factors as regulators of metabolic homeostasis. Biofactors 2009;35(1): 52-60.
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3 Lundåsen T, Hunt MC, Nilsson LM, et al. Pparα is a key regulator of hepatic FGF21. Biochem
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Biophys Res Commun 2007;360(2): 437-40.
4 Kim H, Mendez R, Zheng Z, et al. Liver-enriched transcription factor crebh interacts with
Endocrinology 2014;155(3): 769-82.
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peroxisome proliferator-activated receptor alpha to regulate metabolic hormone FGF21.
ED
5 Cuevas-Ramos D, Almeda-Valdes P, Aguilar-Salinas CA, et al. The role of fibroblast growth
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factor 21 (FGF21) on energy balance, glucose and lipid metabolism. Curr Diabetes Rev 2009;5(4): 216-20.
CE
6 Kharitonenkov A, Shiyanova TL, Koester A, et al. FGF-21 as a novel metabolic regulator. J
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Clin Invest 2005;115(6): 1627-35. 7 Wang C, Dai J, Yang M, et al. Silencing of FGF-21 expression promotes hepatic gluconeogenesis and glycogenolysis by regulation of stat3- socs3 signal. FEBS J 2014; doi: 10.1111/febs.12767. 8 Xu J, Lloyd DJ, Hale C, et al. Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 2009;58(1): 250-9. 9 Vassilopoulos A, Fritz KS, Petersen DR, et al. The human sirtuin family: Evolutionary divergences and functions. Hum Genomics 2011;5(5): 485-96. 19
ACCEPTED MANUSCRIPT 10 Michan S, Sinclair D. Sirtuins in mammals: Insights into their biological function. The Biochem J 2007;404(1): 1-13.
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11 Purushotham A, Schug TT, Xu Q, et al. Hepatocyte-specific deletion of SIRT1 alters fatty acid
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metabolism and results in hepatic steatosis and inflammation. Cell Metab 2009;9(4): 327-38. 12 Caron AZ, He X, Mottawea W, et al. The SIRT1 deacetylase protects mice against the
SC
symptoms of metabolic syndrome. FASEB J 2014;28(3): 1306-16.
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13 Lee J, Hong SW, Chae SW, et al. Exendin-4 improves steatohepatitis by increasing sirt1 expression in high-fat diet-induced obese C57BL/6J mice. PloS one 2012;7(2): e31394.
MA
14 Kim HS, Xiao C, Wang RH, et al. Hepatic-specific disruption of sirt6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis. Cell Metab 2010;12(3):
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224-36.
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15 Yang SJ, Choi JM, Chae SW, et al. Activation of peroxisome proliferator-activated receptor gamma by rosiglitazone increases sirt6 expression and ameliorates hepatic steatosis in rats. PloS
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one 2011;6(2): e17057.
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16 Van Rooyen DM, Larter CZ, Haigh WG, et al. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis. Gastroenterology 2011;141(4): 1393-403. 17 Leite NC, Salles GF, Araujo ALE, et al. Prevalence and associated factors of non‐alcoholic fatty liver disease in patients with type‐2 diabetes mellitus. Liver Int 2008;29(1): 113-9. 18 Misu H, Ishikura K, Kurita S, et al. Inverse correlation between serum levels of selenoprotein p and adiponectin in patients with type 2 diabetes. PloS one 2012;7(4): e34952. 19 Misu H, Takamura T, Takayama H, et al. A liver-derived secretory protein, selenoprotein p, causes insulin resistance. Cell Metab 2010;12(5): 483-95. 20 Zhao Y, Dunbar JD, Kharitonenkov A. FGF21 as a therapeutic reagent. Adv Exp Med Biol 20
ACCEPTED MANUSCRIPT 2012: 214-28. 21 Camporez JP, Jornayvaz FR, Petersen MC, et al. Cellular mechanisms by which FGF21
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improves insulin sensitivity in male mice. Endocrinology 2013;154(9): 3099-109.
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22 Suzuki M, Uehara Y, Motomura-Matsuzaka K, et al. Βklotho is required for fibroblast growth factor (FGF) 21 signaling through FGF receptor (FGFR) 1c and FGFR3c. Mol Endocrinol
SC
2008;22(4): 1006-14.
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23 Kurosu H, Choi M, Ogawa Y, et al. Tissue-specific expression of βklotho and fibroblast
Biol Chem 2007;282(37): 26687-95.
MA
growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21. J
24 Ge X, Wang Y, Lam KS, et al. Metabolic actions of FGF21: Molecular mechanisms and
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therapeutic implications. Acta Pharm Sin B 2012;2(4): 350-7.
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25 Jurynec MJ, Grunwald DJ. SHIP2, a factor associated with diet-induced obesity and insulin sensitivity, attenuates FGF signaling in vivo. Dis Model Mech 2010;3(11-12): 733-42.
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26 Potthoff MJ, Inagaki T, Satapati S, et al. FGF21 induces PGC-1alpha and regulates
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carbohydrate and fatty acid metabolism during the adaptive starvation response. Proc Natl Acad Sci U S A 2009;106(26): 10853-8. 27 Nygaard EB, Vienberg SG, Ørskov C, et al. Metformin stimulates FGF21 expression in primary hepatocytes. Exp Diabetes Res 2012;2012: 465282. 28 Samson S, Sathyanarayana P, Jogi M, et al. Exenatide decreases hepatic fibroblast growth factor 21 resistance in non-alcoholic fatty liver disease in a mouse model of obesity and in a randomised controlled trial. Diabetologia 2011;54(12): 3093-100. 29 Zhang X, Yeung DCY, Karpisek M, et al. Serum FGF21 levels are increased in obesity and are independently associated with the metabolic syndrome in humans. Diabetes 2008;57(5): 1246-53. 21
ACCEPTED MANUSCRIPT 30 Chui PC, Antonellis PJ, Bina HA, et al. Obesity is a fibroblast growth factor 21 (FGF21)resistant state. Diabetes 2010;59(11): 2781-9.
