The Effect Of RAC1 On Differentiation Of Preadipocytes

ObjectiveIn recent years,the incidence of adult obesity(BMI≥30 kg/m~2)increased dramatically and became a global problem.Epidemiology studies have found that obesity was associated with a wide range of chronic diseases,such as diabetes,cardiovascular disease,and cancer.Approximately 4 million people died each year from obesity-related diseases worldwide.Therefore,understanding the possible mechanisms of obesity are important for the prevention and treatment of obesity and related diseases.Our previous population genetics study found that the RAC1 pathway was significantly associated with plasma adiponectin levels,but its biological mechanism is still unclear.RAC1(Ras-related C3 botulinum toxin substrate 1)is a member of the Rho family of guanosine triphosphate phosphohydrolases(GTPases).RAC1 can bind with dguanosine triphosphate(GTP)or guanosine diphosphate(GDP),which leads to RAC1 in active or inactive state,and transformation between the GTP or GDP bound forms regulates various downstream cellular functions,including secretory processing,phagocytosis of apoptotic cells,epithelial cell polarization,neuronal adhesion,migration and differentiation,glucose uptake,and growth factor-induced membrane fold formation.However,whether RAC1 could induce preadipocyte differentiation directly remained enigmatic.The main purpose of this study is to investigate the effects of RAC1 on the differentiation and lipid synthesis of 3T3-L1 cells,and to decipher the molecular mechanism of RAC1 induced adipogenesis through transcriptome profiles.MethodIn our study,using the 3T3-L1 cell as our model system to investigate preadipocyte differeniations.DXMSamethasone(DXMS),3-isobutyl-1-methylxanthine(IBMX)and insulin were used to induce 3T3-L1 cells to differentiate into adipocytes.In this process,the siRNA of Rac1 was used to inhibit the expression of RAC1,overexpress of RAC1 was obtained by transfection with the plasmid of Rac1.EHT1864,a small molecular inhibitor of RAC1,was used to suppress the function of RAC1.After 6days,the cells were stained with Oil-Red-O to evaluate differentiations of 3T3-L1cells.Western Blotting and Quantitative Real-time PCR were used to detected the expression levels of adipose cell markers PPAR-γand Adiponectin(ADPN).Triglyceride Quantitation Kits were used to measure the triglyceride(TG)level in3T3-L1 cells.Glucose content detection kits were used to measure the residual glucose in the culture medium.At last,after induced by DXMS,IBMX and insulin,stimulated with 20μM EHT1864 or water for 6 days,total RNA samples were extracted from 3T3L1 cells,samples were sequenced by RNA-Sequencing at Illumina Hiseq 4000.Sequencing data were analyzed and enriched in Gene Ontology(GO),Reactome Pathway Database(Reactome),and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.Results1.The results of Oil-Red-O staining and triglyceride quantitative detection indicated that siRNA of Rac1 or EHT1864 could promote the storage of triglyceride in 3T3-L1cells when it differentiated into adipose cells.RAC1 protein overexpression could reduce the storage of triglyceride in 3T3-L1 cells when it differentiated into adipose cells.2.The results of Quantitative Real-time PCR showed that EHT1864 could promote the expression of PPAR-γmRNA,while the expression of Rac1 mRNA had little change.3.The results of Western Blotting showed that the treatments of EHT1864 or siRNA of Rac1 could promote the expression of PPAR-γand ADPN,and the overexpression of Rac1 could reduce the expression of PPAR-γand ADPN in differentiating 3T3-L1 cells.4.RNA sequencing showed that a total of 3541 differential expressed genes,including 1940 up-regulated and 1601 down-regulated genes,after 20μM EHT1864treatment for 6 days in 3T3-L1 cells.The results of KEGG enrichment analyses showed when the function of RAC1 was inhibited by EHT1864,biological pathways associated with glycolysis/gluconeogenesis,non-alcoholic fatty liver disease(NAFLD),PPAR-γsignaling pathway,pyruvate metabolism,citrate cycle(TCA cycle),and many other signaling pathways related to cell glucose metabolism and adipogenesis were induced.Meanwhile,GO and Reactome enrichment analyses indicated that the treatment of EHT1864 could affect the biological processes such as fatty acid metabolism,oxidative phosphorylation,and glycosylation.Conclusions1.SiRNA,EHT1864 and the Rac1 overexpression plasmid were used to regulate the expression of RAC1 had direct impacts on the differeniation of adipocytes.Inhibition of RAC1 would promote the differentiation of preadipocytes,while the overexpression of RAC1 would inhibit the differentiation of adipocytes.2.The transcriptional profiling of 3T3-L1 cells revealed that genes associated with lipid metabolism and glucose metabolism were up-regulated when RAC1 had been inhibited by EHT1864.Meanwhile,energy metabolism related pathways were regulated to promote the differentiation of preadipocytes to adipocytes.