Curcumin prevents obesity by targeting TRAF4‐induced ubiquitylation in m 6 A‐dependent manner
2021; Springer Nature; Volume: 22; Issue: 5 Linguagem: Inglês
10.15252/embr.202052146
ISSN1469-3178
AutoresYushi Chen, Ruifan Wu, Wei Chen, Youhua Liu, Xing Liao, Botao Zeng, Guanqun Guo, Fangfang Lou, Yun Xiang, Yizhen Wang, Xinxia Wang,
Tópico(s)Circular RNAs in diseases
ResumoArticle20 April 2021free access Transparent process Curcumin prevents obesity by targeting TRAF4-induced ubiquitylation in m6A-dependent manner Yushi Chen Yushi Chen College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Ruifan Wu Ruifan Wu College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Wei Chen Wei Chen College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Youhua Liu Youhua Liu College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Xing Liao Xing Liao College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Botao Zeng Botao Zeng College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Guanqun Guo Guanqun Guo College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Fangfang Lou Fangfang Lou Jinhua Academy of Agricultural Sciences, Jinhua, China Search for more papers by this author Yun Xiang Yun Xiang Jinhua Academy of Agricultural Sciences, Jinhua, China Search for more papers by this author Yizhen Wang Yizhen Wang College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Xinxia Wang Corresponding Author Xinxia Wang [email protected] orcid.org/0000-0002-2328-7003 College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Yushi Chen Yushi Chen College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Ruifan Wu Ruifan Wu College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Wei Chen Wei Chen College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Youhua Liu Youhua Liu College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Xing Liao Xing Liao College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Botao Zeng Botao Zeng College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Guanqun Guo Guanqun Guo College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Fangfang Lou Fangfang Lou Jinhua Academy of Agricultural Sciences, Jinhua, China Search for more papers by this author Yun Xiang Yun Xiang Jinhua Academy of Agricultural Sciences, Jinhua, China Search for more papers by this author Yizhen Wang Yizhen Wang College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Xinxia Wang Corresponding Author Xinxia Wang [email protected] orcid.org/0000-0002-2328-7003 College of Animal Sciences, Zhejiang University, Hangzhou, China Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China Search for more papers by this author Author Information Yushi Chen1,2,3,4, Ruifan Wu1,2,3,4, Wei Chen1,2,3,4, Youhua Liu1,2,3,4, Xing Liao1,2,3,4, Botao Zeng1,2,3,4, Guanqun Guo1,2,3,4, Fangfang Lou5, Yun Xiang5, Yizhen Wang1,2,3,4 and Xinxia Wang *,1,2,3,4 1College of Animal Sciences, Zhejiang University, Hangzhou, China 2Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, Hangzhou, China 3Key Laboratory of Animal Nutrition and Feed Science (Eastern of China), Ministry of Agriculture and Rural Affairs, Hangzhou, China 4Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Hangzhou, China 5Jinhua Academy of Agricultural Sciences, Jinhua, China *Corresponding author. Tel: +86 13067753599; E-mail: [email protected] EMBO Reports (2021)22:e52146https://doi.org/10.15252/embr.202052146 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Obesity has become a major health problem that has rapidly prevailed over the past several decades worldwide. Curcumin, a natural polyphenolic compound present in turmeric, has been shown to have a protective effect on against obesity and metabolic diseases. However, its underlying mechanism remains largely unknown. Here, we show that the administration of curcumin significantly prevents HFD-induced obesity and decreases the fat mass of the subcutaneous inguinal WAT (iWAT) and visceral epididymal WAT (eWAT) in mice. Mechanistically, curcumin inhibits adipogenesis by reducing the expression of AlkB homolog 5 (ALKHB5), an m6A demethylase, which leads to higher m6A-modified TNF receptor-associated factor 4 (TRAF4) mRNA. TRAF4 mRNA with higher m6A level is recognized and bound by YTHDF1, leading to enhanced translation of TRAF4. TRAF4, acting as an E3 RING ubiquitin ligase, promotes degradation of adipocyte differentiation regulator PPARγ by a ubiquitin–proteasome pathway thereby inhibiting adipogenesis. Thus, m6A-dependent TRAF4 expression upregulation by ALKBH5 and YTHDF1 contributes