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31 Dushay J, Chui PC, Gopalakrishnan GS, et al. Increased fibroblast growth factor 21 in obesity
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and nonalcoholic fatty liver disease. Gastroenterology 2010;139(2): 456-63. 32 Mai K, Schwarz F, Bobbert T, et al. Relation between fibroblast growth factor-21, adiposity,
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metabolism, and weight reduction. Metabolism 2011;60(2): 306-11.
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33 Reinehr T, Woelfle J, Wunsch R, et al. Fibroblast growth factor 21 (FGF-21) and its relation to obesity, metabolic syndrome, and nonalcoholic fatty liver in children: A longitudinal analysis. J
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Clin Endocrinol Metab 2012;97(6): 2143-50.
34 Wang ZQ, Zhang XH, Yu Y, et al. Artemisia scoparia extract attenuates non-alcoholic fatty
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liver disease in diet-induced obesity mice by enhancing hepatic insulin and ampk signaling
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independently of FGF21 pathway. Metabolism 2013;62(9): 1239-49. 35 Yang M, Zhang L, Wang C, et al. Liraglutide increases FGF-21 activity and insulin sensitivity
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e48392.
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in high fat diet and adiponectin knockdown induced insulin resistance. PloS one 2012;7(11):
36 Lee MS, Choi SE, Ha ES, et al. Fibroblast growth factor-21 protects human skeletal muscle myotubes from palmitate-induced insulin resistance by inhibiting stress kinase and NF-kB. Metabolism 2012;61(8): 1142-51. 37 Zhu SL, Zhang ZY, Ren GP, et al. Therapeutic effect of fibroblast growth factor 21 on NAFLD in MSG-iR mice and its mechanism. Yao Xue Xue Bao 2013;48(12): 1778-84. 38 Hou X, Xu S, Maitland-Toolan KA, et al. Sirt1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase. J Biol Chem 2008;283(29): 20015-26. 39 Caton PW, Nayuni NK, Kieswich J, et al. Metformin suppresses hepatic gluconeogenesis 22
ACCEPTED MANUSCRIPT through induction of SIRT1 and GCN5. J Endocrinol 2010;205(1): 97-106. 40 Dominy Jr JE, Lee Y, Jedrychowski MP, et al. The deacetylase Sirt6 activates the
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acetyltransferase GCN5 and suppresses hepatic gluconeogenesis. Mol Cell 2012;48(6): 900-13.
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41 Gerhart-Hines Z, Dominy JE, Jr., Blattler SM, et al. The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD(+). Mol Cell
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2011;44(6): 851-63.
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42 Rodgers JT, Lerin C, Haas W, et al. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature 2005;434(7029): 113-8.
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43 Wang RH, Kim HS, Xiao C, et al. Hepatic sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance. J Clin Invest
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2011;121(11): 4477-90.
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44 Labbé A, Garand C, Cogger VC, et al. Resveratrol improves insulin resistance hyperglycemia and hepatosteatosis but not hypertriglyceridemia, inflammation, and life span in a mouse model
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for werner syndrome. J Gerontol A Biol Sci Med Sci 2011;66(3): 264-78.
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ACCEPTED MANUSCRIPT Tables
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Table 1. Body weight gain, liver weight and hepatic triglycerides in mice.
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Figure legends
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Fig. 1. Exendin-4 increases the expression of FGF21 and its receptors in vivo and in vitro. (A) Mice were fed a low fat diet (control), high fat diet (HF), or HF diet with 1 nmol/kg/day
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exendin-4 ip injection for 10 weeks. Total RNA and protein were extracted from liver tissue, and FGF21, FGFR1, 2, 4, and β-klotho mRNA and FGFR1 protein expression levels were measured
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using quantitative real-time RT-PCR and Western blot. * p
S0026-0495(14)00135-8 doi: 10.1016/j.metabol.2014.04.011 YMETA 53017
To appear in:
Metabolism
Received date: Revised date: Accepted date:
13 January 2014 17 April 2014 29 April 2014
Please cite this article as: Lee Jinmi, Hong Seok-Woo, Park Se Eun, Rhee Eun-Jung, Park Cheol-Young, Oh Ki-Won, Park Sung-Woo, Lee Won-Young, Exendin-4 regulates lipid metabolism and fibroblast growth factor 21 in hepatic steastosis, Metabolism (2014), doi: 10.1016/j.metabol.2014.04.011
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ACCEPTED MANUSCRIPT Exendin-4 regulates lipid metabolism and fibroblast growth factor 21 in hepatic steastosis
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Jinmi Leea, Seok-Woo Honga, Se Eun Parkb, Eun-Jung Rheeb, Cheol-Young Parkb, Ki-Won Ohb,
Institute of Medical Research, Kangbuk Samsung Hospital, Sungkyunkwan University School of
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a
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Sung-Woo Parkb, Won-Young Leeb
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Medicine, Seoul 110-746, Republic of Korea
Department of Endocrinology and Metabolism, Kangbuk Samsung Hospital, Sungkyunkwan
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Correspondence:
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University School of Medicine, Seoul 110-746, Republic of Korea
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Won-Young Lee, MD, PhD
Division of Endocrinology and Metabolism Department of Internal Medicine, Kangbuk Samsung Hospital Sungkyunkwan University School of Medicine # 108 Pyung-Dong, Jongro-Ku, Seoul 110-746, Republic of Korea Tel: 82-2-2001-2579 Fax: 82-2-2001-2049 E-mail: [email protected]
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ACCEPTED MANUSCRIPT Word count of text: 4970, abstract: 265, number of references: 44, and number of tables: 1
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and figures: 4
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Funding
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This study was supported by a grant from Daewoong Pharmaceutical (# MRI-110907-004/5)
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and by the National research Foundation (NRF) grant funded by the Korea government (NRF2013R1A1A2063069). The funders had no role in the study design, data collection and analysis,
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decision to publish, or preparation of the manuscript.
