The Mechanism Of TPT1 Regulating Differentiation Of Sheep Preadipocytes

Mutton is a kind of meat with high protein and low cholesterol,which can supplement amino acids and vitamins needed by human body and is widely popular among consumers.Fat is an important tissue to maintain the balance of energy metabolism,but excessive fat deposition in the tail is not conducive to the increase of mutton yield.TPT1 is a widely expressed gene in various cell types.Previous studies have shown that inhibition of TPT1 during differentiation of porcine preadipocytes can promote lipid droplet formation.However,the mechanism of TPT1 regulating ovine preadipocyte differentiation remains unclear.In this study,seven kinds of tissues were used as the research objects,the sequence characteristics of TPT1 and the structure and function of its encoded protein were revealed by bioinformatics analysis software;the spatial expression of TPT1 was detected by RT-q PCR.Tail,subcutaneous and perirenal fat were used as the research objects,the expression of TPT1 in different adipose tissues were detected by RT-q PCR.Sheep preadipocytes were used as the research objects,the expression trend of TPT1 during ovine preadipocyte differentiation was detected by Western blotting;the subcellular localization of TPT1 in preadipocyte was explored by cell immunofluorescence protein test;the function of TPT1 in ovine preadipocyte was explored by the test of TPT1 overexpression and inhibition mediated Lentivirus packaging;The effect of TPT1 on Fox O1 was detected by RT-q PCR and Western blotting,and the PI3K/Akt pathway inhibition test was used to explore whether this pathway is involved in the differentiation process regulated by TPT1.The purpose of this study is to reveal the function and regulatory mechanism of TPT1 in the differentiation of ovine preadipocytes.The main results are as follows:1.Bioinformatics analysis of sheep TPT1The length of sheep TPT1 is 660 bp,which has the highest similarity with goat(99.7%).It shows that sheep and goat are homologous.TPT1 encodes 219 amino acids,and the total average hydrophilicity of TPT1 protein is-0.374,which shows the protein is hydrophilic.The protein is mainly located in mitochondria and nucleus,suggesting that it may function in different parts of the cell.TPT1 contains 16 potential phosphorylation sites,suggestting that the protein may function by protein phosphorylation.2.Spatiotemporal expression and subcellular localization of sheep TPT1The expression of TPT1 mRNA was the highest in kidney,followed by muscle,liver and fat,and the lowest in heart and spleen.The results showed that TPT1 plays different roles in different tissues.The expression of TPT1 was the highest in perirenal,subcutaneous and tail fat.In the process of preadipocyte differentiation,the expression of TPT1 first increased,then decreased,and then increased.TPT1 was localized in the nucleus of sheep preadipocytes.It is suggested that TPT1 mainly plays its function in the nucleus of adipocytes.3.Sheep TPT1 promotes the differentiation of preadipocytesAfter overexpressing TPT1,the mRNA expression of PPARγ,Adipo Q and FABP4 were significantly up-regulated,But C/EBPα was no significant change,and the lipid droplet production was increased;after inhibiting TPT1,the expression of these genes was decreased,and the lipid droplet production was decreased.These results indicate that TPT1 can regulate the expression of key genes in adipogenic differentiation,thus promoting the differentiation of adipocytes and the formation of lipid droplets.4.TPT1 promotes sheep preadipocyte differentiation through Fox O1Overexpression of TPT1 significantly down-regulated Fox O1 and Fox O1 protein;on the contrary,inhibition of TPT1 significantly increased Fox O1 and Fox O1 protein.These results suggest that TPT1 can promote the differentiation of sheep preadipocytes by down regulating FoxO1.5.TPT1 promotes the differentiation of sheep preadipocytes through PI3K/Akt pathwayAfter overexpression of TPT1,the expression of p-Akt was significantly up-regulated,and the opposite result was observed after inhibition of TPT1;after treatment with LY294002,the expression of PPARγ,C/EBPα,Adiponectin and FABP4,which are key genes of adipogenic differentiation,were significantly down regulated,and lipid droplets were reduced.TPT1 can promote the differentiation of preadipocytes by activating PI3K/Akt pathway.In conclusion,TPT1 is indispensable for the differentiation of sheep tail adipocytes,and participates in the process of adipogenesis by regulating Fox O1 and PI3K/Akt dependent pathways.This study provides a theoretical basis for further exploring the effect of TPT1 on lipid deposition and lipogenesis.