Study Of Latexin On Obesity Induced By High-Fat Diet In Mice And Its Related Mechanism

Obesity is characterized by an excessive expansion of white adipose tissue(WAT)that relies on hypertrophy of preexisting adipocytes and the generation of mature adipocytes through growth and differentiation of preadipocyte after adipogenesis.Early studies have shown that peroxisome proliferator activated receptor-γ(PPAR-γ)is the main regulatory molecule of adipogenesis.A key finding in the field of PPARγ is that fat mass is almost proportional to PPARγ activity.And PPARγ/RXR forms heterodimers and regulates the transcription of genes involved in insulin action,adipocyte differentiation,lipid metabolism and inflammation.Latexin(LXN)is the only mammalian carboxypeptidase inhibitor with 222 amino acids in length,which was frst identifed in the lateral neocortex of rats.Aagaard et al.reported that LXN is implicated in the infammatory response.At the same time,it has been shown that Latexin acts endogenously in HSCs to negatively regulate their population size by enhancing apoptosis and by decreasing self-renewal.There are multiple LXN promoter regions PPARA: RXRA Binding sites.These results suggest that LXN gene expression may be regulated by PPAR and retinoic acid.Therefore,we speculate whether LXN can affect the obesity induced by high-fat diet in mice by regulating the differentiation of preadipocytes.First,we analyzed the expression of LXN in obese mice by Western Blot and tissue section staining.Firstly,the expression of LXN in liver and adipose tissue of WT mice and ob / ob mice was detected.Western Blot results showed that LXN expression in liver and adipose tissue of ob /ob mice was significantly increased.Compared with normal diet mice,the expression of LXN in liver,heart and adipose tissue of wild-type high-fat diet induced obese mice increased significantly,and the expression of LXN in liver and adipose tissue increased with the increase of high-fat feeding time.The above results indicate that LXN can participate in the occurrence and development of obesity in mice.In order to further study the effect of LXN on high-fat diet induced obesity,we constructed high-fat diet induced obesity models in wild-type C57 BL / 6(WT)mice and LXN gene knockout(KO)mice.Compared with WT mice,the weight of KO mice increased slowly,and the volume and weight of white adipose tissue in liver and body were significantly reduced.The adipose tissue of each part was selected to make paraffin section and stained with H&E.It was observed that the adipocyte area of KO mice was significantly reduced.The results of oil red O staining showed that compared with WT mice,the accumulation of lipid droplets in KO mice was decreased.RT-PCR was used to detect the expression of inflammatory related factors(i NOS,IL-1 β and TNF-α)in the liver of KO mice.We found that the mRNA level of inflammatory related factors in the liver of KO mice was significantly decreased.These results confirm that LXN deficiency alleviates nonalcoholic fatty liver disease caused by obesity.At the same time,glucose tolerance and insulin resistance experiments showed that KO mice could significantly improve grape tolerance and insulin resistance.These results suggest that LXN deficiency can improve obesity induced by highfat diet in mice.Secondly,we studied the relationship between LXN and preadipocyte differentiation and its related mechanism.The primary preadipocytes of WT and KO mice were obtained and differentiated into mature adipocytes.Oil red O staining showed that LXN knockout could significantly inhibit the differentiation of preadipocytes into mature adipocytes.At the same time,we used 3T3-L1 as a preadipocyte.Overexpression of LXN can promote the differentiation of preadipocytes,whereas knockdown of LXN can inhibit the differentiation,which is consistent with the results of primary preadipocytes.Western Blot analysis showed that LXN and PPARγwere closely related to the differentiation of preadipocytes.At the same time,the phosphorylation levels of mTOR and AKT were increased.To further clarify the signal pathway of LXN expression regulation,we used Perifosine(mTOR inhibitor),rapamycin(AKT inhibitor)and GW9662(PPARγ inhibitor)respectively treatment of 3T3-L1 cells were induced to differentiate.The results showed that mTOR and PPARγ were inhibited.It can inhibit the expression of LXN more significantly.At the same time,PA452(RXR inhibitor)also inhibited PPARγ expression and LXN expression efficiently in 3T3-L1 cells.These results suggest that the mTOR/RXR/PPARγ signaling pathway mediates the increase of LXN expression during the differentiation of preadipocytes.To further investigate the mechanism of LXN affecting preadipocyte differentiation,we overexpressed or knocked down LXN in 3T3-L1 cells.Western Blot analysis showed that the stable expression of flag LXN in 3T3-L1 cells resulted in a significant increase of PPARγ protein level,whereas the loss of LXN resulted in a decrease of PPARγ protein level.However,RT-PCR results showed that the mRNA level of PPARγ did not change with the knockdown of LXN,suggesting that LXN may affect the expression of PPARγ at the protein level.Studies have shown that FABP4 negatively regulates PPARγ by promoting ubiquitination and proteasome degradation of PPARγ.Therefore,we speculate that LXN may down regulate PPARγ through FABP4 mediated ubiquitination.Western Blot results showed that the expression of FABP4 was significantly upregulated and the expression of PPARγ was down regulated when LXN was knocked down.Under the same conditions,the expression of PPARγ increased after 3T3-L1 cells were treated with BMS(FABP4 inhibitor).These results suggest that the regulatory effect of LXN on PPARγ is mediated by FABP4.In vitro ubiquitination experiments showed that the loss of LXN in 3T3-L1 cells increased the ubiquitination of PPARγ,and the ubiquitination of PPARγ was inhibited by the addition of BMS.These results suggest that LXN deletion can inhibit the differentiation of preadipocytes by increasing the expression of FABP4 and promoting the ubiquitination of PPARγ.In conclusion,this project aims to study the effect of LXN on high-fat diet induced obesity in mice,analyze the regulatory mechanism of LXN on preadipocyte differentiation,and provide new ideas for the prevention and control of obesity and related syndromes.