Study On The Regulation And Mechanism Of Wisp2 Gene Knockout On Adipose Tissue Of Obese Mice Induced By High-fat Diet

Objectives1.To explore the effect of Wisp2 gene knockout on the phenotype of obesity in mice induced by high-fat diet.2.To explore the possible molecular mechanisms of Wisp2 gene knockout to improve the adipose tissue dysfunction induced by high-fat diet.Materials and MethodsWe established four groups of models in this study,named the normal diet wild group(ND Wisp2^+/+),the normal diet Wisp2 knockout group(ND Wisp2^-/--),the high-fat diet wild group(HFD Wisp2^+/+)and the high-fat diet Wisp2 knockout group(HFD Wisp2^-/--).The treatment time of each group was 24 weeks.Adipose derived mesenchymal stem cells and preadipocytes 3T3L1 cells were used in the in vitro experiments.The cells overexpressed Wisp2 were obtained by lentivirus infection,mature adipocytes were obtained by induction of differentiation,and then overload lipids to get hypertrophic adipocytes.1.Immunohistochemistry:to detect the expression of Lac Z protein in mouse adipose tissue.2.Nuclear Magnetic Resonance:to detect the body fat rate of mice.3.HE staining:to detect the cell morphology of adipose and liver tissues.4.Oil red O staining:to detect the fat content of liver.5.Cell Titer-Lumi?luminescence method and crystal violet staining method:to detect the activity and proliferation of adipose precursor cells.6.Western blot method:to detect the expression of Lac Z,Flag,and proteins related to apoptosis,adipocytes differentiation,and lipid droplet synthesis,etc.7.Real-time PCR:to detect m RNA expression of Wisp2 and other genes related to adipocytes differentiation,lipid droplet decomposition,and adipose tissue inflammation.ResultsFirstly,we verified Wisp2 knockout mice at DNA level,RNA level,and protein level.The results showed that wild mice(Wisp2^+/+)didn’t express lac Z,while Wisp2knockout mice expressed lac Z but not Wisp2.These indicated that the construction of Wisp2 knockout mice was successful.In addition,we also verified the cell model of overexpressing Wisp2.The results of Real-time PCR and Western Blot proved that we had successfully constructed Wisp2 overexpressed 3T3L1 cells.After 6 months of high-fat diet,the phenotype of Wisp2 knockout mice induced by high-fat diet was as follows:the weight gain of Wisp2^-/-mice was significantly lower than that of Wisp2^+/+mice（P<0.05）,the body fat rate and e WAT/i WAT ratio were also significantly lower than those of Wisp2^+/+mice at the same time（0.342±0.002 vs 0.385±0.001,P<0.05;1.127±0.020 vs 1.545±0.025,P<0.01）.The paraffin sections and frozen sections of e WAT and i WAT were stained with HE dyeing.It was found that under normal diet conditions,whether in e WAT or in i WAT,the adipocytes size of Wisp2^-/-mice was significantly smaller than that of Wisp2^+/+mice（109.3±2.0 vs 57.7±0.5 cells per 200×field,P<0.001;118.4±0.8 vs 61.6±0.5 cells per 200×field,P<0.001）.Adipocytes was obviously hypertrophic after high-fat diet,however,Wisp2 gene knockout significantly reduced adipocytes hypertrophy induced by energy excess（71.0±1.0 vs21.3±0.5 cells per 200×field,P<0.001;68.6±0.6 vs 35.2±0.4 cells per 200×field,P<0.001）;oil red O staining showed that high-fat diet increased lipid droplets in the liver of Wisp2^+/+mice,and lipid deposits in the liver of Wisp2^-/-mice were significantly reduced compared with Wisp2^+/+mice.To explore the mechanism under the phenotype,we found that:Wisp2 gene knockout inhibited not only the proliferation of adipose-derived mesenchymal stem cells（P<0.05）,but also the apoptosis of them by decreasing the expression of Caspase3 and Bax;Wisp2 gene knockout promoted adipose differentiation significantly by increasing the expression of peroxisome proliferator-activated receptorγ（PPARγ）and glucose transporter 4（GLUT4）（P<0.05）,and the adipocytes of Wisp2^-/-mice were obviously smaller than those of wild mice,so Wisp2 gene knockout presented a role of promoting adipogenesis;we also found Wisp2 gene knockout inhibit the expression of acetyl-Co A carboxylase（ACC）in adipose tissue which reduced lipid synthesis,and increase the expression of adipose triglyceride lipase（ATGL）which promoted lipid breakdown（P<0.001）,thereby improving the abnormal lipid metabolism and inhibiting lipid accumulation in high-fat diet conditions;finally,we tested the effect of Wisp2 on adipose tissue inflammation,the results showed that Wisp2 gene knockout inhibited the inflammation of adipose tissue by reducing the expression of inflammatory factors tumor necrosis factorα（TNFα）and interleukin 6（IL-6）（P<0.05）,while overexpression of Wisp2 had the opposite results.Conclusions1.Wisp2 gene knockout improves high-fat diet induced mice obesity.2.Wisp2 gene knockout improves high-fat diet induced adipose tissue dysfunction by promoting adipogenesis,inhibiting adipocytes hypertrophy;improving lipid metabolism,reducing lipid accumulation;and inhibiting adipose tissue inflammation.