to curcumin-induced obesity prevention. Our findings provide mechanistic insights into how m6A is involved in the anti-obesity effect of curcumin. SYNOPSIS Curcumin exerts its anti-obesity effect by increasing the ALKBH5-mediated m6A modification of TRAF4 mRNA, and enhancing TRAF4 translation in an YTHDF1-dependent manner. TRAF4 in turn promotes ubiquitination of PPARγ and inhibits adipogenesis. Curcumin feeding prevents HFD-induced obesity and metabolic dysfunction in mice. Curcumin feeding increases m6A modified RNA levels in iWAT and eWAT and decreases ALKBH5 levels in mice. Decreased ALKBH5 protein results in higher m6A modification of TRAF4 mRNA, which is recognized and bound by YTHDF1, leading to enhanced translation of TRAF4 in adipocytes. TRAF4, acting as an E3 RING ubiquitin ligase, promotes ubiquitination of PPARγ, leading to lower PPARγ level and inhibits adipogenesis in adipocytes. Introduction Obesity is a complex pathophysiology and is closely associated with various chronic metabolic disorders, and it has become a major health burden that has rapidly prevailed over the past several decades worldwide (Tsai et al, 2011; Jung et al, 2017). Thus, to develop effective therapeutic strategies to reduce obesity is urgent. Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a polyphenol derived from the rhizome of Curcuma longa (turmeric), which has been widely consumed daily throughout Asian countries over centuries without reported toxicity (Ejaz et al, 2009). Curcumin has attracted attention in the medical field because of its potentially diverse health-promoting effects such as antioxidant (Wafi et al, 2019), anti-inflammation (Ikram et al, 2019), and anti-cancer properties (Mirzaei et al, 2018). An increasing body of evidence shows that curcumin can prevent and (or) alleviate various chronic diseases like neurodegenerative disorders (Ghosh et al, 2015), inflammatory diseases (Hanai & Sugimoto, 2009), and cardiovascular diseases (Wongcharoen & Phrommintikul, 2009). In recent years, studies demonstrated that curcumin also had beneficial effects in obesity and diabetes (Ghosh et al, 2015). For instance, dietary supplementation with curcumin to 80 mg/kg body weight decreased fasting plasma glucose and improved insulin sensitivity in obese rats (El-Moselhy et al, 2011). Similar results also have been reported in mice (Shao et al, 2012) and humans (Ghorbani et al, 2014). It has been shown that curcumin inhibits adipogenesis by blocking mitotic clonal expansion process, upregulating adipocyte energy metabolism and apoptosis (Ejaz et al, 2009; Kim et al, 2011; Shao et al, 2012), and targeting PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα), and KLF5, as well as some enzymes involved in fatty acid metabolism like CPT-1, FAS, and GPAT-1 (Ejaz et al, 2009; Kim et al, 2011; Yan et al, 2018). In addition, Wnt/β-catenin signaling pathway has been reported to participate in curcumin-mediated suppression of adipogenesis in 3T3-L1 cells (Ahn et al, 2010; Tian et al, 2017). However, the underlying mechanisms of curcumin’s anti-obesity effect are not fully understood. Recent evidence showed EGCG (epigallocatechin gallate), also a polyphenol, inhibited adipogenesis in an mRNA m6A-YTHDF2-dependent manner (Wu et al, 2018b). N6-methyladenosine (m6A) is the most plentiful internal RNA modifications in eukaryotes (Wang et al, 2015b). It has been proposed that m6A could regulate adipogenesis through mediating mRNA splicing (Zhao et al, 2014), mitotic clonal expansion (Wu et al, 2018a), JAK2-STAT3-C/EBPβ (Wu et al, 2019b), and autophagy pathway (Wang et al, 2020b). Curcumin has been shown to change the abundance of m6A in various tissues of mammal (Lu et al, 2018; Gan et al, 2019), which raises the question of whether the anti-obesity effect of curcumin is also related to RNA methylation. In the present study, we revealed that curcumin inhibited ALKBH5 expression, leading to increased m6A methylation of TRAF4 and enhanced expression of TRAF4 proteins mediated by YTHDF1. TRAF4, acting as an E3 ligase of PPARγ, induced ubiquitylation of the PPARγ protein. Consequently, PPARγ protein reduced and adipogenesis was further inhibited, suggesting a completely new way in which m6A mediated the anti-obesity effect of curcumin. Results Curcumin prevents HFD-induced obesity and related metabolic dysfunctions To assess the effect of curcumin on obesity, the mice were fed with NCD, HFD, and HFD+ curcumin (CUR) for 12 weeks. The results showed that HFD group exhibited rapid increases in mice body weight despite eating significantly less