成纤维细胞生长因子(FGF)受体-2参与FGF-21介导的糖代谢活性

Fibroblast Growth Factor Receptor-2 Involved in FGF-21-mediated Glucose Metabolism

最近发现,FGF-21具有很强的调节血糖和血脂的作用,已经成为糖尿病研究领域的新热点,但是其功能受体和作用机制还不清楚.前期结果表明,FGF-21促进3T3L1脂肪细胞代谢葡萄糖,对前脂肪细胞无作用,说明脂肪细胞表达FGF-21功能受体.以3T3L1脂肪细胞为靶标,旨在寻找FGF-21的功能受体.结果表明,FGF-21可与3T3L1脂肪细胞膜蛋白形成FGF-21/受体复合物,免疫检测结果发现,FGF-21/受体复合物中含有FGF受体-2(FGFR-2).为明确FGF-21/FGFR-2的特异性关系,系统研究了FGFR-2对FGF-21刺激后的酪氨酸磷酸化反应.结果表明,虽然前脂肪细胞和脂肪细胞均表达FGFR-2,但是FGF-21只诱导脂肪细胞中表达的FGFR-2磷酸化,对前脂肪细胞表达的FGFR-2无作用,与葡萄糖吸收试验相符.FGF-21不仅可使原位表达的FGFR-2磷酸化,还可使异位表达的FGFR-2磷酸化.克隆后测序分析结果表明,FGFR-2Ⅲc是3T3L1脂肪细胞表达的主要FGFR-2类型.这些结果提示,FGFR-2Ⅲc是FGF-21的功能受体,参与FGF-21在脂肪细胞介导的糖代谢活性.此外,系统分析了FGFR-2在3T3L1分化过程中的差异表达,为FGF-21在前脂肪细胞和脂肪细胞中的功能差异提供了依据.

FGF-21 is a new member of FGF family and reported to regulate glucose metabolism, as well as lipid homeostasis, therefore, FGF-21 is a potential therapeutics for treatment of diabetes and metabolic syndrome. However, little is known about its functional receptor（s） and mechanism of action. The aim of the paper is to investigate the functional receptor（s） for FGF-21 in order to understand the mechanism of action of the molecule. Previous result showed that FGF-21 could stimulate glucose uptake in differentiated 3T3L1 adipocytes, but not in pre-adipocytes, suggesting that differentiated 3T3L1 adipocytes express functional receptor （s） for FGF-21. Therefore, the differentiated 3T3L1 adipocyte is a perfect target for searching receptor（s） of FGF-21. 3T3L1 pre-adipocytes were differentiated into adipocytes. The cell membrane of the life adipocytes were incubated with FGF-21 which was labeled with either Flag-tag or biotin, the FGF-21/receptor complexes were cross-linked once they interacted by cross-link reagent BS3. The FGF-21/receptor complexes were immunoprecipitated by either Flag antibody or streptavidin and analyzed by SDS-PAGE. The results showed that FGF-21 could form putative FGF-21/receptor （s） complexes with surface membrane protein of differentiated 3T3L1 adipocytes, not pre-adipocytes. The molecular mass of the complexes is proximate 300~400 ku. The complexes were formed by the FGF-21 labeled with either Flag-tag or biotin, suggesting the complex formation was not influenced by different labeling. To understand the component of the FGF-21/receptor（s） complexes, Western blot assay was used to examine the interaction of the complexes with known antibodies. FGFR-2 was detected within the FGF-21/receptor complexes by specific antibody for FGFR-2. To confirm the specific relationship between FGF-21 and FGFR-2, FGF-21-induced FGFR-2 tyrosine-phosphorylation was studied in 3T3L1 adipocytes. The results showed that although FGFR-2 was expressed in both pre-adipocytes and adipocytes FGF-21 could only enable adipocyte-expressed FGFR-2 tyrosine phosphorylation, which was consistent with glucose-uptake by FGF-21 in this two cell types. FGF-21 not only phosphorylated in situ adipocyte-expressed FGFR-2, but also ectopically expressed FGFR-2 in mouse pre-B cell line BaF3 cells. Sequencing analysis of FGFR-2 cDNA from adipocytes revealed that FGFR-2 Ⅲ c was the only subtype of FGFR-2 expressed in adipocytes, suggesting that FGFR-2 Ⅲc is at least one of the functional receptors for FGF-21 and involved in glucose metabolism in the cells. In order to understand the reason why the same FGFR-2 only functions in adipocyte and not in pre-adipocytes, differential expression of FGFR-2 before and after 3T3L1 cell differentiation was systematically analyzed, the result showed that adipocytes expressed high level precursors of FGFR-2, which were not observed in pre-adipocytes, suggesting FGFR-2 turn-over rate could be a key factor to determine the function the molecule. Alternatively, adipocyte-specific molecules could be another key factor participating the signal transduction of FGF-21.