FGF9 inhibits browning program of white adipocytes and associates with human obesity

in Journal of Molecular Endocrinology
Correspondence should be addressed to J Wang or J Hong: wangjq@shsmu.edu.cn or hongjie13d@hotmail.com

*(Y Sun and R Wang contributed equally to this work)

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Browning of white adipose tissue has been proven to be a potential target to fight against obesity and its metabolic commodities, making the exploration of molecules involved in browning process important. Among those browning agents reported recently, FGF21 play as a quite promising candidate for treating obesity for its obvious enhancement of thermogenic capacity in adipocyte and significant improvement of metabolic disorders in both mice and human. However, whether other members of fibroblast growth factor (FGF) family play roles in adipose thermogenesis and obese development is still an open question. Here, we examined the mRNA expression of all FGF family members in three adipose tissues of male C57BL/6 mice and found that FGF9 is highly expressed in adipose tissue and decreased under cold stress. Furthermore, FGF9 treatment inhibited thermogenic genes in the process of beige adipocytes differentiation from stromal vascular fraction (SVF) in a dose-dependent manner. Similar results were obtained with FGF9 overexpression. Consistently, knockdown of FGF9 in SVF cells by using lentiviral shRNA increased thermogenic genes in differentiated beige adipocytes. RNA sequencing analysis revealed a significant increment of hypoxia-inducible factor (HIF) pathway in the early stage of beige adipocytes differentiation under FGF9 treatment, which was validated by real-time PCR. FGF9 expression was increased in subcutaneous WAT of obese human and mice. This study shows that adipose-derived FGF9 play as an inhibitory role in the browning of white adipocytes. Activation of hypoxia signaling at early stage of adipose browning process may contribute to this anti-thermogenic effect of FGF9.

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  • Supplementary Figure 1. A-H, mRNA level of adipogenic genes in response to different FGF9 treatment during white adipogenesis. I-J, mRNA level of FGF9 in full-differentiated mice SVF isolated from iWAT (I) and eWAT (J) in response to CL316243.
  • Supplementary Figure 2. A Cluster images of global sample distribution and relationships analyzed by t-distributed stochastic neighbor embedding. B-D, Volcano plots of differential genes in response to FGF9 treatment. E, Differential genes induced by FGF9 at the very beginning of differentiation stage (Day 0). F, Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis based on the downregulated genes in Fig. 5H. G, KEGG pathway enrichment analysis based on consistently downregulated genes in day 1 and day 2 after differentiation.

 

      Society for Endocrinology

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    FGF9 was highly expressed in adipose tissues. (A, B and C) mRNA levels of FGF family members in brown adipose tissue (n = 3) (A), inguinal white adipose tissue (n = 5) (B) and epididymal white adipose tissue (n = 6) (C). (D) mRNA levels of Ucp1 expression in fully differentiated SVF (day 8 after incubation) isolated from inguinal white adipose tissue of mice (n = 4) under browning incubation in response to treatment of FGF9, FGF10, and FGF23. (E) mRNA level of FGF9 in multiple tissues of mice (n = 3–6).

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    FGF9 inhibits adipose thermogenesis in a dose-dependent manner. (A) mRNA level of FGF9 expressed in iWAT in mice (n = 3) under cold condition (4°C). (B) mRNA level of FGF9 expressed in eWAT in mice (n = 10) in response to CL316243 (1.5 mg/kg, 10 days injection). (C) Oil Red staining of full-differentiated SVF (8th day after incubation) toward brown in the absence and presence of FGF9 (100 ng/mL). (D, E, F, G, H, I, J and K) mRNA of thermogenic biomarkers (D, E, F and G) and adipogenic biomarkers (H, I, J and K) in full-differentiated SVF isolated from mice iWAT in response to gradient concentration of FGF9. (L and M) Protein level of UCP1 and oxygen consumption rate (OCR) of full-differentiated SVF in response to FGF9 treatment (100 ng/mL). (N) mRNA level of browning genes in full-differentiated human SVF under different dose of FGF9. (O) mRNA level of thermogenic and adipogenic genes profile under condition of FGF9 overexpression. (P) Protein level of UCP1 and PGC1a in response to FGF9 overexpression. (Q) Oxygen consumption rate in response to FGF9 overexpression.

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    FGF9 suppression promotes browning of white adipocytes. (A) mRNA expression of Fgf9 in different time points during browning differentiation under two Fgf9-shRNA treatments. (B) Morphological changes of full-differentiated WAT-derived SVF in response to knockdown of endogenous FGF9 by two shRNAs. (C) mRNA changes of thermogenic biomarkers, adipogenic biomarkers and FGF9 in full-differentiated iWAT-derived SVF under condition of knockdown FGF9. (D) Protein change of UCP1 and PGC1a in response to FGF9 knockdown. (E and F) Dynamic (E) and quantitative (F) change of OCRs in response to FGF9 knockdown.

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    FGF9 suppresses thermogenic genes from the early stage of adipocytes differentiation, following a provocation of HIF1α pathway. (A, B, C, D and E) Dynamic change of thermogenic genes over the whole process of browning in the presence and absence of FGF9. (F) Schematic design of RNA sequencing to explore underlying mechanism of FGF9, in which 100 ng/mL FGF9 was added in full course over browning process, and the differentiating cells in three time points, including day 0, day 1 and day 2, were collected and undergone sequencing analysis. (G) Cluster images of global sample distribution and relationships analyzed by hierarchical clustering. (H) Heatmap generated using the genes exclusively upregulated by FGF9 treatment in day 1 and day 2 after differentiation initiated. (I) Visualization of the upregulated pathway produced by KEGG enrichment analysis based on the differential genes in Fig. 5H. (J) KEGG enrichment analysis of genes consistently and exclusively upregulated by FGF9 in day 1 and day 2 after differentiation. (K) mRNA level of genes related to HIF1a pathway in the presence and absence of FGF9 during the browning differentiation process.

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    Adipose-derived FGF9 expression increases in subcutaneous adipose tissue of obese human and mice. (A and B) mRNA level of FGF9 in subcutaneous adipose tissue (sWAT) and visceral adipose tissue (vWAT) isolated from patients with obesity (n = 21) and lean subjects (n = 11). (C and D) mRNA level of FGF9 in iWAT and eWAT isolated from ob/ob mice (n = 11) and their counterpart control (n = 11). (E and F) mRNA level of FGF9 expressed in iWAT and eWAT isolated from mice fed with HFD (n = 3) and normal chow (n = 3). (G and H) mRNA level of FGF9 expressed in mature adipocyte (AD) and SVF in mice fed with HFD and normal chow.

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