than NCD group (Fig 1A–C). However, HFD-induced weight gain was suppressed by CUR administration (Fig 1A and B). No difference was observed in food intake in mice between HFD and HFD+CUR groups (Fig 1C). In addition, the fat mass of the subcutaneous inguinal WAT (iWAT) and visceral epididymal WAT (eWAT) was significantly lower in HFD+CUR mice than those in HFD mice at 16 weeks of age (Fig 1D and E). In agree with this, histological analysis showed that mice in HFD+CUR group had markedly reduced lipid contents and adipocyte diameter in WAT compared with those in HFD group (Fig 1F and G), suggesting the less weight gain with administration of CUR was mainly associated with fewer fat mass. H&E staining of liver showed that the administration of curcumin dramatically reversed HFD-induced hepatic steatosis (Fig 1H). We next examined the effect of curcumin on glucose tolerance and insulin sensitivity. HFD+CUR mice showed enhanced glucose disposal ability and increased insulin sensitivity (Fig 1L–O), suggesting curcumin preserves glucose homeostasis. The serum of HFD+CUR mice showed lower levels of glucose, total triglyceride, and free fatty acid (FFA) than those of HFD mice (Fig 1I–K). These results demonstrate that curcumin can protect against HFD-induced obesity by regulating glucose homeostasis. Figure 1. Curcumin prevents HFD-induced obesity A–C. Appearance, body weight trajectories, and food intake of the mice. D. Representative pictures of iWAT and eWAT. E. iWAT, eWAT, and liver weights at termination of study. F, G. H&E staining and quantification of size of adipocytes of fixed iWAT and eWAT (scale bar = 200 μm). H. Changes in the morphologies of livers and H&E staining depicted steatosis as circular white gaps caused when the dehydration process leaches the fat out of fixed livers (scale bar = 200 μm). I–K. Fasting blood glucose, serum triglyceride, and FFA levels of mice after 12 weeks on HFD. L–O. The blood glucose level of mice after intraperitoneal injection of glucose or insulin for glucose (GTT) (L) and insulin tolerance tests (ITT) (N), and AUC, area under the curve (M, O). Data information: Data are representative of the mean ± s.d. of n = 6 mice per group and were analyzed using two-way ANOVA and Tukey’s multiple comparison test; *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. Download figure Download PowerPoint Curcumin inhibits adipogenesis through attenuating ALKBH5 expression To gain insight into the role of curcumin on adipogenesis, we incubated primary stromal vascular fraction (SVF) or 3T3-L1 cells with various concentrations of curcumin throughout the 8-day-long differentiation and processed for analysis of lipid accumulation and gene expression by ORO and qPCR, respectively. ORO analysis showed that adipogenesis was inhibited by curcumin in a concentration-dependent manner with maximum effects observed at 20 μM (Fig 2A and C). qPCR analysis showed that the expression of adipogenesis master regulators, including PPARγ, C/EBPα, and fatty acid-binding protein 4 (FABP4), was all attenuated by curcumin also in a dose-dependent manner (Fig 2B and D). Because a recent study showed that the protective effect of curcumin on LPS-induced hepatic lipid metabolism disorders might be because of the increased m6A RNA level (Lu et al, 2018), we prompted to assess whether curcumin affects m6A level in adipocytes. We measured total m6A modified mRNA levels using HPLC–QqQ–MS/MS. The results showed both iWAT and eWAT in mice from HFD+CUR group or curcumin-treated 3T3-L1 cells exhibited elevated m6A level upon curcumin treatment (Fig 2E and H). To further analyze which m6A regulator contributes to the enhanced m6A level, the mRNA and protein expression of methyltransferases (MTETTL3 and METTL14) and demethylases (FTO and ALKBH5) were examined. Intriguingly, we observed that the mRNA and protein expression of ALKBH5, not METTL3, METTL14 or FTO, were significantly reduced in iWAT from mice in HFD+CUR group and SVF/3T3-L1 cells upon curcumin treatment (Figs 2F, G, I and J, and EV1A). These results indicated that the elevated m6A levels might be a result of curcumin-induced ALKBH5 deficiency. To further verify the ALKBH5 mediated the effect of curcumin on adipogenesis, we performed rescue experiment and found that forced expression of ALKBH5 restored adipogenesis and triglyceride accumulation in cells inhibited by curcumin (Fig 2K). Consistently, the mRNA and protein expression of adipocyte key regulator genes, including PPARγ, C/EBPα, and FABP4, were remarkably reduced in curcumin-treated cells, which could