Identification of hepatic fibroblast growth factor 21 as a mediator in 17β‐estradiol‐induced white adipose tissue browning
2018; Wiley; Volume: 32; Issue: 10 Linguagem: Inglês
10.1096/fj.201800240r
ISSN1530-6860
AutoresLun Hua, Yong Zhuo, Dandan Jiang, Jing Li, Xiaohua Huang, Zhu Yingguo, Zhen Li, Lijun Yan, Chao Jin, Xuemei Jiang, Lianqiang Che, Zhengfeng Fang, Yan Lin, Shengyu Xu, Jian Li, Bin Feng, De Wu,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe FASEB JournalVolume 32, Issue 10 p. 5602-5611 ResearchFree Access Identification of hepatic fibroblast growth factor 21 as a mediator in 17β-estradiol-induced white adipose tissue browning Lun Hua, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, China These authors contributed equally to this work.Search for more papers by this authorYong Zhuo, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, China These authors contributed equally to this work.Search for more papers by this authorDandan Jiang, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorJing Li, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorXiaohua Huang, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorYingguo Zhu, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorZhen Li, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorLijun Yan, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorChao Jin, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorXuemei Jiang, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorLianqiang Che, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorZhengfeng Fang, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorYan Lin, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorShengyu Xu, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorJian Li, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorBin Feng, Corresponding Author fengbin@sicau.edu.cn Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Correspondence: Sichuan Agricultural University, 211 Huimin Rd., Wenjiang District, Chengdu 611130, China. E-mail: fengbin@sicau.edu.cn Correspondence: Sichuan Agricultural University, 211 Huimin Rd., Wenjiang District, Chengdu 611130, China. E-mail: wude@sicau.edu.cnSearch for more papers by this authorDe Wu, Corresponding Author wude@sicau.edu.cn Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Correspondence: Sichuan Agricultural University, 211 Huimin Rd., Wenjiang District, Chengdu 611130, China. E-mail: fengbin@sicau.edu.cn Correspondence: Sichuan Agricultural University, 211 Huimin Rd., Wenjiang District, Chengdu 611130, China. E-mail: wude@sicau.edu.cnSearch for more papers by this author Lun Hua, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, China These authors contributed equally to this work.Search for more papers by this authorYong Zhuo, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, China These authors contributed equally to this work.Search for more papers by this authorDandan Jiang, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorJing Li, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorXiaohua Huang, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorYingguo Zhu, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorZhen Li, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorLijun Yan, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorChao Jin, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorXuemei Jiang, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorLianqiang Che, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorZhengfeng Fang, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorYan Lin, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorShengyu Xu, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorJian Li, Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, ChinaSearch for more papers by this authorBin Feng, Corresponding Author fengbin@sicau.edu.cn Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Correspondence: Sichuan Agricultural University, 211 Huimin Rd., Wenjiang District, Chengdu 611130, China. E-mail: fengbin@sicau.edu.cn Correspondence: Sichuan Agricultural University, 211 Huimin Rd., Wenjiang District, Chengdu 611130, China. E-mail: wude@sicau.edu.cnSearch for more papers by this authorDe Wu, Corresponding Author wude@sicau.edu.cn Institute of Animal Nutrition and Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Key Laboratory for Animal Disease-Resistant Nutrition, Ministry of Education of China, Sichuan Agricultural University, Chengdu, China Correspondence: Sichuan Agricultural University, 211 Huimin Rd., Wenjiang District, Chengdu 