The Mechanism Of FTO On Suppressing Mice Adipocytes Apoptosis By Reducing Mitochondrial UPR

Fat and obesity-related genes?FTO?is a member of the non-heme dioxygenase superfamily.FTO protein is involved in many biological processes,such as cell proliferation,apoptosis,differentiation and cell cycle.Obesity is the most important risk factor for type?diabetes,and FTO mutants affect the risk of obesity and type?diabetes.By studying the FTO gene and BMI of a large number of people of different ages and BMI in Europe,predicting the association between overweight and obesity shows that FTO is closely related to BMI and aging from birth to aging?Frayling et al.,2007?.In addition,multiple potentially functional SNP loci contained in the FTO sequence are highly correlated with early-onset and severe obesity characteristics in the European population.Therefore,the impact of the FTO gene on energy balance still needs to be studied?Dina et al.,2007?.Studies have shown that proliferation and differentiation of porcine intramuscular fat precursor cells are enhanced after overexpression of FTO?Zhang et al.,2015?.It is unclear whether apoptosis is an important physiological activity of cells regulated by FTO.This study aims to explore the mechanism of FTO on suppressing mice adipocytes apoptosis by reducing UPR^mt,and the assay methods include that qPCR,Western blotting,immunofluorescence,transcriptome sequencing,immunohistochemistry.These results provide some experimental evidence for deep disclose of the interaction between UPR^mt and apoptosis and the prevention of lipodystrophy disorders.This study obtained the following results:1.FTO inhibits adipocytes apoptosis by activating the JAK2/STAT3 signaling pathway.Cycloleucine?CL?-treated 3T3-L1 cells significantly up-regulated FTO mRNA level?P<0.05?,but significantly down-regulated the mRNA levels of apoptosis-related genes Caspase-3 and Bax,and significantly up-regulated Bcl-2,these were reversed by betaine?Bet?treatment.Transcriptome sequencing results shown that overexpression of FTO up-regulated the transcription levels of Bax and XAF1,these were verified by qPCR?P<0.05?.The mRNA levels of cell proliferation related genes p27 and p53 and apoptosis-related marker genes Caspase-3,Caspase-9 and Bax were significantly down-regulated by overexpression of FTO-treated 3T3-L1 cells?P<0.05?,and the mRNA levels of PCNA,CyclinE and Bcl-2 were significantly increased?P<0.05?.Induction of apoptosis model by palmitic acid treatment revealed that FTO further inhibited pro-apoptosis genes mRNA and protein levels,up-regulated anti-apoptosis gene mRNA and protein level?P<0.05?.TUNEL staining and immunofluorescence showed overexpression of FTO inhibited apoptosis of adipocytes?P<0.05?.It was found that overexpression of FTO activated phosphorylation of JAK2/STAT3 signaling pathway and inhibited Caspase-3 cleavage by the pathway inhibitor SD1008 treatment?P<0.05?.2.FTO inhibits UPR^mt by reducing m6A methylation levels of HSP60 mRNA.The UPR^mt model was constructed by treating adipocytes with nicotinamide ribose?NR?,the results showed the mitochondrial respiratory chain marker genes were detected by FTO treatment in UPR^mt model and the mRNA levels of TFAM and COX2 were significantly down-regulated?P<0.05?.Overexpression of FTO had no significant effect on the mRNA level of HSP60,but ClpP mRNA level was significantly inhibited?P<0.05?.The mRNA and protein levels of HSP60 and ClpP increased significantly in the UPR^mt model?P<0.05?,but overexpression of FTO significantly inhibited the protein levels of HSP60 and ClpP?P<0.05?.In order to further study the molecular mechanism of FTO regulating UPR^mt in adipocytes,the results showed that CL and Bet had no significant effect on HSP60 and ClpP mRNA levels,but CL treatment inhibited HSP60 protein level?P<0.05?,Bet treatment up-regulated the protein level of HSP60?P<0.05?,then predicted the m6A modification site of HSP60 mRNA and mutated,and treated adipocytes showed no significant change in HSP60mRNA level.The HSP60 protein level was significantly down-regulated in the mutant group compared with the control group?P<0.05?.The overexpression of FTO after mutation did not affect HSP60 protein level.These results indicate that FTO inhibits adipocytes UPR^mt by decreasing m6A levels of HSP60 mRNA.3.FTO suppresses apoptosis of adipocytes by inhibiting mitochondrial UPR.PKR inhibitor 2-AP and FTO treatment of adipocytes found that 2-AP significantly inhibited the activation of PKR/eIF-2?signaling pathway?P<0.05?,while overexpression of FTO further down-regulated phosphorylation of PKR/eIF-2?signaling pathway?P<0.05?.ATF5 protein level in the mitochondria was significantly downregulated?P<0.05?,but it in the nucleus was significantly upregulated?P<0.05?.ATF5 translocation was significantly inhibited treatment with overexpression of FTO?P<0.05?.ATF5 and Bax mRNA levels were significantly decreased in interference with ATF5-treated adipocytes?P<0.05?,they were further down-regulated with the treatment by overexpression of FTO?P<0.05?.Overexpression of FTO and NR co-treatment in adipocytes and adipose tissues showed that overexpression of FTO significantly inhibited the mRNA and protein levels of pro-apoptosis genes in the UPR^mtt model?P<0.05?,the mRNA and protein level of anti-aopotosis gene Bcl-2 were significantly upregulated?P<0.05?.HE staining found the number of lipid droplets increased with the treatment of pc-FTO?P<0.05?,and it was decreased after NR treatment?P<0.05?.Immunohistochemistry showed that the expression of Bcl-2 in adipose tissue was significantly increased after overexpression of FTO?P<0.05?,and the up-regulation of Bcl-2was alleviated by NR treatment?P<0.05?,Bax was the opposite of Bcl-2.The results of this experiment showed that FTO inhibits the expression of UPR^mt-mediated proapoptotic genes in adipose tissue.These results demonstrate that FTO promotes activation of the JAK2/STAT3 signaling pathway to inhibit adipocytes apoptosis and inhibits UPR^mt by reducing m6A levels of HSP60 mRNA.These results disclose the function of UPR^mt in the regulation of adipocytes apoptosis,and provide a theoretical basis for fierce study of lipid metabolism and function,meanwhile,provide research ideas for the treatment of metabolic syndrome such as obesity.