Effect Of Adipose Tissue-specific Knockout Of YAP Gene On Obesity In Mice And Its Mechanism

Objective:Obesity has become an important social public health issue,not only cardiovascular disease,diabetes,tumors and other high-risk causative factors.obesity complications such as impaired glucose tolerance,hyperlipidemia,hypertension,atherosclerosis,etc.are also clinical treatment problems.Obesity refers to the hypertrophy and/or increase of fat cells caused by a variety of factors,and the deposition of triglycerides is the main cause of fat cell enlargement.The Hippo pathway is a signaling pathway found in drosophilid for the first time to maintain apoptosis balance and proliferation,and control organ size,mainly by regulating the transcriptional activity of its downstream transcription factor YAP.At present,there are few studies on the role of YAP in adipose tissue and the research conclusions are inconsistent,most of them focus on the effect of YAP in the process of obesity,and lack of corresponding exploration of other functions of YAP in fat metabolism.In previous studies,it was found that the expression of YAP was downregulated during the adipogenic differentiation of 3T3 cells,which indicated that YAP may be closely related to lipid metabolism.So we used adipose tissue-specific YAP knockout mice to study the effect of YAP on lipid metabolism at the animal level and explore some of the mechanisms.Method:1.Construction and reproduction of YAPKO mice(1)YAPflox mice and adipoq-cre tool mice were used to construct adipose tissue YAP specific knockout mice(YAPa KO mice).(2)Verify that mice are knocked out successfully with Westerm Blot 、RT-q RCR and agarose gel electrophoresis.2.Mice of YAPa KO promote obesity and impaired glucose tolerance(1)A model of dietary obesity in mice was constructed using 60% high-fat feed,and the weight curve and feed consumption curve of mice during the period were recorded to determine the effect of YAPa KO on the obesity process of mice.(2)Collecting mouse fat tissue,recording mouse adipose tissue weight,and determining YAPa KO will lead to an increase in mouse adipose tissue weight.(3)The mouse glucose tolerance experiment was used to compare the glucose tolerance ability of the two groups of mice.3.YAPa KO promotes obesity by inhibiting adipose tissue lipolysis in mice(1)Using fasting to construct a model of increased lipolysis in mice,YAPa KO or YAPflox mice were collected serum after fasting for 48 hours,and the level of free fatty acids in the serum of the two groups of mice after fasting was detected.(2)After collecting mouse adipose tissue,this study examined the expression of adipolysis gene(ATGL,HSL,LPL)and lipolysis protein ATGL in mouse adipose tissue,and looked for the mechanism of inhibition of lipolysis after YAPa KO.Result:1.Construction and breeding of YAPKO mice(1)A group of mice with no treatment for about 9 weeks were collected from white adipose tissue、brown adipose tissue、liver、kidney and heart,and YAPKO mice were detected to have downregulated YAP expression at the protein level and gene level in white adipose tissue and brown adipose tissue.There was no difference in expression on the heart、kidneys and liver.2.YAPa KO promoted obesity in mice,resulting in increased adipose tissue weight,increased abdominal blood glucose and impaired glucose tolerance in mice(1)In the process of 60% high-fat diet,YAPa KO promoted the obesity of mice,and the weight of subcutaneous adipose tissue increased significantly after collecting YAPa KO,and the epididymal adipose tissue tended to increase.(2)Through IPGTT experiments,it was found that the fasting blood glucose of YAPa KO mice was elevated and glucose tolerance was impaired,but it had no effect on random blood glucose.3.After YAPa KO,the expression of adipose tissue was inhibited by inhibiting the expression of lipolytic genes(ATGL,HSL,LPL),thereby promoting obesity in mice(1)Using fasting to build a model to increase adipose tissue lipolysis in mice,it was found that the level of FFA in the serum of mice was downregulated after YAPa KO,and the weight of adipose tissue in the YAPa KO group was heavier than that in the YAPflox group.(2)Western Blot and RT-q PCR detected the expression of adipolysis genes in mouse adipose tissue,and found that YAPa KO downregulated the lipolysis genes ATGL,HSL and LPL in mouse subcutaneous adipose tissue.It was proved that the obesity in mice after YAPa KO was caused by reduced lipolysis.4.YAPa KO down-regulates adipose tissue lipolytic function and leads mice to intolerance to cold exposure under fasting(1)After increasing lipolylysis of mice using fasting,mice were placed in a 4°C freezer during which the temperature of mice was measured at a frequency of 1time/h,and it was found that YAPa KO downregulated the temperature of mice,which proved that the mice could not tolerate cold exposure under starvation after YAPa KO.In the case of normal diet,YAPa KO had no effect on tolerance to cold exposure,indicating that YAPa KO did not affect the function of BAT.Conclusion:(1)YAPa KO promoted obesity in mice and impaired glucose tolerance in mice,which may be related to YAPa KO reducing the expression of ATGL,HSL and LPL in adipose tissue and inhibiting the lipolysis of adipose tissue.