611130, China. E-mail: fengbin@sicau.edu.cn Correspondence: Sichuan Agricultural University, 211 Huimin Rd., Wenjiang District, Chengdu 611130, China. E-mail: wude@sicau.edu.cnSearch for more papers by this author First published: 02 May 2018 https://doi.org/10.1096/fj.201800240RCitations: 1 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat ABSTRACT Both ovarian E2 and hepatic fibroblast growth factor 21 (FGF21) are critical for energy homeostasis and white adipose tissue browning. Estrogen receptor α (ERα) is abundantly expressed in liver. However, whether FGF21 has a role in E2-induced white adipose tissue browning remains uncertain. In this study, we showed that hepatic Fgf21 expression and secretion during estrus cycle changed with the tetradian oscillatory secretion of circulation E2 in adult, female mice, with their peak expressions and secretions at the proestrus. In addition, exogenous E2 robustly stimulated liver Fgf21 expression and elevated serum FGF21 concentrations, which induced browning gene expression and reduced the tissue weight in subcutaneous white adipose in mice with ovariectomies. The inhibitor of mammalian target of rapamycin (mTOR) and of ERα blocked the induction effect of E2 on the expression of Fgf21 in primary hepatocytes, which revealed that E2 might stimulate FGF21 expression via the ERα-mTOR pathway. Furthermore, FGF21 liver-specific deficiency abolished E2-induced white adipose browning in mice with ovariectomies. This study indicates that ovarian E2 increased liver FGF21 expression directly, which in turn, functioned as an endocrine signal to influence inguinal white adipose tissue browning.—Hua, L., Zhuo, Y., Jiang, D., Li, J., Huang, X., Zhu, Y., Li, Z., Yan, L., Jin, C., Jiang, X., Che, L., Fang, Z., Lin, Y., Xu, S., Li, J., Feng, B., Wu, D. Identification of hepatic fibroblast growth factor 21 as a mediator in 17β-estradiol-induced white adipose tissue browning. FASEB J. 32, 5602–5611 (2018). www.fasebj.org ABBREVIATIONS BAT brown adipose tissue Dio2 iodothyronine deio-dinase 2 ERα estrogen receptor α Fasn fatty acid synthase FFA free fatty acid FGF21 fibroblast growth factor 21 FGF21LKO FGF21 liver- specific knockout Hsl hormone-sensitive lipase ICI ICI182, 780 iWAT inguinal white adipose tissue KLB β-klotho mTOR mammalian target of rapamycin OVX ovariectomy PGC-1α peroxisome proliferator- activated receptor-γ coactivator 1 α Ppar α peroxisome proliferator activated receptor α Prdm16 PR domain containing 16 S6 ribosomal protein S6 UCP-1 uncoupling protein 1 WT wild type The female reproductive system has been accepted recently as a critical driver in the formation of a differential susceptibility to metabolic disturbances compared with that of males (1). The fact that menopause induces metabolic syndrome strongly suggests that ovarian E2 is a critical regulator of energy homeostasis (2). In addition, evidence from both humans and laboratory animals has revealed that a substantial reduction of circulating E2 after menopause or ovariectomy (OVX) is associated with aberrant energy expenditure and weight gain (3, 4). However, E2 replacement therapy prevents OVX-induced obesity by decreasing food intake and increasing energy expenditure through central or peripheral action to affect adipose-tissue thermogenesis (5), lipid metabolism (6), and mitochondrial function (7, 8). However, the exact mechanisms by which E2 modulates adipose metabolism has not been well established, and in particular, the target of E2 action remains controversial. Notably, estrogens are primarily metabolized in the liver via hydroxylation by cytochrome P450 enzymes. Estrogen receptor α (ERα) is abundantly expression in the liver (9, 10), and recent studies have revealed that the transcriptional activity of hepatic ERα is strictly associated with the reproductive cycle (6) and nutritional status (11) and have a profound influence on liver lipid metabolism (12). These reports reveal that the liver is an important target organ for estrogen. If that is true, then there should be an estrogen- dependent secretion of hepatokines to regulate the metabolic activities in other tissues, which remains uncertain. Recently, fibroblast growth factor 21 (FGF21), an atypical member of the FGF family, was revealed as one of the most important hepatokines to act as an autocrine, paracrine, and endocrine factor, exerting profound effects on energy homeostasis, apoptotic process, anti-inflammation, and even reproduction (13). The expression and secretion of hepatic FGF21 is induced by protein restriction, ketogenic