The Effects Of Fibroblat Growth Factor-21 In Mice On Metabolism In Vitro

PARTⅠCONSTRUCTION OF FIBROBLAT GROWTH FACTOR-21 SHRNA RECOMBINATED VECTOR IN MICEObjective: To establish fibroblast growth factor-21(FGF-21) shRNA recombinated vector in mice.Methods: Two reverse repeated motifs targeting of FGF-21 gene were designed and synthesized respectively and inserted into plasmid pGenesil1.2 for generate the recombinated vectors. The effective rate of shRNA-vector (pGenesil-FGF21) was verified by Double-Fluorescence Reporter assay system.Results: Fluorescence ratio of GFP/RFP in two groups were lower than that in HK group (P<0.05), and the lowest in the pGenesil-FGF21-1 group.Conclusion: FGF-21 shRNA recombinated vector, pGenesil-FGF21, was established successfully. PARTⅡTHE EFFECTS OF FIBROBLAT GROWTH FACTOR-21 ON METABOLISM IN 3T3-L1 ADIPOCYTESObjective: To investigate the effects of FGF-21 on glucose turnover, triglyceride metabolism and the underlying mechanisms in 3T3-L1 adipocytes.Methods: The two vectors, pGenesil-FGF21 and pcDNA-FGF-21, were transfected into the differentiated 3T3-L1 adipocytes, respectively.The FGF-21 mRNA expressions were measured by real-time quantitative PCR and FGF-21 protein contents were detected by Western Blotting.The uptake rates of 2-Deoxy[3H]-D-glucose (glucose uptake rate, GUR) were evaluated by liquid scintillation counting method (LSCM).The cellular triglyceride (TG) content was measured with a colorimetric assay. The mRNA expressions of betakloth, FGFRs, GLUT-1, GLUT-4, IRS-1,PPAR-α, PPAR-γ, HSL, ATGL, ap2, adiponectin, visfatin and Leptin were detected by real-time quantitative PCR.Results: In 3T3-L1 adipocytes, pcDNA-FGF21 significantly increased FGF-21 expression (both P<0.05). Moreover, pGenesil-FGF21 caused FGF-21 down-regulation by 77.8% in mRNA expression and 75.3% in protein contents (both P<0.05). The upregulation of FGF-21 markedly increased GUR and decreased intracellular TG content (both P<0.05). The betaknotho and FGFR1 mRNA expressions were increased in FGF-21 upregulated adipocytes (both P<0.05). Meanwhile, the upregulation of GLUT-1, IRS-1, PPARγ, PPARα, ATGL, HSL, ap2 and leptin mRNA expressions were also observed (P<0.05). The reverse changes happened in FGF-21 deficient adipocyte except IRS-1.Conclusion: In 3T3-L1 adipocytes, the effect of FGF-21 expression change is mainly through betaklotho and FGFR1. FGF-21 may be related to glucose homeostasis and accommodate intracellular fat content in adipocyte.PARTⅢTHE EFFECTS OF FIBROBLAT GROWTH FACTOR-21 ON METABOLISM IN HEPA1-6 HEPATOCYTESObjective: To investigate the roles of FGF-21 on glucose metabolism, cholesterol metabolism and the underlying mechanisms in Hepa1-6 hepatocytes.Methods: The two vectors, pGenesil-FGF21 and pcDNA-FGF-21, were transfected into Hepa1-6 hepatocytes, respectively. The FGF-21 mRNA expressions were measured by real-time quantitative PCR and FGF-21 protein contents were detected by Western Blotting.The uptake rates of 2-deoxy-[3H]-D-glucose (GUR) were evaluated by liquid scintillation counting method. The mRNA expressions of betaklotho, FGFRs, GLUT-1, GLUT-4, IRS-1,PPARα, PPARγ, PEPCK, HMGR, LDLr and visfatin were detected by real-time quantitative PCR.Results: In Hepa1-6 hepatocytes, pcDNA-FGF21 significantly increased FGF-21 expression (both P<0.05). Moreover, pGenesil-FGF21 caused FGF-21 down-regulation by 86.3% in mRNA expression and 77.3% in protein contents (both P<0.05). FGF-21 has no effect on GUR. The betaknotho and FGFR4 mRNA expressions were increased in FGF-21 upregulated hepatocytes (both P<0.05). Meanwhile, FGF-21 over-expression reduced PPARγ, PPARα, HMGR and PEPCK mRNA expression and increased LDLr expression (P<0.05), but had no effect on GLUT-1, GLUT-4, IRS-1, GK and visfatin expression in Hepa1-6 hepatocytes.Conclusion: The effect of FGF-21 expression change is mainly through betaklotho and FGFR4 in hepa1-6 hepatocytes. FGF-21 maybe plays an important role in cholesterol metabolism and liver gluconeogenesis, but no glucose uptake.