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Conflicts of interest
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All authors declare that there are no conflicts of interest.
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ACCEPTED MANUSCRIPT Abstract
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OBJECTIVE: Hepatokine fibroblast growth factor (FGF) 21 takes part in the regulation of
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lipid metabolism in the liver and adipose tissue. We investigated whether exendin-4 regulates the expression of FGF21 in the liver, and whether the effects of exendin-4 on the regulation of
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FGF21 expression are mediated via silent mating type information regulation 2 homolog (SIRT)1
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or SIRT6.
MATERALS/METHODS: The C57BL/6J mice were fed a low fat diet, high fat diet, or high
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fat diet with 1 nmol/kg/day exendin-4 intraperitoneal injection for 10 weeks. HepG2 used in vitro study was treated with palmitic aicd (0.4 mM) with or without exendin-4 (100 nM) and FGF21
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(50 nM) for 24 hours. The change of FGF21 and its receptors expression by exendin-4 were
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measured using quantitative real-time RT-PCR and Western blot. The intracellular lipid content in HepG2 cells was evaluated by Oil Red O staining. Inhibition of FGF21, SIRT1 and SIRT6, by
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lipid metabolism.
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10 nM siRNA was performed to establish the signaling pathway of exendin-4 action in hepatic
RESULTS: Exendin-4 increased the expression of FGF21 and its receptors in high fat dietinduced obese mice. In addition, recombinant FGF21 treatment reduced lipid content in palmitic acid-treated HepG2 cells. We also observed significantly decreased expression of peroxisomal proliferator-activated receptor (PPAR) α and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in hepatocytes transfected with FGF21 siRNA. In cells treated with exendin-4, inhibition of SIRT1, but not SIRT6, by siRNA significantly repressed the expression of FGF21 mRNA, whereas decreased SIRT1 expression by inhibition of FGF21 was not observed.
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ACCEPTED MANUSCRIPT CONCLUSIONS: These data suggest that exendin-4 could improve fatty liver by increasing
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Keywords: Exendin-4; SIRT1; FGF21; FGFRs; fatty acid oxidation
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SIRT1-mediated FGF21.
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Abbreviation: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; SIRT1,
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silent mating type information regulation 2 homolog 1; NAD, nicotinamide adenine dinucleotide; HF, high-fat; PA, palmitic acid; Ex-4, exendin-4; GLP-1, glucagon-like peptide; NAFLD, non-
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alcoholic fatty liver disease; TG, triglycerides; PPARα, peroxisomal proliferator-activated receptor α; MCAD, medium-chain acyl-coenzyme A dehydrogenase; GLUT1, glucose
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transporter 1; AMPK, AMP-activated protein kinase; PGC-1α, PPAR γ coactivator protein-1α;
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PKA, protein kinase A.
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ACCEPTED MANUSCRIPT 1. Introduction
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The fibroblast growth factor (FGF) family consists of 22 members that have important roles in
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several cell types to regulate diverse physiologic functions. In particular, some members of the FGF19-family, including FGF19, FGF21, and FGF23, function as regulatory hormones of
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glucose, lipid, bile acid, phosphate, and vitamin D metabolism [1, 2]. Moreover, hepatokine
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FGF21 was found as a novel therapeutic agent for diabetes. Hepatic FGF21 expression is regulated by cyclic-AMP-responsive-element-binding protein H (CREBH) and peroxisomal
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proliferator-activated receptor (PPAR) α, and regulates lipid homeostasis [3, 4]. FGF21 is secreted from the liver, functions as a potent activator of glucose uptake in adipocytes via glucose
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transporter (GLUT)-1 [5]. In both ob/ob and db/db mice, administration of FGF21 was shown to
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decrease plasma glucose and triglycerides (TG) [6], whereas liver-specific knockdown of FGF21 led to hepatic insulin resistance by increase of gluconeogenesis and glycogenolysis [7]. In
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mice [8].
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addition, FGF21 treatment improved hepatic steatosis and insulin resistance in diet-induced obese
The silent mating type information regulation 2 homolog (sirtuin, SIRT) family is nicotinamide adenine dinucleotide (NAD)+-dependent enzymes that modify multiple target proteins by deacetylation. Until now, seven members of the sirtuin family have been identified in humans, [9, 10]. In particular, SIRT1 and SIRT6 of seven sirtuins are crucial in metabolic homeostasis. SIRT1 regulates hepatic fatty acid oxidation through positive regulation of AMPactivated protein kinase (AMPK) and PPARα, which are involved in fatty acid β-oxidation in the liver and skeletal muscle [11]. In contrast, the SIRT1 deacetylase-deficient mice on a high-fat diet have excess hepatic lipid accumulation and insulin resistance [12]. In our previous study, we 5
ACCEPTED MANUSCRIPT observed that increased SIRT1 by exendin-4 protected mice against diet-induced obesity and steatohepatitis by increased fatty acid oxidation [13]. Kim et al. [14] reported that SIRT6
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expression is positively regulated by SIRT1 in the liver, and liver-specific knockout of SIRT6
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causes fatty liver via an increase of glycolysis and reduction of β-oxidation. Also, increased SIRT6 expression was shown in rosiglitazone-mediated amelioration of hepatic steatosis [15]. In
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this study, we investigated whether exendin-4 regulates the expression of FGF21 in hepatocytes,
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and whether this mechanism is mediated via SIRT1 or SIRT6.