diets, overfeeding, fasting, and cold exposure (14–20). FGF21 signals via the FGF receptor 1c and the coreceptor β-klotho (KLB), expressed in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) (21), and FGF21 simulates thermogenesis by up-regulating the expression of thermogenic marker peroxisome proliferator-activated re- ceptor-γ coactivator 1α (PGC-1α) and uncoupling protein 1 (UCP1) (22, 23). Several lines of evidence have implicated the close relationship between E2 and FGF21. Firstly, hepatic secretion of FGF21 was regarded as a major endocrine signal for amino acid intake levels (15, 18), whereas amino acids can robustly up-regulate the activity of ERα (11). Secondly, the beneficial effect of protein restriction on the secretion of FGF21 and energy expenditure requires an intact, female reproductive system (24). Thirdly, E2 treatment was observed to promote lipid oxidation and increase energy expenditure, which is associated with a serial of changes in hormonal secretions, including hepatic FGF21 secretion (25). Therefore, the objective of the current study was to test whether E2 could directly influence hepatic FGF21 secretion to influence white adipose tissue metabolism. MATERIALS AND METHODS Animal experiments All animal procedures in this study were handled in accordance with Guide for the Care and Use of Laboratory Animals (National Research Council, Bethesda, MD, USA), and the Institutional Animal Care and Research Committee of Sichuan Agricultural University (SICAU-2015–033). Mice were kept in temperature- controlled facilities with a 12-h light/dark cycle and had free access to food and water. FGF21Liver+/-, Alb-cre mice were generated by mating FGF21loxp/loxp mice (022361; The Jackson Laboratory, Bar Harbor, ME, USA) with Alb-Cre mice (J003574; Model Animal Research Center, Nanjing University, Nanjing, China) transgenic mice. FGF21Liver-/-, Alb-Cre mice were generated by crossing FGF21Liver+/-, Alb>Cre mice with FGF21loxp/loxp mice. Littermates of FGF21loxp/loxp mice were used as control. Identification of the estrus cycle stage Examination of vaginal smears was performed at 9:00 AM, when the mice were killed, unless otherwise stated. Stages of the estrus cycle were assigned by the following criteria, as described by Singhal et al. (26): predominantly nucleated epithelial cells, in the absence of leukocytes, as proestrus; sheets of nonnucleated, squamous, corni- fied cells, in the absence of leukocytes, as estrus; equal distribution of leukocytes and cornified and nucleated epithelial cells as metestrus; and a mixture of epithelial cells and leukocytes, with a predominance of leukocytes, as diestrus. Surgical procedures for ovariectomy and E2 administration Bilateral ovariectomy was performed on 8-wk-old mice. The mice were anesthetized with 50 mg/kg pentobarbital sodium. Abdominal incisions were made longitudinally and bilaterally in the region below the last lumbar vertebra. The ovary, oviduct, and top of the fallopian tubes were tied and removed. The abdominal wall and the skin were then sutured. After a 2-wk recovery, mice received a single subcutaneous injection of vehicle (corn oil, C8267; MilliporeSigma, Billerica, MA, USA) or E2 (E8875; MilliporeSigma) at a dosage of 0.5 mg/kg, which was able to achieve 5 times greater circulation of E2 than that at proestrus (27). Cell culture and treatments Primary hepatocytes were isolated from 8-wk-old, female C57BL/6 mice with 2-step perfusion method (28). Briefly, mouse liver was perfused with perfusion buffer for 5 min through the portal vein, followed by collagenase buffer perfusion for about 5 min until liver cells were dispersed. Cells were then filtered through a 150-mesh tissue sieve, followed twice by washing with the culture medium. Dissociated cells were plated at a density of 3 × 105 cells/well onto a type I collagen-precoated, 12-well plate (A004174; BD Biosciences, Franklin Lakes, NJ, USA). Cells were allowed to adhere for 6 h in 10% fetal bovine serum (10099141; Thermo Fisher Scientific, Waltham, MA, USA) and penicillin/ streptomycin (10378016; Thermo Fisher Scientific) in phenol red-free DMEM medium (21063029; Thermo Fisher Scientific). Unadherent cells were washed away with DMEM medium. After a 12-h incubation in dextran-coated, charcoal-stripped 10% fetal bovine serum (C6241; MilliporeSigma), cells were treated with E2 and with or without the inhibitors ICI182 780 (ICI), inhibitor for ERα (1286650; MilliporeSigma); and rapamycin (S1039; Selleck Chemicals, Houston, TX, USA), inhibitor for mTOR in phenol red-free DMEM medium. Cells were pretreated