The Regulatory Mechanism Of Fibroblast Growth Factor 21 And AdipoRon On Adipogenensis And Lipid Metabolism

Pork is the major meat product consumed in China.With the development of modern life,people are increasingly preferring high quanlity meat products,such as more red and better flavor,which leads to molecular breeding appears.And it is important to find the high meat quanlity-related DNA makers and artificial small molecules.Fibroblast growth factor 21(FGF21),a kind of peptide hormone,is synthesized and secreted from different tissues and plays an important role in regulation of energy homeostasis.Numbers of exciting date suggested the metabolic beneficial effects of FGF21 has been documented,such as weight loss and improvement of glycemia.In the liver tissue,FGF21 is involved in regulation of fatty acid oxidation in mice under fast and high-fat fed state.FGF21 also plays an important role in glucose metabolism and improves the browning effects in white adipose tissues.However,the studies about the effects of FGF21 on intramuscular fat content and feed additives beneficial to improving the content of intramuscular fat are rarely reported.AdipoRon,an agonist of adiponectin receptors,is involved in lipid and enrgy metabolism,improves insulin sensitivity,and is a candidate for insulin resistance related disease.In our study,we focused on the regulatory mechanism of FGF21 and small artificial melocular AdipoRon on lipid metabolism.The results are as follows: 1.In order to understand the role of FGF21 in IMF deposition,we isolated the intramuscular fat(IMF)cells by ―speed difference adhesion‖.The cells were similar to fibroblasts and there were no lipid droplets in the cytoplasm.2.The cells in good state were was induced on the 8th day by IBMX + DEX + insulin,and then the oil red staining was performed.A significant amount of lipid droplets generated in the cytoplasm was observed under the inverted microscope which provided direct evidence that the cells we isolated were intramuscular preadipocyte cells.3.The stable transfection was performed,and we acquired the FGF21 stable monoclone(FM)after 10 days of screening by G418.The qRT-PCR analysis revealed that FM cells had higher FGF21 mRNA level than that in the control,and western blot analysis showed that the FGF21 protein level was up-regulated significantly in FM cells.4.The oil red staining was performed in both FM and control groups which were induced to mature for 8 days,and the results showed less lipid droplets in FM cells than that in the control.5.In order to measure the difference of the adipogenesis genes expression between FM and Control groups,the cDNA and total protein were made.The q-PCR data suggested the mRNA expression of the key genes PPARγ was down-regulated dramatically in FM compared with the control.The mRNA expressions of CEBPα involved in regulation of PPARγ expression was also decresed significantly.Moreover,PPARγ and CEBPα proteins are delined dramatically.Despite of no change in CEBPδ mRNA,the protein level was significantly lower.There were no significant changes in the mRNA levels of KLF2,SIRT1,SIRT2 and FOXO1,which could regulate PPARγ expression.There was no significant difference in the expression of COUP-TF II,which could regulate PPARγ expression,between FM and control.Unexpectedly,there was a higher level in KLF3 mRNA and a lower level in KLF5 mRNA.The ratio of FAS and HSL was down-regulated although it did not reach a significant level.6.We proved FABP4 and PLIN1 declined dramatically in mRNA level.However,there was no significant change in GLUT4 and ADIPOQ.Western blot analysis demonstrated FABP4 and ADIPOQ protein were rapidly decreased.The qPCR analysis demonstrated FGF21 did not change the Wnt/β-catenin pathway.No significant difference of methylation level between FM and the control was found in the region from-172 to +2 bp in CEBPA promoter with the Quantification Tool for Methylation Analysis on line.The EMSA and CHIP analysis demonstrated CEBPβ can bind to FGF21 promoter to down-regulate FGF21 mRNA expression.7.We further explored the regulation of a small molecule AdipoRon on PPARα signaling resulting in increasing FGF21 concentration of blood circulation,which led to whole body insulin sensitivity.Our data suggested there was no difference in the total lipid content was after 10mg/kg AdipoRon treatment for 10 days in C57BL/6 regular chow diet-fed wild type mice.In addition,there was no significant change in lipolysis related protein expression in white adipose tissue.Moreover,10mg/kg AdipoRon treatment suppressed lipolysis-related proteins expression in high fat diet-fed mice,leading to reductions of palmitate and glycerol turnover.Due to the reduction of acetyl CoA from fatty acid β oxidation,the activity of PC was decreased,resulting in the decrease of endogenous glucose production.Interstingly,8.The Western blot was carried out to measure the protein of PPARα in liver tissues.The data suggested AdipoRon activated PPARα signaling to increase FGF21 expression and secretion,leading to 1.36 times increase in plasma FGF21 concentration,which suppressed lipolysis in white adipose tissue(WAT).Our study focoused on the regulatory mechanism of FGF21 on lipid metabolism in IMF and further suggested AdipoRon improved insulin sensitivity by activating FGF21 expression in liver,leading to redistribution of lipid in vivo.It’s important to high intramuscular fat-pig breeding.