| ObjectiveObesity is closely related to chronic diseases,especially Diabetes mellitus type 2(T2DM).Obesity can lead to Insulin resistance(IR),which further causes T2 DM and seriously affects human health.Liraglutide(Lira),as a long-acting Glucagon-like peptide 1(GLP1)analogue,can improve IR and reduce body weight.It has attracted more and more attention as a new generation of hypoglycemic drugs.Irisin is encoded by transmembrane fibronectin type III domain-containing protein 5(FNDC5),which is mainly secreted by skeletal muscle cells and acts on various parts of the body with blood circulation.Irisin plays a role in bone remodeling,neuroprotection,and promotion of brown adipose-like development.Our previous study found that Lira could improve serum irisin levels in obese T2 DM patients.However,whether Lira is involved in the browning of White adipose tissue(WAT)is still unclear.AMPKα is related to the maintenance of homeostasis and energy regulation,and its downstream ACC1 is the rate-limiting enzyme for fat synthesis.Ampkα-ACC1 signaling pathway is crucial in fat metabolism.In this study,animal experiments of obese T2 DM rats,C2C12(Mouse myoblast)and 3T3-L1(Mouse embryonic fibroblasts)cell experiments and RNA sequencing were used to explore the specific mechanism of Lira inducing WAT browning by promoting the expression of myogenic irisin,so as to provide new theoretical support for its improvement of glucose and lipid metabolism and weight loss.Methods1.Forty male SD rats aged 7 weeks were divided into randomly control group(CON)and high-fat diet group(HFD).The CON group rat were fed with normal diet(320kcal/100g),however the rats in HFD group were fed with high-fat diet(460kcal/100 g,content of 45% fat to provide high fat and high heat)for 12 weeks.The rats in the HFD group were intraperitoneally injected with streptozotocin(STZ)at a dose of 30mg/kg to establish the obese T2 DM rat model.After the model was successfully established,the HFD group was randomly divided into DM group and Lira group.Rats in DM group and Lira group were injected with the same amount of saline intraperitoneally and Lira(0.4 mg/kg/ day)twice in the morning and evening for 8 weeks.Body weight was measured weekly.At the end of the treatment,the blood and adipose tissue of each group were collected for subsequent detection of serological indicators,immunohistochemistry,epididymal adipose tissue(EAT)and subcutaneous adipose tissue(SAT)and HE staining2.C2C12 were mouse myoblast,which were used for subsequent experiments after induction and maturation.Mature skeletal muscle cells(C2C12 cells)were divided into at random CON group,PA group(0.4m M),PA+Lira group(0.4m M PA+100n M Lira),and PA+Lira+Exendin939 group(0.4m M PA+100n M Lira+100n M Exendin939).WB and q RT-PCR were used to test the expression of PGC1α and FNDC5,and ELISA was used to further measure the level of irisin in the supernatant.3.3T3-L1 cells were mouse embryonic fibroblasts,which were used for subsequent experiments after induced differentiation and maturation.Mature adipocytes were treated with irisin alone or Lira-treated C2C12 supernatant.WB and q RT-PCR were used to detect the browning of adipocytes and the expression of key proteins in AMPKα-ACC1 signaling pathway.AMPKα inhibitor compound C(20μM)and si AMPKα(100μM)were used to further verify whether AMPKα-ACC1 signaling pathway was involved in the browning of adipocytes.Lipid accumulation variation in 3T3-L1 cells deal with irisin tested by Oil red O staining and TG content in the cells.4.Mature 3T3-L1 cells were divided into CON group and Irisin group(using the concentration of 50ng/m L)randomly.Totally three samples were repeated in each group for RNA sequencing and transcriptome analysis,and the genes related to fat synthesis were verified by q RT-PCR.Results1.Animal experiments showed that compared with the CON group,the body weight and serum TC,TG and FBG levels of the DM group were significantly increased,while the related serum indexes and body weight of the Lira group were obviously improved after Lira treatment.According to the results of HE staining,compared with the CON group,the DM group had a strong increase in the size of EAT and SAT,while the Lira group had a significant reduction in the volume of adipocytes after Lira treatment.Immunohistochemistry showed that compared with the CON group,the expression of p-AMPKα,p-ACC1 and UCP1 in the EAT and SAT of the DM group decreased,while the expression of p-AMPKα,p-ACC1 and UCP1 increased strongly after Lira treatment.2.C2C12 myotubes were induced with 2% horse serum for 4 days and then used for subsequent experiments.Westernblot,q RT-PCR and ELISA showed that the expression of PGC1α and FNDC5 were visibly down-regulated in the PA group,compared with the CON group.Nevertheless the expression of PGC1α and FNDC5 were visibly up-regulated in the PA+Lira group after Lira treatment.However,PA+Lira+Exendin939 significantly inhibited the expression of PGC1αand FNDC5 after inhibiting GLP1 R.3.3T3-L1 cells could be used for subsequent experiments after 8 days of induced differentiation until significant lipid droplets appeared.After co-culturing with irisin alone or supernatant,the expression of p AMPKα/AMPKα and p ACC1/ACC1 was clearly increased in the protein and RNA levels.Inhibition of AMPKαexpression by AMPKα inhibitors Compound C and si AMPKα dramatically reduced the browning of adipose tissue and the expression of classic genes in AMPKα-ACC1 signaling pathway.4.After RNA sequencing and transcriptome analysis of the cell samples in the CON group and Irisin group,6 samples met the sequencing requirements and the sequencing data were of high quality,which were suitable for bioinformatics analysis.A total of 3170 differentially expressed genes(DEGs)were tested.There are 1862 genes were strongly increased and 1308 genes were strongly decreased.According to the heat map results,the expression of genes related to adipose browning was enhanced while decreased the expression of genes related to adipose synthesis.KEGG enrichment analysis revealed that DEGs were closely related to AMPKα-ACC1,PPAR,PI3K-AKT signaling pathway and insulin resistance.GO and Wiki Pathway enrichment analysis showed that differentially expressed genes were mainly refered to the process of lipid biosynthesis,lipid catabolism and brown adipocyte differentiation.The sequencing results also supported the previous animal and cell experiments.Conclusion1.Lira can improve the body weight and glucose and lipid metabolism in obese T2 DM rats,and activate AMPKα-ACC1 signaling pathway to promote WAT browning.2.Lira can secrete irisin in skeletal muscle cells,which can further activate AMPKα-ACC1-UCP1 pathway and promote lipolysis and inhibit fat synthesis in adipocytes,providing a theoretical basis for the curement obesity or diebetes and alleviate glucose and lipid metabolism. |
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