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ACCEPTED MANUSCRIPT 2. Materials and methods
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2.1. Animals
Male C57BL/6J mice of 6 weeks of age were obtained from Central Laboratory (Shizuoka
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Laboratory Animal Center, Shizuoka, Japan) and bred in standard conditions under a 12 hour
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light/dark cycle. All animal experiments were conducted in accordance with the Ethics Committee for Animal Experiments of the Sungkyunkwan University Kangbuk Samsung
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Hospital. Mice were randomly assigned to a low-fat diet (control, 10 kcal % fat consist of 5.6 kcal % lard and 4.4 kcal % soy bean oil, 20 kcal % protein, and 70 kcal % carbohydrate), high-fat
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(HF) diet (HF, 45 kcal % fat consist of 39.4 kcal % lard and 4.4 kcal % soy bean oil, 20 kcal %
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protein, and 35 kcal % carbohydrate), or a HF diet plus 1 nmol/kg/day exendin-4 (Sigma-Aldrich Corp., St. Louis, MO, USA) via intraperitoneal (IP) injection. During the 10-week treatment
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period, mice of each group were injected every other day with exendin-4 or saline (control) and
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allowed to access to their specific diet and water ad libitum. Body weight was checked twice per week. At the end of treatment, animals were sacrificed; liver tissues were collected and stored at 80 °C until required for this study.
2.2. Cell culture and transfection
A hepatocellular carcinoma cell line, HepG2, was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and was grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum (Gibco, 7
ACCEPTED MANUSCRIPT Grand Island, NY, USA), 1 % penicillin/streptomycin (Gibco, Grand Island, NY, USA) at 37 °C in 5 % CO2. To examine the effects of exendin-4 and FGF21 on hepatic steatosis, HepG2 cells
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were treated with palmitic acid (PA) (0.4 mM) with or without Ex-4 (100 nM) and FGF21 (50
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nM) (Sigma-Aldrich Corp., St. Louis, MO, USA) for 24 hours. Specific siRNAs of SIRT1 [1137490], SIRT6 [1137529], FGF21 [1052655], FGFR1 [100382] (Bioneer, Daejeon, Korea),
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and negative control (Invitrogen, Carlsbad, CA, USA) were purchased and transiently transfected
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into HepG2 cells using Lipofectamine RNAiMAX reagent, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). After incubation for 24 hours, cells were harvested
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to assay for gene knockdown.
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2.3. Total RNA isolation and real-time RT-PCR
Total RNA was isolated from the liver tissue and cultured hepatocytes using Trizol reagent
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(Invitrogen, Carlsbad, CA, USA). Complementary DNA was generated from 2 μg total RNA
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using moloney murine leukemia virus reverse transcriptase (MMLV-RT) and oligo (dT)12-18 primer (Invitrogen, Carlsbad, CA, USA) as described previously [13]. Analysis of mRNA was conducted by real-time PCR (Light-Cycler 480; Roche, Lewis, UK) using SYBR green (Roche, Lewis, UK) and specific primers (Bioneer, Daejeon, Korea) (Supplementary Table 1.) according to the manufacturer’s instructions. After pre-incubation at 95 °C for 10 minutes, thermal cycling conditions consisted of 40 cycles of 94 °C for 15 seconds, 55 °C for 10 seconds, and 72 °C for 20 seconds. Data were normalized using β-actin as an internal control and calculated using the comparative Ct method (2-delta delta Ct).
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ACCEPTED MANUSCRIPT 2.4. Western blot analysis
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Cultured hepatocytes were homogenized at 4 °C in RIPA buffer (Cell Signaling Technology,
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Danvers, MA, USA) supplemented with a cocktail of protease and phosphatase inhibitors, and protein was obtained by centrifugation at 13,000 rpm for 20 minutes at 4 °C. The concentration
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of the sample was determined using the Bradford protein assay (Bio-Rad Protein Assay, BioRad,
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Hercules, CA) with bovine serum albumin standard (Thermo Scientific, Rockford, IL, USA). After the assay, 20 μg of each sample was separated on 4-12 % bis-Tris Nupage gels (Invitrogen,
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Carlsbad, CA, USA) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Chalfont St. Giles, UK). The membranes were blocked and incubated overnight at 4°C with the
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following primary antibodies: FGFR1 (#9740; Cell Signaling Technology, Danvers, MA, USA)
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and β-actin (#4967; Cell Signaling Technology, Danvers, MA, USA). After being washed, the membranes were reacted with horseradish peroxidase conjugated secondary antibodies.
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Antibody–antigen complexes were visualized with enhanced chemiluminescence Western
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blotting detection reagents (Invitrogen, Carlsbad, CA, USA).
2.5. Lipid detection
For analysis of hepatic triglycerides, total hepatic lipids were extracted from frozen tissue. Total TG content was determined by enzymatic assays (TR0100, Sigma-Aldrich Corp., St. Louis, MO, USA) and normalized to protein concentration. The intracellular lipid content in HepG2 cells was evaluated by Oil Red O staining. Briefly, cells were washed 3 times with PBS, fixed for 30 minutes with 4 % paraformaldehyde in PBS, stained with Oil-Red O for 1 hour at room 9
ACCEPTED MANUSCRIPT temperature, and then rinsed with distilled water. Finally, cell images were captured under a microscope (magnification, x400). For quantitative analysis of lipid accumulation, 1 mL of
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isopropanol was added to the stained culture plate, and the absorbance of extracted dye was
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measured at 540 nm.
