Anti-obesity Effect Of Wisp2 Gene Deficiency On Obese Mice Induced By High Fat Diet And Its Potential Mechanism

Objective The aim of the present study was to investigate the effects of Wisp2on obesity and to elucidate the potential underlying mechanism.Our results provided new molecular mechanism and experimental basis for obesity treatment.Materials and MethodsWisp2 knockout(Wisp2^-/-)mice were used in this study.Wild type（WT）mice and Wisp2^-/-mice were fed with normal chow diet（ND）or high-fat diet（HFD）.Changes in body weight were measured weekly,when HFD WT mice reached the obesity index（more than 20%of normal body weight）,the above four groups of mice were killed at the same time to obtain experimental materials.1.Genotyping identification of newborn mice Taking the tail tip tissue from mice,used the commercial kit（Beyotime D7283M,Nanjing,Jiangsu,China）to extract the DNA,after PCR reaction,loading the sample to perform agarose gel electrophoresis detection.2.Identification of Wisp2 knockout effect Extracted the RNA of iWAT and eWAT by trizol,and detected the relative expression of Wisp2 m RNA by qPCR.Extracted the protein of iWAT,western blot was used to detect the expression of Lac Z.Cryosections of iWAT were subjected to immunohistochemistry to observe the expression of Lac Z.3.Effect of Wisp2 knockout on obesity characterization in mice Changes in body weight were measured weekly.The percentage per whole body weight was calculated for every iWAT and eWAT tissue samples.Body fat content of mice were assessed by nuclear magnetic resonance（NMR）.4.Effect of Wisp2 knockout on lipid metabolism Serum total cholesterol,low-density lipoprotein（LDL）,and high-density lipoprotein（HDL）were carried out using HITACHI 7100 Automatic Analyzer（HITACHI）.The cryosections of liver were detected by oil red O staining.5.Effect of Wisp2 on adipocyte differentiation iWAT cryosections and eWAT paraffin sections were used by haematoxylin-eosin（H&E）staining to observe the morphology of adipocyte.Primary mesenchymal stem cells of Wisp2^+/+（normal control）and Wisp2^-/-were isolated from iWAT of WT and Wisp2^-/-mice.Infected3T3L1 cells with negative control（NC）lentivirus and overexpressed Wisp2 lentivirus and selected the corresponding 3T3L1 cell line by antibiotic screening.Cell Titer-Lumi^TMluminescence method was used to detect the proliferation and oil red O staining was used to observe the differentiation of the two groups of cells.6.Effect of Wisp2 knockout on diet Changes in food intake were monitored weekly.Detected Mrap2 m RNA expression level in hypothalamus,iWAT and eWAT by qPCR.ResultsWe first used PCR to identify genotypes of newborn mice,and then used Real-time qPCR to verify the selected Wisp2 gene knockout mice.The result showed that in normal diet and high-fat diet,Wisp2 m RNA in the iWAT and eWAT of knockout mice was almost undetectable.Western blot and immunohistochemical results showed that Lac Z was hardly expressed in WT mice,but expressed abundantly in Wisp2^-/-mice.After five months of high-fat diet,the weight gain of Wisp2^-/-mice was significantly lower than WT mice（P<0.05）,the ratio of iWAT to eWAT of Wisp2^-/-mice was significantly higher than WT mice（0.859±0.136 vs 0.722±0.138,P<0.05）,and the body fat rate was significantly lower than WT mice（0.336±0.02 vs 0.381±0.009,P<0.01）.Blood lipid test results showed that Wisp2 knockout reduced plasma total cholesterol（TC）,low-density lipoprotein（LDL）,high-density lipoprotein（HDL）levels compared with WT mice after high-fat diet（2.643±0.702 mmol/L vs 3.083±0.629 mmol/L,NS;0.329±0.042 mmol/L vs 0.415±0.112 mmol/L,P<0.05;1.702±0.523 mmol/L vs 2.11±0.248 mmol/L,P<0.05）.Oil Red O staining suggested that high-fat diet caused a large increase in lipid droplets in the liver of WT mice,the lipid deposition in the liver of Wisp2^-/-mice was significantly lower than WT mice.We performed HE staining on frozen sections and paraffin sections of iWAT and eWAT,and found that in normal diet,whether in iWAT or eWAT,the adipocyte morphology of Wisp2^-/-mice was significantly smaller than that of WT mice（68.8±2.168 cells vs35.2±1.924 cells,P<0.05,field of vision（200X）;34±1.581 cells vs 18±1 cells,P<0.05,field of vision（200X））.After high-fat diet,the cells became larger,but there was no difference within the group（14.4±1.817 cells vs 24.4±1.14 cells,11±2.345cells vs 16.8±1.643 cells,NS,field of vision（200X））.Wisp2 knockout inhibited the proliferation and differentiation of adipose-derived mesenchymal stem cells,overexpression of Wisp2 promoted the proliferation and differentiation of adipose precursor cells.Finally,we observed the food intake of the mice,the result showed that in normal diet,there was no significant difference in weekly energy intake between WT mice and Wisp2^-/-mice,while in high-fat diet,the weekly energy intake of WT mice was significantly higher than that Wisp2^-/-mice（109.957±6.714 kcal vs105.172±6.765 kcal,P<0.05）.After high-fat diet,the expression of Mrap2 in iWAT,eWAT and hypothalamus of Wisp2^-/-mice were significantly increased compared with WT mice（P<0.01,P<0.01,P<0.05）.Conclusions1.Wisp2 gene knockout resisted high-fat diet induced weight increase,up-regulated iWAT/eWAT ratio,and significantly reduced body fat rate.2.Wisp2 gene knockout reduced the content of plasma TC and LDL in high-fat diet.3.Wisp2 gene knockout reduced the deposition of lipid in liver in high-fat diet.4.Wisp2 gene knockout made iWAT and eWAT cells smaller in normal diet.5.Wisp2 gene knockout inhibited he proliferation and differentiation of adipose precursor cells.6.Wisp2 gene knockout reduced the food intake in high-fat diet,and up-regulated the expression levels of Mrap2 in iWAT,eWAT and hypothalamus.