be elevated to normal level by the overexpression of ALKBH5 (Figs 2L and EV1B). Collectively, ALKBH5 acts as a mediator of curcumin-inhibited adipogenesis. Figure 2. Curcumin inhibits adipogenesis through attenuating ALKBH5 expression A. ORO staining of SVF cells isolated from iWAT of male mice. Differentiation was induced with curcumin up to day 8 (scale bar = 500 μm). B. mRNA expression of adipogenesis-related factors of SVF cells isolated from iWAT of male mice. C. ORO staining of 3T3-L1 cells. Differentiation was induced with curcumin up to day 8 (scale bar = 500 μm). D. mRNA expression of PPARγ, C/EBPα, and FABP4 expression of 3T3-L1 cells treated with curcumin. E. LC-MS/MS quantification of the m6A/A in mRNA of iWAT of mice fed with HFD and HFD+CUR groups. F, G. mRNA and protein expression levels of m6A regulator in iWAT from mice in HFD and HFD+CUR groups. H. LC-MS/MS quantification of the m6A/A in mRNA of 3T3-L1 cells. I, J. mRNA and protein expression levels of m6A regulator in 3T3-L1 cells were analyzed by qPCR. K. Oil Red O staining of vector or OE-ALKBH5-transfected 3T3-L1 cells. Differentiation was induced with curcumin up to day 8 (scale bar = 500 μm). L. mRNA expression of PPARγ, C/EBPα, and FABP4 expression of cells described in (K). Data information: Data are shown as the means ± s.d. from three independent experiments and analyzed by t-test or one-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The expression levels of m6A regulator in differentiated SVFs upon curcumin treatment A. Western blot analysis of m6A regulator in primary SVFs and the quantification of protein levels normalized to β-actin expression. B. Western blot analysis of PPARγ and C/EBPα protein levels in cells described in Fig 2K and quantification of protein levels normalized to β-actin expression. Data information: Data are shown as the means ± s.d. from three independent experiments and analyzed by t-test or one-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. Download figure Download PowerPoint ALKBH5 modulates adipogenesis via regulating TRAF4 expression To further explore how ALKBH5 regulates adipogenesis, we conducted bioinformatics analysis of the potential target gene of ALKBH5 from GEO database, which provides RNA profile data of 3T3-L1 cells differentiation (GSE69313, GSE6794). TRAF4, a member of the TRAF family, attracted our attention, which are involved in numerous cellular physiological processes. A recent study showed that the downregulation of TRAF4 during adipogenesis of the mesenchymal stem cells (MSCs) harvested from human bone marrow was regulated by ALKBH5 (Cen et al, 2020). Thus, we hypothesized that ALKBH5 might regulate adipogenesis of mouse 3T3-L1 cells via TRAF4 expression. To this end, we extracted the mRNA expression of ALBKH5 and TRAF4 from these 34 samples and carried out Pearson's correlation analysis. Analysis revealed that during the process of adipogenesis, TRAF4 was negatively correlated with ALKBH5 expression in 3T3-L1 cells (Fig 3A). To further confirm this, we first detect TRAF4 expression in iWAT from mice in HFD+CUR group and SVF, 3T3-L1 cells, and the experiments showed a profound increase in TRAF4 protein expression upon curcumin-treated (Fig EV2A–C); next, we performed rescue experiment and observed that knockdown of TRAF4 could partially rescue the inhibition of adipogenesis and triglyceride accumulation caused by curcumin (Fig EV2D–F). To further substantiate the hypothesis, we conducted Western blot assays in 3T3-L1 cells and found that ALKBH5 depletion promoted TRAF4 protein expression (Fig 3B), and, reversely, ALKBH5 overexpression reduced protein abundance of TRAF4 in 3T3-L1 cells (Fig 3C), indicating the inverse correlation between ALKBH5 and TRAF4. To validate their upstream–downstream relationship, we silenced TRAF4 and found the protein abundance of ALKBH5 was unchanged by siTRAF4 (Fig 3D), suggesting ALKBH5 was the upstream of TRAF4. We next sought to address whether TRAF4 mediated the effect of ALKBH5 on adipogenesis. We performed rescue experiment and observed that ALKBH5 depletion inhibited lipid accumulation, whereas silencing of TRAF4 could restore the siALKBH5-induced poor adipogenesis (Fig 3E). Consistently, the mRNA and protein expression of PPARγ and C/EBPα could be restored upon depletion of TRAF4 in ALKBH5-deleted cells (Fig 3F and G). Taken together, these results demonstrate that ALKBH5 modulates adipogenesis via regulating TRAF4 expression. Figure 3. ALKBH5 modulates adipogenesis via regulating TRAF4 A. The relationship between ALKBH5 and TRAF4 was