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2.6. Statistical analysis
All statistical analyses were performed using PASW Statistics 17 (SPSS Inc., Chicago, IL,
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USA). The data are presented as the mean ± standard error. For the evaluation of the significant differences between the mean values in the experimental groups, the one-way analysis of
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variance (ANOVA) test was used. Multiple comparisons between the experimental groups were
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adjusted with the Bonferroni correction. Null hypotheses that showed no differences between the
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groups were rejected if the p-values were less than 0.05.
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ACCEPTED MANUSCRIPT 3. Results
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3.1. Effects of exendin-4 on the expression of FGF21 and its receptors
To investigate whether exendin-4 regulates FGF21 and its receptors in the liver tissue of mice,
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the expression levels of hepatic FGF21 and its receptors such as FGFR1, 2, and 4, and co-
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receptor β-klotho were measured by real-time PCR and Western blot. In the exendin-4-treated group, the expression levels of FGF21, FGFR1, FGFR2, FGFR4, and β-klotho mRNA and
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FGFR1 protein were significantly increased compared with those in the HF group (Fig. 1A). Regulation of FGF21 by exendin-4 in liver tissues was consistently shown in HepG2 hepatocytes.
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Exendin-4 increased the expression of FGF21 mRNA in a dose-dependent manner, and markedly
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increased the expression of FGFR1 mRNA and protein in hepatocytes (Fig. 1B). Therefore, these
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results suggest that exendin-4 regulates the expression of FGF21 and FGFRs in hepatocytes.
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3.2. Relationship between FGF21 and hepatic lipid accumulation
The body weight gain, liver weight and hepatic TG of the HF group were higher than those of the control group; those of the exendin-4-treated group were significantly lower compared with those of the HF group (Table 1). No differences in food intake were observed between the exendin-4-treated group and HF group (data not shown). We investigated whether FGF21 as well as exendin-4 were associated with inhibition of hepatic lipid accumulation by applying in vitro models. Palmitic acid (PA) was treated in HepG2 cells for 24 hours to induce lipid accumulation. Exendin-4 and recombinant hepatic FGF21 were then added to cells pretreated with or without 11
ACCEPTED MANUSCRIPT PA for 24 hours, and intracellular lipids were detected by Oil red O staining (Fig. 2A). As shown in Fig. 2B, PA induced an increase of intracellular lipid content, and exendin-4 and FGF21
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significantly decreased PA-increased lipid accumulation. In addition, FGF21 inhibition by siRNA
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induced a decrease in the expression levels of PPARα and medium-chain acyl-coenzyme A dehydrogenase (MCAD) mRNA, which are key regulators of fatty acid oxidation, in HepG2 cells
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treated with exendin-4 (Fig. 2C). Therefore, these data suggest that increased expression of
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hepatic FGF21 may be associated with the protective effects of exendin-4 on hepatic lipid accumulation. In addition, to investigate whether exendin-4 regulates an autocrine effect of
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FGF21 via FGFR1, HepG2 cells were transfected with siRNA for FGFR1. FGFR1 inhibition induced decreased expression levels of FGF21 and PPARα in cells treated with exendin-4 (Fig.
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2D).
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3.3. Regulation of FGF21 by SIRT1 in HepG2 cells
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In the previous study, we found that SIRT1 is regulated by exendin-4 in hepatocytes [13]. Moreover it was observed significant increase of SIRT6 expression by exendin-4 in the liver of high fat diet-fed mice (Supplementary Fig. 1). Therefore, we investigated the relationship between FGF21 and sirtuin in hepatocytes treated with exendin-4. The expression levels of SIRT1, SIRT6, and FGF21 mRNA were monitored in HepG2 cells treated with exendin-4, after pretransfection with siRNA targeting SIRT1, SIRT6, and FGF21 for 24 hours. Inhibition of SIRT1, but not SIRT6, decreased the expression of exendin-4-mediated FGF21 mRNA (Fig. 3AB). Conversely, we examined whether the expression of hepatic SIRT1 or SIRT6 could be affected by FGF21. No change in the expression of SIRT1 and SIRT6 was observed in FGF21 12
ACCEPTED MANUSCRIPT siRNA–transfected cells (Fig. 3C). These data suggest that the increase of FGF21 by exendin-4 is
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mediated via SIRT1 in hepatocytes.
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ACCEPTED MANUSCRIPT 4. Discussion
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In this study, hepatic FGF21 and it receptors were increased by pharmacological dose of
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exendin-4 in diet-induced obese mice. In addition, recombinant FGF21 treatment resulted in improved lipid accumulation in HepG2 cells treated with PA due to increased fatty acid oxidation.
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Furthermore, our findings reveal that the expression of FGF21 is positively regulated by
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increasing SIRT1, but not SIRT6, which has beneficial effects on lipid and glucose homeostasis in the liver.
