Study On The Mechanism Of Regulating The Metabolism Of Chicken Primary Hepatocytes And Preadipocytes By Affecting Mitochondrial Function And Screening Of Effective Genetic Markers

Adipose tissue is an important endocrine organ and energy storage organ of an organism,and plays a crucial role in the energy metabolism balance of the organism.Fat in poultry is mainly synthesized in the liver,and then transferred to adipose tissue for utilization and storage.In this series of processes,many transcription factors related to lipid metabolism play a significant regulating part in coordination with each other.Studies have indicated that the dysfunction of these transcription factors will lead to disorders of lipid metabolism;at the cellular level,the excessive proliferation of pro-adipocytes or their differentiation into mature adipocytes is one of the causes of fat deposition.As the "supply station" of cellular energy,mitochondria are closely connected to the metabolism of lipids.Therefore,the normal function of mitochondria is vital for maintaining the energy balance of body.Sex-linked dwarf(SLD)chickens are widely used in production due to their advantages of low feed consumption,resilient stress resistance and adaptability,but SLD chickens are also prone to excessive fat deposition.Previous studies have found that the mitochondrial function of SLD chicken’livers has changed compared to normal chickens,which may explain their excessive fat deposition.Nevertheless,how the changes in the functions of mitochondrial affect the specfic regulatory mechanism of lipid metabolism remians unknown.The purpose of this study is to elucidate the connection between lipid metabolism and mitochondrial function in poultry.Firstly,SLD chickens were used as the research object.The liver lipid levels of SLD chickens and normal chickens were detected by pathological methods,as well as the differences in mitochondrial-related functions.Secondly,we isolated chicken primary liver cells and primary precursor adipocytes,overexpressed and interfered with GHR genes in the cells.q RT-PCR was applied to detect the expression of AMPK-PGC-1α-PPARγ pathway genes,and the cell lipid levels and mitochondria were also tested.In the final analysis,the effect of abnormal GHR gene expression on preadipocyte differentiation was explored by oil red O staining.The results showed that the distribution area of lipid droplets in the livers of 15-and14-day-old SLD chickens was larger than that of normal chickens,and the triglyceride content in livers of SLD chickens was higher than that of normal chickens.The results of mitochondrial function tests showed that,compared with normal chickens,SLD chickens had lower mitochondrial membrane potential,lower ATP levels,and higher MDA levels.First,the over-expression vector and interfering fragment of GHR gene were constructed and transfected in two kinds of cells.The over-expression vector and interfering fragment detected by q RT-PCR were proved effective for both cells.Next,we overexpressed and interfered with GHR genes in two primary cells and utilized q RT-PCR to detect the m RNA levels of lipid metabolism-related transcription factors and mitochondrial oxidative phosphorylation-related genes.We figured out that GHR genes were overexpressed and lipid metabolism-related transcription factor genes were expressed and down-regulated,besides,mitochondrial oxidative phosphorylation-related genes were also down-regulated;Given interference with GHR genes,the lipid metabolism-related transcription factor gene and mitochondrial oxidative phosphorylation-related genes were both up-regulated.The results were consistent in both cells.We also examined the GHR mitochondrial-related functions of the cells,which were interfered with the GHR gene in both cells.The mitochondrial membrane potential decreased,the ATP level decreased,and the MDA level increased,which is consistent with the results of living level.Finally,the gene was overexpressed and interfered in the precursor adipocytes,and the preadipocytes were induced to differentiate.The oil triglyceride content was detected by oil red O staining.It was found that the overexpression of GHR reduced the cell triglyceride content and the degree of cell differentiation lower;on the contrary,interference with GHR increased the triglyceride content of cells and gained higner degree of cell differentiation.In summary,GHR gene affects the expression of mitochondrial oxidative phosphorylation related genes and lipid metabolism related transcription factors FAS,SREBP-1c,A-FABP and other genes through the expression of AMPK-PGC-1α-PPARγsignaling pathway gene,thereby regulating chicken lipid metabolism.Therefore,the GHR gene is of great significance for maintaining the normal function of mitochondria and the stability of lipid metabolism in chickens.