The Mechanism Of Glucagon-like Peptide-1 Receptor Agonists Regulates Visceral Adipose Tissue To Lose Weight Via MiR-130a-3p

ObjectiveObesity is a high risk factor for many chronic diseases,which aggravates the occurrence and development of type 2 diabetes mellitus,hypertension,coronary atherosclerotic heart disease and other diseases.Glucagon-like peptide-1 receptor agonists(GLP-1RAs)not only have hypoglycemic effects,but also have significant weight loss and protection of cardiac and renal functions.However,the mechanisms of weight loss of GLP-1RAs have not been fully clarified.Recent years,some of the miRNAs involved in lipid metabolism and energy balance have been found to be involved in the development of obesity by regulating gene expression.In this study,the effect of liraglutide on miRNAs in epididymal white adipose tissue was analyzed by transcriptome sequencing analysis and bioinformatics to investigate whether GLP-1RAs regulate lipid metabolism by influencing miRNAs,regulate the expression of target genes in lipid metabolism-related signaling pathways and thus exert a weight-loss effect.Furthermore,the study helps predict the mechanism of its target gene in the occurrence and development of obesity and provide a theoretical basis for clarify the weight loss mechanism of GLP-1RA and finding new targets for weight loss therapy.MethodsThirty C57BL/6 male mice were randomly divided into a normal diet group(ND group,n=10)and a high-fat diet group(HFD group,n=20).The ND group was fed The HFD group was fed a 60% high-fat diet for 8 weeks to establish an obesity model,and the obese mice that met the weight requirements were further divided into two groups: a simple obesity group(HFD+NS)and a liraglutide intervention group(HFD+LIRA),which continued to be fed a60% high-fat diet and intervened.The HFD+LIRA group was treated with 0.6mg/kg liraglutide once daily by subcutaneous abdominal injection for 8 weeks.Body weights of the mice were measured weekly during this period.At the end of the 8th week after the intervention,intraperitoneal glucose tolerance test(IPGTT)and insulin tolerance test(IPITT)were performed to assess glucose metabolism,triglyceride(TG),total cholesterol(TC)and low density lipoprotein cholesterol(LDL-C)were detected to assess lipid metabolism.The adipose tissue of mouse epididymis was stained with HE and liver oil red O to observe the morphological changes of adipose tissue and liver;three white adipose tissues of each mouse epididymis were randomly selected for transcriptome sequencing.The differential miRNAs among ND+NS,HFD+NS and HFD+LIRA groups were screened and validated using quantitative real-time PCR;target genes were searched in the miRNA database and the crtical genes were subjected to KEGG pathway analysis to find the pathways related to lipid metabolism.The differential expression of transcription factors and target genes on the relevant signaling pathways was verified using quantitative real-time and protein blotting assays.Results(1)After 8 weeks of high-fat diet intervention,the weight of the HFD group was significantly higher than that of the ND group(>20%,P<0.05).After 8 weeks of liraglutide intervention,the weight of the HFD+LIRA group was significantly higher than that of the HFD+NS group(P<0.01).The weight of the HFD+LIRA group was significantly lower than that of the ND+NS group(P<0.05).(2)The results of IPGTT showed that blood glucose levels were significantly increased at all points in the high-fat fed mice(vs.ND+NS group,P< 0.01);after 8 weeks of liraglutide intervention,blood glucose levels were significantly reduced(vs HFD+NS group,P<0.01).The results of IPITT showed that blood glucose levels were significantly increased in the high-fat fed mice(vs.ND+NS group,P< 0.01 or 0.05),after 8 weeks of liraglutide intervention,blood glucose levels were significantly reduced(vs HFD+NS group,P<0.01).(3)Compared with the ND+NS group,the serum TG,TC and LDL-C levels of HFD+NS group increased significantly(P< 0.01 or 0.05).After 8 weeks of liraglutide intervention,the serum TG,TC and LDL-C levels(P<0.01 or 0.05)decreased significantly in the HFD+LIRA group(vs HFD+NS group).(4)The oil red O of the liver tissues was sever in HFD+NS group compared with the ND+NS group.After 8 weeks of liraglutide intervention,oil red O of the liver tissues showed that the lipid deposition in HFD+LIRA group was alleviated compared with that in HFD+NS group.(5)Transcriptome sequencing results shows that a total of 20 miRNAs were differentially expressed between the ND+NS and HFD+NS groups using P<0.05 and |log2FC|>1 as the screening threshold,of which 8 were upregulated and 12 were downregulated.A total of 186 miRNAs were differentially expressed between the HFD+NS and HFD+LIRA groups,of which 67 were up-regulated and 119 were down-regulated.A total of 11 miRNAs were differentially expressed between the ND+NS and HFD+NS groups and between the HFD+NS and HFD+LIRA groups,of which two were upregulated in obesity and downregulated after liraglutide intervention(miR-21 c,miR-467a-3p)and nine were downregulated in obesity and upregulated after liraglutide intervention(miR-130a-3p,miR-193b-3p,miR-3072-3p,miR-3073b-5p,miR-365-3p,miR-378a-3p,miR-378a-5p,miR-378 d,miR-6392-5p).Further KEGG analysis revealed that the enriched signaling pathways contained signaling pathways related to lipid metabolism,such as the MAPK,AMPK and PI3K/AKT signaling pathway.Using Target Scan to predict target genes,the results showed that miR-130a-3p had binding sites to Pik3 cb and Pparg in the PI3K/AKT signaling pathway;miR-378a-3p had binding sites to Akt1;miR-193b-3p had binding sites to Ppargc1a;miR-378 d had binding sites to Srebf1 has a binding site.(6)The results of real-time quantitative PCR showed that miR-130a-3p expression levels were reduced in the HFD+NS group compared to the ND+NS group(P< 0.01),miR-130a-3p expression levels were significantly increased after liraglutide intervention(P< 0.01)and were higher than normal levels(P< 0.01).Compared with the ND+NS group,the expression levels of transcription factor SREBP-1 were significantly higher in the HFD+NS group(P< 0.01),and the expression levels of its target genes ACC and FAS were significantly higher(P< 0.05),while the expression levels of SREBP-1 were significantly lower after liraglutide intervention(P< 0.01),and the expression levels of its target genes ACC and FAS were significantly lower(P< 0.01).The results of protein blotting experiments showed that the p-AKT/t-AKT level increased(P< 0.01)and the SREBP-1,ACC and FAS levels decreased after liraglutide intervention compared with the HFD+NS group(P< 0.01 or 0.05).ConclusionLiraglutide reduced body weight and blood lipid levels in obese mice significantly,while improving glucose tolerance.GLP-1RAs may upregulate miR-130a-3p to inhibit the expression of SREBP-1,ACC and FAS in white adipose tissue to reduce visceral fat,thus lead to lose weight.