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Disruption of energy balance causes various liver diseases such as non-alcoholic fatty liver disease (NAFLD) which is strongly associated with obesity and type 2 diabetes [16, 17]. In this
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study, we presented in vivo and in vitro models of hepatic steatosis induced by high fat diet and
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PA, respectively, and this was confirmed through a comparison of lipid contents. The liver plays a central role in lipid and glucose homeostasis and is a major organ for the
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production of secretory proteins. Similar to adipokines, hepatokines act as a biomarker in insulin
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resistance and type 2 diabetes [18, 19]. Hepatokine FGF21 is a novel therapeutic agent known to attenuate obesity and diabetes [20, 21]. In the present study, we found that pharmacological treatment of exendin-4 increased the expression of hepatic FGF21, FGFR1, FGFR2, FGFR4 and β-Klotho mRNA and FGFR1 protein in mice as well as FGF21 mRNA and FGFR1 mRNA and protein in vitro models. The affinity between FGF21 and its receptors appear different in each tissue, and upon the presence of the co-receptor β-Klotho, FGF21 binds to FGFR1 with higher affinity compared to others [22, 23]. Interestingly, during inhibition of FGFR1 by siRNA, an increase of FGF21 and PPARα expression by exendin-4 was not shown (Fig. 2D), suggesting that an autocrine effect of FGF21 stimulated by exendin-4 treatment in hepatocytes is mediated by 14
ACCEPTED MANUSCRIPT FGFR1. We confirmed that recombinant FGF21 in hepatocytes increases the expression of FGF21 mRNA in a dose-dependent manner (data not shown) and decreases TG accumulation
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(Fig. 2A-B). Previous studies reported that liver-derived FGF21 may act in an endocrine or
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antucrine/ paracrine manner to regulate ketogenesis, gluconeogenesis and fatty acid oxidation [24], and that FGF signaling is inhibited with the FGFR inhibitor [25]. However, we consider that
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more detailed further research is needed to clarify the possible mechanism for the down-
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regulation of the expression of FGF21 and PPARα mRNA by inhibition of FGFR1. FGF21 regulates fatty acid metabolism through induction of PPAR γ coactivator protein-1α
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(PGC-1α) in the liver [26]. Also, the expression of FGF21 was increased in primary hepatocytes treated with metformin which is known to have a protective effect against NAFLD and insulin
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resistance through activation of AMPK [27]. In accordance with this finding, we observed that
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inhibition of FGF21 in HepG2 cells treated with exendin-4 induces decreased expression of PPARα and MCAD, which are regulators of fatty acid β-oxidation. Thus, these results suggest
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that the decrease in body weight gain and hepatic TG levels in exendin-4 treated mice may be due
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to increased FGF21-mediated fatty acid oxidation. Meanwhile, modulation of FGF21 by exendin-4 has been previously reported. Samson et al. [28] found that exendin-4 decreases serum- and hepatic FGF21 levels in NAFLD in a diet-induced obese mouse model, which is different from our present results. Although, FGF21 acts importantly on improvement of insulin sensitivity, glucose tolerance and hepatic steatosis, they reported that since metabolic syndromes such as obesity and type 2 diabetes are state of FGF21 resistance, plasma FGF21 levels increased under these disorders and FGF21 resistance in the liver was improved by exendin-4 treatment. We believe that these conflicting results may be attributable to the differences in the fat content of the diet, treatment period and concentration of 15
ACCEPTED MANUSCRIPT exendin-4, and in the age and number of animal models. In our study, the duration of exendin-4 administration was much longer than that in the previous report, and the age of the animal model
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used in this study was younger. Further studies are needed to assess this point.
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In addition, upon metabolic dysfunction, expression patterns of FGF21 are controversial. Zhang X et al. [29] reported that serum FGF21 levels are increased in individuals with metabolic
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syndrome. They suggested that their models of metabolic syndromes are in FGF21 resistant state-
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[28, 30, 31], whereas, in clinical and animal study, multiple studies reported no correlation between FGF21 and weight loss [32], metabolic syndrome, NALFD [33] and adiponectin, which
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is a biomarker of the metabolic syndrome [34]. In addition, glucagon-like peptide (GLP)-1 analogue liraglutide leads to an improvement of FGF21 and insulin resistance induced by a
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combination of HFD, ApoE deficiency and adiponectin knockdown via increasing FGF21
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activity [35] and FGF21 treatment in skeletal muscle cells treated with palmitic acid and in MSGIR mice improved insulin resistance [36, 37]. Therefore, to clarify the effect of exendin-4 on the
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regulation of hepatic FGF21, additional studies on diet-induced obesity conditions in human and
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animal models should be undertaken. SIRT1 and SIRT6, members of the sirtuin family, are NAD+-dependent deacetylases that act in the modification of lipid and glucose metabolism in the liver [14, 38-40]. In our previous study, we demonstrated that exendin-4 increases the expression of SIRT1 in the liver of mice [13], and that the protein kinase A (PKA) pathway which mediates the GLP-1-dependent action activates SIRT1 to maintain energy homeostasis [41]. Knockdown of hepatic SIRT1 and SIRT6 leads to insulin resistance and fatty liver [14, 42, 43], whereas resveratrol, a possible activator of SIRT1, improves insulin resistance, hyperglycemia and hepatic steatosis [44]. In this study, the expression of SIRT1 and FGF21 was increased by exendin-4. In addition, SIRT1 inhibition 16
ACCEPTED MANUSCRIPT decreased the expression of FGF21 in cells treated with exendin-4, whereas the expression of SIRT1 was not affected by knockdown of FGF21. Thus, these data suggest that the effect of
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exendin-4 on the regulation of hepatic FGF21 may be mediated by SIRT1, indicating that SIRT1
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is an upstream of FGF21 in hepatocytes.
In other words, the strength of this study is the observation that the expression of FGF21 and
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PPAR-alpha, which are associated with fatty acid catabolism, was down-regulated by FGFR1
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inhibition, and that SIRT1 was identified as an upstream regulator of FGF21. On the other hand, there are a few limitations in our study. First, in addition to SIRT1, a variety of factors may
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contribute to the regulation of FGF21. Since this study was only investigated using specific siRNA in vitro to clarify the relationship between SRIT1 and FGF21, we have not identified the
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effects of other regulators in the regulation of FGF21. Second, after inhibition of FGFR1 by
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siRNA transfection, change in interaction between FGF21 and FGFR1 was not directly tested. Since a decrease of FGFR1 expression was observed, we expected that the binding between two
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proteins may be reduced. These are needed to confirm through further experiments.