analyzed in GEO data base. Pearson's correlation analysis indicated TRAF4 and ALKBH5 had negative relationship (Cor = −0.70). B. Western blot analysis of TRAF4 protein levels in control and ALKBH5-depleted cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression. C. Western blot analysis of TRAF4 protein levels in control and ALKBH5-overexpressing cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression. D. Western blot analysis of PPARγ and C/EBPα protein levels in control and TRAF4-depleted cells after MDI-induced for 48 h and quantification of protein levels normalized to β-actin expression. E. Oil Red O staining of control, ALKBH5-depleted, and ALKBH5+TRAF4-depleted cells after induced for 8 days (scale bar = 500 μm). F. qPCR analysis of PPARγ, C/EBPα, and FABP4 expression in cell described in (E). β-Actin was used as an internal control. G. Western blot analysis of PPARγ and C/EBPα protein levels in cells described in (E) and quantification of protein levels normalized to β-actin expression. Data information: Data are shown as the means ± s.d. from three independent experiments and analyzed by t-test or one-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. TRAF4 expression contributes to curcumin-induced obesity prevention A. Protein expression levels of TRAF4 in iWAT of mice fed with HFD and HFD+CUR. B. Protein expression levels of TRAF4 in primary SVFs treated with curcumin. C. Protein expression levels of TRAF4 in 3T3-L1 adipocytes treated with curcumin. D. Oil Red O staining of rescue experiment of TRAF4 on day 8 of differentiation and the quantification of ORO staining (scale bar = 500 μm). E. Western blot analysis of PPARγ and C/EBPα protein levels in cells described in (D) and the quantification of protein levels normalized to β-actin expression. F. qPCR analysis of PPARγ, C/EBPα, and FABP4 expression in cell described in (D). β-Actin was used as an internal control. Data information: Data are shown as the means ± s.d. from three independent experiments and analyzed by t-test or one-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. Download figure Download PowerPoint ALKBH5 decreases TRAF4 protein expression by increasing its m6A level We next investigated how ALKBH5 regulates TRAF4 protein expression. Since ALKBH5 is one of the demethylases, we raise a question whether the m6A demethylase activity of ALKBH5 is necessary for suppressing TRAF4 expression. Wild-type (ALKBH5-WT) and catalytic mutant ALKBH5H204A (ALKBH5-MUT) vectors (Zhang et al, 2017) were constructed. The impact of ectopic expression of ALKBH5-WT or ALKBH5-MUT on cellular m6A level was detected by LC-MS/MS (Fig 4A). Overexpression of ALKBH5-WT decreased TRAF4 protein expression, whereas ALKBH5-MUT had no effect on TRAF4 expression compared to the empty vector (Fig 4B), implying that ALKBH5 modulated TRAF4 protein expression in a demethylase activity-dependent manner. According to the published m6A-seq data of 3T3-L1 (Zhao et al, 2014), two m6A sites were found at CDS (site #1) and 3ʹ UTR (site #2) of TRAF4 mRNA (Fig 4C). Knockdown of ALKBH5 significantly increased the m6A levels on site #2, but not site #1, of TRAF4 mRNA, indicating m6A site #2 was the target of ALKBH5 (Fig 4D). Furthermore, as expected, compared with ALKBH5-MUT or the empty vector, ALKBH5-WT overexpression markedly reduced the m6A level on site #2 of TRAF4 mRNA (Fig 4E). Next, to explore whether the m6A site on TRAF4 3’UTR was essential for ALKBH5-mediated TRAF4 expression, a dual-luciferase reporter and site-directed mutagenesis assay was performed. Dual-luciferase assays showed that the overexpression of ALKBH5-WT, but not ALKBH5-MUT, substantially increased the luciferase activity of reporter constructs containing wild-type 3ʹUTR fragments of TRAF4, compare to the control (Fig 4F). This increase was abrogated when the m6A sites were mutated (A was replaced with T) (Fig 4F). To sum up, these results illustrate that ALKBH5 targets TRAF4 and increases its m6A level at 3’UTR of TRAF4 transcript. Figure 4. ALKBH5 regulates the protein expression of TRAF4 via m6A-YTHDF1-dependent manner A. LC-MS/MS quantification of the m6A/A in mRNA of control, wild-type (WT), and mutant (MUT) ALKBH5-overexpressing cells. B. Western blot analysis of ALKBH5 and TRAF4 in control, WT, and MUT ALKBH5-overexpressing cells and quantification of protein levels normalized to β-actin expression. C. Integrative genomics viewer (IGV) plots of m6A peaks at TRAF4 mRNAs. The y-axis shows sequence read number, blue boxes represent exo
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