Down-regulation Of Osteopontin Inhibits Browning Of White Adipose Tissue In C57BL/6 Mice And Its Mechanism

Objective: Currently,browning of white adipose tissue has positive significance in the treatment of obesity and its associated metabolic diseases.In our previous study,we have found that OPN achieves induction of brown adipocytes.In this study,specific adenoviruses(Ad-aP2-OPN-shRNA)were used to infect normal and obese mice to down-regulate OPN levels in fat metabolism-related sites,thereby explored the effects of OPN on the browning of white adipose tissue in mice and the underlying mechanisms.Methods: First,40 healthy male C57BL/6 mice were randomly divided into two groups,one of which was fed with high-fat diet(HFD).After 12 weeks,the obesity model was successfully established,and the other group was given a normal chow as a control group.Subsequently,15 obese mice were randomly divided into 3 groups and received tail vein injection of phosphate buffered solution(PBS),Vehicle and Ad-aP2-OPN-shRNA,respectively.The normal mice also performed the above operations.Finally,this study were grouped into the Ctrl group,Vehicle group,sh-OPN group,Obese group,Obese+Vehicle group,and Obese+ sh-OPN group.We finally detected the expression levels of iOPN and sOPN by Western Blotting and ELISA to determine the down-regulation of OPN expression in mice infected with Ad-aP2-OPN-shRNA.The following indicators were subsequently tested : 1)weight,length,Lee’s index and fat index(FI);2)the weight of inguinal white adipose tissue(IWAT),epididymal white adipose tissue(EWAT),perirenal white adipose tissue(PWAT),rachial white adipose tissue(RWAT),liver,heart,spleen,lungs and kidneys;3)HE staining was used to observe the cell morphology of IWAT,EWAT,PWAT and RWAT;4)fasting plasma glucose(FPG),serum insulin(FINS),triglyceride(TG),total cholesterol(TCH),insulin resistance index(HOMA-IR)and serum resistin;5)lipid accumulation in liver by oil red O staining;6)expression of PGC-1α,PRDM16 and UCP-1 in IWAT by Immunohistochemistry;7)Western Blotting assay was used to detect the protein expression levels of PI3 K,AKT,AKT-pS473 and PPARγ in IWAT.Results: 1)The iOPN levels in adipose tissue and the sOPN levels in serum of mice droped after infection with Ad-aP2-OPN-shRNA.2)After OPN was down-regulated,the mice figure and the morphology of adipose tissue in each site were larger,and the obese group was more obvious than the normal group.3)After OPN was down-regulated,there was no significant difference in weight,length,Lee’s index,and FI in normal mice,but weight and Lee’s index were significantly lower in obese mice.4)The results of HE staining showed that the cell morphology of adipose tissue in each site became larger after OPN down-regulated.5)After OPN was down-regulated,the weights of adipose tissue and organs in each part of the mice increased,but only IWAT and liver showed significant differences in the obese group.6)After the OPN was down-regulated,biochemical indicators such as FPG,FINS,TG,TCH,HOMA-IR and Resistin showed an upward trend in mice.7)The results of oil red O staining showed that the amount of lipids in the liver elevated after the OPN down-regulated.8)Immunohistochemical results showed that the expression of PGC-1α,PRDM16,and UCP-1 in IWAT impaired after OPN downregulation.9)According to Western Blotting results,the expression levels of PI3 K,AKT-pS473,and PPARγ in IWAT were reduced.Conclusion: The OPN was down-regulated in C57BL/6 mice infected with Ad-aP2-OPN-shRNA,and the browning of white adipose tissue in mice inhibited after OPN down-regulation.At the same time,we found that down-regulation of OPN inhibit the browning of white adipose tissue through PI3K-AKT pathway mediates the inhibition of PPARγ.