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In conclusion, the present study demonstrated that exendin-4 increases FGF21-mediated fatty acid oxidation by increasing SIRT1 in hepatocytes (Fig. 4). Therefore, the protective effect of exendin-4 in an in vivo hepatic steatosis model may also be mediated through the SIRT1-FGF21 pathway, and hepatic FGF21 may serve as a therapeutic target for the treatment of fatty liver disease associated with metabolic diseases, such as obesity and type 2 diabetes mellitus.
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ACCEPTED MANUSCRIPT Author contributions
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Conceived and designed the experiments: JL SWH WYL. Performed the experiments: JL SWH.
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Analyzed the data: JL SWH SEP EJR CYP KWO SWP WYL. Contributed
reagents/materials/analysis tools: JL SWH SEP EJR CYP KWO SWP WYL. Wrote the paper: JL
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EJR WYL.
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ACCEPTED MANUSCRIPT References
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1 Kharitonenkov A. FGFs and metabolism. Current opinion in pharmacology 2009;9(6): 805-10.
RI P
2 Kurosu H, Kuro-o M. Endocrine fibroblast growth factors as regulators of metabolic homeostasis. Biofactors 2009;35(1): 52-60.
SC
3 Lundåsen T, Hunt MC, Nilsson LM, et al. Pparα is a key regulator of hepatic FGF21. Biochem
NU
Biophys Res Commun 2007;360(2): 437-40.
4 Kim H, Mendez R, Zheng Z, et al. Liver-enriched transcription factor crebh interacts with
Endocrinology 2014;155(3): 769-82.
MA
peroxisome proliferator-activated receptor alpha to regulate metabolic hormone FGF21.
ED
5 Cuevas-Ramos D, Almeda-Valdes P, Aguilar-Salinas CA, et al. The role of fibroblast growth
PT
factor 21 (FGF21) on energy balance, glucose and lipid metabolism. Curr Diabetes Rev 2009;5(4): 216-20.
CE
6 Kharitonenkov A, Shiyanova TL, Koester A, et al. FGF-21 as a novel metabolic regulator. J
AC
Clin Invest 2005;115(6): 1627-35. 7 Wang C, Dai J, Yang M, et al. Silencing of FGF-21 expression promotes hepatic gluconeogenesis and glycogenolysis by regulation of stat3- socs3 signal. FEBS J 2014; doi: 10.1111/febs.12767. 8 Xu J, Lloyd DJ, Hale C, et al. Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 2009;58(1): 250-9. 9 Vassilopoulos A, Fritz KS, Petersen DR, et al. The human sirtuin family: Evolutionary divergences and functions. Hum Genomics 2011;5(5): 485-96. 19
ACCEPTED MANUSCRIPT 10 Michan S, Sinclair D. Sirtuins in mammals: Insights into their biological function. The Biochem J 2007;404(1): 1-13.
T
11 Purushotham A, Schug TT, Xu Q, et al. Hepatocyte-specific deletion of SIRT1 alters fatty acid
RI P
metabolism and results in hepatic steatosis and inflammation. Cell Metab 2009;9(4): 327-38. 12 Caron AZ, He X, Mottawea W, et al. The SIRT1 deacetylase protects mice against the
SC
symptoms of metabolic syndrome. FASEB J 2014;28(3): 1306-16.
NU
13 Lee J, Hong SW, Chae SW, et al. Exendin-4 improves steatohepatitis by increasing sirt1 expression in high-fat diet-induced obese C57BL/6J mice. PloS one 2012;7(2): e31394.
MA
14 Kim HS, Xiao C, Wang RH, et al. Hepatic-specific disruption of sirt6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis. Cell Metab 2010;12(3):
ED
224-36.
PT
15 Yang SJ, Choi JM, Chae SW, et al. Activation of peroxisome proliferator-activated receptor gamma by rosiglitazone increases sirt6 expression and ameliorates hepatic steatosis in rats. PloS
CE
one 2011;6(2): e17057.
AC
16 Van Rooyen DM, Larter CZ, Haigh WG, et al. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis. Gastroenterology 2011;141(4): 1393-403. 17 Leite NC, Salles GF, Araujo ALE, et al. Prevalence and associated factors of non‐alcoholic fatty liver disease in patients with type‐2 diabetes mellitus. Liver Int 2008;29(1): 113-9. 18 Misu H, Ishikura K, Kurita S, et al. Inverse correlation between serum levels of selenoprotein p and adiponectin in patients with type 2 diabetes. PloS one 2012;7(4): e34952. 19 Misu H, Takamura T, Takayama H, et al. A liver-derived secretory protein, selenoprotein p, causes insulin resistance. Cell Metab 2010;12(5): 483-95. 20 Zhao Y, Dunbar JD, Kharitonenkov A. FGF21 as a therapeutic reagent. Adv Exp Med Biol 20
ACCEPTED MANUSCRIPT 2012: 214-28. 21 Camporez JP, Jornayvaz FR, Petersen MC, et al. Cellular mechanisms by which FGF21
T
improves insulin sensitivity in male mice. Endocrinology 2013;154(9): 3099-109.
RI P
22 Suzuki M, Uehara Y, Motomura-Matsuzaka K, et al. Βklotho is required for fibroblast growth factor (FGF) 21 signaling through FGF receptor (FGFR) 1c and FGFR3c. Mol Endocrinol
SC
2008;22(4): 1006-14.
NU
23 Kurosu H, Choi M, Ogawa Y, et al. Tissue-specific expression of βklotho and fibroblast
Biol Chem 2007;282(37): 26687-95.
