The Functions And Mechanisms Of Liraglutide In The Regulation Of Adipocyte Differentiation And White Fat Browning

Objective: Liraglutide,as a glucagon-like peptide-1(GLP-1)long-lasting analogue has been used for the treatment of T2 DM,which,besides its well-known anti-diabetic properties,has been also recommended for the treatment of obesity.Recent studies showed that liraglutide played an important role in regulating adipocyte differentiation and activating brown adipose tissue thermogenesis,however,the exact signaling pathway involved is still not completely understood.Preadipocyte 3T3-L1 cells and high-fat-fed KKAy mice were used in our study,the effects of liraglutide on differentiation of adipocytes and white adipose tissue browning were studied and the possible mechanisms in this process were exposed in the research.Methods: 1.Preadipocyte 3T3-L1 cells were cultured in were cultured in adipocyte-inducing medium and treated with liraglutide.Subsequently,cell proliferation was determined by CCK8 assay and cell immunofluorescence for Ki67,and apoptosis was assessed by flow cytometry.Lipid droplet production was detected by Oil red O staining.The expression levels of the master transcription factors and adipocyte-specific genes were measured by RT-QPCR and immunoblotting analysis.Next,the expression levels of the core components in the YAP signaling pathway as well as YAP-specific target genes were measured.Finally,short interfering RNAs of MST1,a key proteinkinase in the Hippo/YAP pathway were used to determine whether liraglutide regulated adipogenic differentiation via the Hippo-YAP pathway.2.KKAy mice were fed with a high-fat diet and treated with liraglutide or the same volume of PBS by peripheral injection for 12 weeks.The body weight of mice in each group was recorded.The perirenal renal white fat tissue(prWAT)and inguinal white fat tissue(iWAT)were collected from vehicle and liraglutide treated HFD fed mice respectively after 12 weeks.Lipid droplet production was detected by Oil red O staining and HE staining.The expression levels of the lipolytic activity marker genes,brown fat marker genes and mitochondrial associated genes were measured by RTQPCR,immunoblotting analysis,immune histochemistry and immunofluorescence.Next,the expression levels of the core components in the sGC signaling pathway were measured by RT-QPCR,immunoblotting analysis and immunefluorescence.In addition,3T3-L1 cells were cultured in adipocyte-inducing medium for 3 days,and then treated with sGC activators or sGC Inhibitor accompany with adding liraglutide or not.Relative mRNA expression levels and protein expression levels of sGC-mediated pathway important genes,mitochondrial marker gene,brown fat marker gene were measured by RT-QPCR and immunoblotting analysis.Results: 1.Compared with the normal control group,liraglutide promotes adipogenic differentiation of 3T3-L1 cells at the early phase of adipogenic differentiation.Liraglutide up-regulate the expression level of PPAR?,C/EBP?and aP2.Liraglutide suppressed proliferation in a dose-dependent manner,did not affect apoptosis of 3T3-L1 cells.Liraglutide promoted adipogenic differentiation,suppressed proliferation,and activated the Hippo/YAP signaling pathway at the initial stage of adipogenesis.Silencing of MST1 counteracted the effect of increasing adipogenesis by liraglutide.2.Compared with the control group,liraglutide significantly decreases body weight of mice and reduces the size of white adipocytes.ATGL and HSLwere significantly up-regulated in iWAT and prWAT from liraglutide-treated mice.Liraglutide increased the expression of Cidea,PPAR?,PRDM16,UCP1.As well as the expression of CytoC,PGC1?,TFAM also were up regulated in iWAT and prWAT.Compared with the control group,sGC and PKGI were up regulated by liraglutide in vivo and in vitro,stimulation of sGC elevated expression of brown fat tissue marker genes and PKGI,which suggested that liraglutide induced WAT browning via sGC-dependent pathway.Conclusion: 1.Liraglutide may activate the Hippo/YAP signaling pathway leading to the inhibition of proliferation of preadipocyte 3T3-L1 cells,and result in cells achieving transformation into mature adipocytes sooner.2.Liraglutide induced WAT browning via sGC dependent pathway.Taken together,this study expands our knowledge on the mechanism of liraglutide inducing white fat tissue browning,and provides a theoretical support for clinical usage of liraglutide on obesity treatment.