鼠源成纤维细胞生长因子-21对脂肪细胞糖代谢的作用

Cloning, Expression and Purification of Mouse Fibroblast Growth Factor-21 and Its Function in Adipocyte Glucose Metabolism

成纤维细胞生长因子-21(FGF-21)是FGF家族的成员之一.近年发现FGF-21是一种新的代谢调节因子.从小鼠肝脏克隆FGF-21cDNA,经测序正确后亚克隆至具有羟胺切割位点的小泛素相关修饰物表达载体上,转化宿主菌Rosetta,得到的转化子经IPTG诱导后获得稳定、高效、可溶的表达产物.表达产物经羟胺切割、透析、复性、柱层析纯化后,在每升宿主菌中可获得4mg纯度为95%的成熟鼠源FGF-21蛋白,利用葡萄糖氧化酶-过氧化物酶(POD-GOD)法在小鼠3T3-L1脂肪细胞中进行生物学活性检测.结果表明,鼠源FGF-21具有促进脂肪细胞吸收葡萄糖的作用,短期作用(1h)与胰岛素相似,长期作用(8和12h)明显优于胰岛素.这一结果为以鼠源FGF-21为模型进一步研究FGF-21的生物学活性及其在糖代谢方面的作用机理奠定了基础.

Fibroblast growth factor （FGF）-21 is a new member of FGF family. Recently, it is discovered as a potent glucose regulator and a potential drug candidate for treatment of type 2 diabetes mellitus. However the mechanism of action is not known. Mouse FGF-21 （mFGF-21） is the best model for study of the mechanism of action of human counterpart, but function of mFGF-21 has not been reported. The aim of this paper is to study the function of mFGF-21 for glucose regulation. For efficiently production of bioactive FGF-21, a Sumo-His expression vector for efficient expression of soluble recombinant proteins was constructed. A hydroxylamine cleavage site was used to substitute the Sumo protease cleavage site for economic purpose. The mFGF-21 cDNA was cloned from mouse liver and sub-cloned into the Sumo-His expression vector. The mFGF-21 was stably expressed in Rosetta host cells, the expressed products were water-soluble. The Sumo-His-mFGF fusion protein was purified by Ni-NTA Column and subsequently subjected to cleavage with hydroxylamine solution to remove the Sumo-His tag; the mature mFGF-21 was dialyzed against 20 mmol/L Tris buffer （pH 8.0） for re-nature. The mature protein with high purity was obtained. The sequencing result indicated that the mature protein consisted of 182 amino acids. SDS-PAGE gel analysis showed that the protein molecular mass was 24 ku, which was recognized by the polyclonal antibody against FGF-21. The amino acid sequence of mFGF-21 had 80% homology with that of human counterpart which was consistent with the published sequence. To examine the glucose regulation activity of mFGF-21, 3T3-L1 pre-adipocytes were differentiated into adipocytes, glucose up-take activity of mFGF-21 was examined by Glucose Oxidase and Peroxidase （GOD-POD） assay at the 14th day after differentiation when 90% of preadipocytes were differentiated into adipocytes. To validate the glucose uptake assay system commercial available human insulin was used to test the assay system. The result showed that insulin could stimulate glucose uptake of 3T3L1 adipocytes in dose-dependent manner, suggesting the glucose-uptake assay system is valid, mFGF-21 was subsequently tested in this system, the result showed that like human FGF-21 and insulin, mouse FGF-21 could also stimulate glucose uptake of 3T3-L1 adipocytes in dose-dependent manner. To examine time of action of mFGF-21, 1000 nmol/L of mFGF-21 were used to treat differentiated adipocytes for 1, 4, 8 and 12 h respectively, together with the same concentration of insulin and BSA as a positive and negative control. Glucose consumption of the medium was examined. The result showed that both insulin and mFGF-21 had tendency to increase glucose up-take of adipocytes with increment of action time. However, mFGF-21 was more potent and showed stronger time-dependent action, which was in agreement with the function of human FGF-21 as reported previously. BSA did not show any glucose uptake activity as expected. It was conclude that mouse FGF-21 is similar to human FGF-21 and possesses strong bioactivity for glucose homeostasis in 3T3-L1 adipocytes, and therefore can be used as a model for study mechanism of action of human FGF-21. The function of mFGF-21 in glucose metabolism at animal level is remained to be studied.