with inhibitors 30 min before E2 treatment and were treated with E2 plus inhibitors for 12 h. Serum analysis Serum free fatty acid (FFA) levels were measured on an automatic biochemical analyzer (7020; Hitachi, Tokyo, Japan) with analysis kits (GS191Z; Beijing Strong Biotechnologies, Beijing, China), according to the manufacturer's instructions. Serum estradiol (KGE014) and FGF21 (MF2100) levels were measured with commercial ELISA kits, according to the manufacturer's instructions (R&D Systems; Bio-Techne, Minneapolis, MN, USA). RNA extraction and gene expression analysis RNA was isolated with Trizol reagent (15596018; Thermo Fisher Scientific) and RNeasy Mini Kit (RR037A; Takara Bio, Kusatsu, Japan), according to the manufacturer's instruction. RNA (1 μg) was reverse transcribed to cDNA with reagents from Takara Bio. The mRNA levels were then analyzed with a 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific) using SYB Green Real-Time PCR regent (RR820A; Takara Bio). Gene expression levels were normalized to β-actin expression levels. The cycle threshold (2−ΔΔCt) method was used to calculate the relative gene expression. The sequences of the primers were β-actin, forward 5′-GGCTGTATTCCCCTC- CATCG-3′ andreverse5′-CCAGTTGGTAACAATGCCATGT-3′ fatty acid synthase (Fasn), forward 5′-GGCTCTATGGAT- TACCCAAGC-3′ and reverse 5′-CCAGTGTTCGTTCCTCG- GA-3′; Ucp-1, forward 5′-GCCAAAGTCCGCCTTCAGAT-3′ and reverse 5′-CAGTTTCGGCAATCCTTCTGTT-3′; Fgf21, forward 5′-CTGCTGGGGGTCTACCAAG-3′ and reverse 5′-CTGCGC- CTACCACTGTTCC-3′; peroxisome proliferator-activated receptor α (Pparα), forward 5′ -TACTGCCGTTTTCACAAGTGC-3′ and reverse 5′-AGGTCGTGTTCACAGGTAAGA-3′ Pgc-1α, forward 5′-TATGGAGTGACATAGAGTGTGCT-3′ and reverse 5 ′-CCACTTCAATCCACCCAGAAAG-3′; Klb, forward 5′-CAA- CCCACTCCCATCTCGG-3′ and reverse 5′-AGCACAGCTCA- GCGTAGTCC-3′; PR domain containing 16 (Prdml6), forward 5′-ACAAGTCCTACACGCAGTTCTC-3′ and reverse 5′- ATGCTGCCAGGCGTGTAATG-3′; iodothyronine deiodinase 2 (Dio2), forward 5′-GCTTACGGGGTAGCCTTTGA-3′ and reverse 5′-TGTAGGTTATAGCTGAAGGGGC-3′; and hormonesensitive lipase (Hsl), forward 5′-TGAAGCCAAAGATGA- AGTGAGAC-3′ and reverse 5′-CTTGACTATGGGTGACG- TGTAGAG-3′. Western blotting Western blotting was performed as described in Feng et al. (29). Briefly, samples were homogenized in lysis buffer (P0013B; Beyotime Institute of Biotechnology, Jiangsu, China) supplemented with protease inhibitor cocktail (04693132001; Roche, Basel, Switzerland). The homogenates were then centrifuged at 12, 000 g for 30 min at 4°C. The supernatant was collected, and protein concentration was measured with BCA Protein Assay Kit (23227; Thermo Fisher Scientific) on a plate reader. Proteins were separated on 10% SDS-PAGE gel after boiling at 95°C for 5 min and were then transferred to a polyvinylidene fluoride membrane (1620177; Bio-Rad Laboratories, Hercules, CA, USA). The membrane was washed in Tris-buffered saline containing Tween and blocked with 1% bovine serum albumin in Tris-buffered saline containing Tween at room temperature for 1 h with gentle shaking. Then, the membranes were incubated overnight at 4°C with the respective primary antibodies. Antibodies against PGC-1α (54481) and FGF21 (171941) were purchased from Abcam (Cambridge, United Kingdom); antibodies against β-actin (4970S), p-mTOR (2971S), mTOR (2972S), p-S6 (4858T), ribosomal protein S6 (S6; 2217S), and UCP-1 (14670S) were obtained from Cell Signaling Technology (Danvers, MA, USA); and the antibody against KLB (AF2619) was from Bio-Techne. The next morning, membranes were incubated with the respective secondary antibodies (7074 and 7076; Cell Signaling Technology) at room temperature for 1 h, after 5 washes. After further thorough washing, protein signals were detected by ECL Western blotting detection reagent (1705060; Bio-Rad Laboratories) on a Molecular Imager ChemiDoc XRS+ System (Bio-Rad Laboratories). Blots were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Statistical analysis Data were analyzed with Prism 6 software (GraphPad Software, La Jolla, CA, USA). Unpaired t test was used to analyze the difference between 2 groups; 1-way ANOVA was applied to analyze the difference among 3 or more groups. For the experiments involving FGF21 liver-specific knockout mice, data were analyzed by the 2-way ANOVA test or the Holm-Bonferroni correction for multiple comparisons to determine differences between each group, where appropriate. Data are presented as means ± sem.Statistical significance was set at P < 0.05. RESULTS Liver FGF21 expression underwent cyclic changes with the E2 