MA
growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21. J
24 Ge X, Wang Y, Lam KS, et al. Metabolic actions of FGF21: Molecular mechanisms and
ED
therapeutic implications. Acta Pharm Sin B 2012;2(4): 350-7.
PT
25 Jurynec MJ, Grunwald DJ. SHIP2, a factor associated with diet-induced obesity and insulin sensitivity, attenuates FGF signaling in vivo. Dis Model Mech 2010;3(11-12): 733-42.
CE
26 Potthoff MJ, Inagaki T, Satapati S, et al. FGF21 induces PGC-1alpha and regulates
AC
carbohydrate and fatty acid metabolism during the adaptive starvation response. Proc Natl Acad Sci U S A 2009;106(26): 10853-8. 27 Nygaard EB, Vienberg SG, Ørskov C, et al. Metformin stimulates FGF21 expression in primary hepatocytes. Exp Diabetes Res 2012;2012: 465282. 28 Samson S, Sathyanarayana P, Jogi M, et al. Exenatide decreases hepatic fibroblast growth factor 21 resistance in non-alcoholic fatty liver disease in a mouse model of obesity and in a randomised controlled trial. Diabetologia 2011;54(12): 3093-100. 29 Zhang X, Yeung DCY, Karpisek M, et al. Serum FGF21 levels are increased in obesity and are independently associated with the metabolic syndrome in humans. Diabetes 2008;57(5): 1246-53. 21
ACCEPTED MANUSCRIPT 30 Chui PC, Antonellis PJ, Bina HA, et al. Obesity is a fibroblast growth factor 21 (FGF21)resistant state. Diabetes 2010;59(11): 2781-9.
T
31 Dushay J, Chui PC, Gopalakrishnan GS, et al. Increased fibroblast growth factor 21 in obesity
RI P
and nonalcoholic fatty liver disease. Gastroenterology 2010;139(2): 456-63. 32 Mai K, Schwarz F, Bobbert T, et al. Relation between fibroblast growth factor-21, adiposity,
SC
metabolism, and weight reduction. Metabolism 2011;60(2): 306-11.
NU
33 Reinehr T, Woelfle J, Wunsch R, et al. Fibroblast growth factor 21 (FGF-21) and its relation to obesity, metabolic syndrome, and nonalcoholic fatty liver in children: A longitudinal analysis. J
MA
Clin Endocrinol Metab 2012;97(6): 2143-50.
34 Wang ZQ, Zhang XH, Yu Y, et al. Artemisia scoparia extract attenuates non-alcoholic fatty
ED
liver disease in diet-induced obesity mice by enhancing hepatic insulin and ampk signaling
PT
independently of FGF21 pathway. Metabolism 2013;62(9): 1239-49. 35 Yang M, Zhang L, Wang C, et al. Liraglutide increases FGF-21 activity and insulin sensitivity
AC
e48392.
CE
in high fat diet and adiponectin knockdown induced insulin resistance. PloS one 2012;7(11):
36 Lee MS, Choi SE, Ha ES, et al. Fibroblast growth factor-21 protects human skeletal muscle myotubes from palmitate-induced insulin resistance by inhibiting stress kinase and NF-kB. Metabolism 2012;61(8): 1142-51. 37 Zhu SL, Zhang ZY, Ren GP, et al. Therapeutic effect of fibroblast growth factor 21 on NAFLD in MSG-iR mice and its mechanism. Yao Xue Xue Bao 2013;48(12): 1778-84. 38 Hou X, Xu S, Maitland-Toolan KA, et al. Sirt1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase. J Biol Chem 2008;283(29): 20015-26. 39 Caton PW, Nayuni NK, Kieswich J, et al. Metformin suppresses hepatic gluconeogenesis 22
ACCEPTED MANUSCRIPT through induction of SIRT1 and GCN5. J Endocrinol 2010;205(1): 97-106. 40 Dominy Jr JE, Lee Y, Jedrychowski MP, et al. The deacetylase Sirt6 activates the
T
acetyltransferase GCN5 and suppresses hepatic gluconeogenesis. Mol Cell 2012;48(6): 900-13.
RI P
41 Gerhart-Hines Z, Dominy JE, Jr., Blattler SM, et al. The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD(+). Mol Cell
SC
2011;44(6): 851-63.
NU
42 Rodgers JT, Lerin C, Haas W, et al. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature 2005;434(7029): 113-8.
MA
43 Wang RH, Kim HS, Xiao C, et al. Hepatic sirt1 deficiency in mice impairs mTorc2/Akt signaling and results in hyperglycemia, oxidative damage, and insulin resistance. J Clin Invest
ED
2011;121(11): 4477-90.
PT
44 Labbé A, Garand C, Cogger VC, et al. Resveratrol improves insulin resistance hyperglycemia and hepatosteatosis but not hypertriglyceridemia, inflammation, and life span in a mouse model
AC
CE
for werner syndrome. J Gerontol A Biol Sci Med Sci 2011;66(3): 264-78.
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ACCEPTED MANUSCRIPT Tables
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Table 1. Body weight gain, liver weight and hepatic triglycerides in mice.
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Figure legends
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Fig. 1. Exendin-4 increases the expression of FGF21 and its receptors in vivo and in vitro. (A) Mice were fed a low fat diet (control), high fat diet (HF), or HF diet with 1 nmol/kg/day
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exendin-4 ip injection for 10 weeks. Total RNA and protein were extracted from liver tissue, and FGF21, FGFR1, 2, 4, and β-klotho mRNA and FGFR1 protein expression levels were measured
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using quantitative real-time RT-PCR and Western blot. * p