during estrus cycle To investigate the expression of Fgf21 in liver during the estrus cycle, 10-wk-old, female mice were harvested at different phases of the estrus cycle. The phases were confirmed by serum E2 levels and uterus weight (Fig. 1A, B), which showed cyclic changes across the estrus cycle with the highest E2 level and uterus weight at proestrus and the nadir at metestrus and diestrus (Fig. 1A, B). Both mRNA and protein levels of FGF21 in liver and the circulating FGF21 levels underwent cyclic changes during the estrus cycle (Fig. 1C-E), with the highest level at proestrus, similar to the changes of circulating E2 level. However, such cyclic changes of Fgf21 expression were not observed in BAT, iWAT, epididymal adipose tissue, perirenal adipose tissue, and skeletal muscle (Fig. 1C). Furthermore, the expression of browning markers Ucp-1 and Pgc-lα (Fig. 1F, G), showed its greatest level of the estrus cycle at proestrus, in the iWAT. These results demonstrated a potential correlation between ovarian E2, liver FGF21 expression, and the browning of iWAT. Figure 1Open in figure viewer Liver FGF21 expressions underwent cyclic changes with circulating E2. Circulating E2 concentration (A), uterine weight (B), Fgf21 mRNA expression in various tissues (C), liver FGF21 protein expression (D), circulating FGF21 concentration (E), and mRNA expression of Pgc-1α (F) and Ucp-1 (G) in inguinal adipose tissue at the estrus cycle of proestrus (P), estrus (E), metestrus (E), and diestrus (D) were detected. eWAT, epididymal adipose tissue; pWAT, perirenal adipose tissue; n = 9–10/group. Statistical significance was evaluated by unpaired Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001. Exogenous E2 robustly increased hepatic FGF21 expression in OVX mice OVX mice, which are a model without fluctuations of ovarian E2 production during the estrus cycle, are good for studying the function of E2. To test the effect of E2 on hepatic Fgf21 expression, OVX mice were administered a single subcutaneous injection of E2. OVX mice treated with E2 showed an acute increase in circulating E2 levels at 6, 12, and 24 h after injection compared with the mice treated with vehicle (Fig. 2A). In addition, liver Fgf21 mRNA and protein levels were up-regulated by E2 administration (Fig. 2B, C). Furthermore, serum FGF21 concentrations were increased by 148, 592, and 493% compared with control mice at 6, 12, and 24 h after E2 injection, respectively (Fig. 2D). White adipose tissue is the main target of FGF21 (30); thus, we tested whether the E2-induced FGF21 level could act as an endocrine modulator of subcutaneous adipose tissue metabolism. E2 administration induced a substantial reduction in iWAT weight at 12 and 24 h after injection (Fig. 2E), accompanied by an increase in gene expression of Pgc-lα and Ucp-1/2, critical marker genes for adipose tissue browning, in E2-administrated iWAT (Fig. 2F, G). However, mRNA levels of Fgf21 in iWAT were not affected (Fig. 2H), suggesting that E2 does not directly increase the Fgf21 expression in iWAT. Protein levels of KLB, the obligatory coreceptor of FGF21 receptor, were up-regulated at 24 h after E2 administration in iWAT (Fig. 2I), indicating enhanced FGF21 signaling in subcutaneous adipose tissue. Further, protein expression of PGC-1α (Fig. 2J) and UCP-1 (Fig. 2K) was detected and their expression was significantly increased by the E2 treatment. Serum FFA was increased at 24 h after injection (Fig. 2L), and the Hsl mRNA expression in iWAT was increased 24 h after E2 injection (Fig. 2M). Notably, food intake was not affected by E2 treatment as compared with that of vehicle-treated mice (data not shown); thus, the reduction of iWAT weight can be attributed to an increase in thermogenesis and lipolysis. These results indicated that E2-induced hepatic FGF21 might act as an endocrine hepatokine regulating the thermogenesis of white adipose tissue. Figure 2Open in figure viewer E2 treatment increased FGF21 production in vivo. Circulating E2 concentration (A); liver Fgf21 mRNA (B) and protein expression (C); circulating FGF21 concentration (D); weight of iWAT (E); mRNA expression of Pgc-1< (F), Ucp-1 (G), and Fgf21 (H) in iWAT; protein expression of KLB (I), PGC-1a (J), and UCP-1 (K); serum free fatty acids (L); and Hsl mRNA expression (M) were detected in 3-mo-old, female OVX mice, at 6, 12, and 24 h after a single E2 injection (0.5 mg/kg); n = 6–7/group. Statistical significance was evaluated by unpaired Student's t test. *P < 0.05, **P < 0.01, ***P < 0.
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