| Objectives:The prevalence of obesity is increasing worldwide and has become a major global public health problem.Obesity is a chronic metabolic disease characterized by the accumulation of adipose tissue and weight gain.Adipose tissue is classified into white adipose tissue(WAT)and brown adipose tissue(BAT).WAT stores energy in the form of triglycerides,while BAT is rich in mitochondria and can release heat through specific uncoupling protein 1(UCP1),thereby reducing body weight and improving metabolism.Increasing BAT activity in human is an effective way to combat obesity.The amount of classical brown adipocytes is little in the adult,but cells in WAT can be characterized as brown adipocytes under the conditions of cold and sympathetic stimulation,etc.These cells are defined as "beige adipocytes",and the process of differentiation or transformation of beige adipocytes is defined as "adipose tissue browning".Irisin is a myogenic cytokine secreted by skeletal muscle during exercise,which can induce adipose tissue browning and increase BAT activity in animals and humans,and plays an important role in regulating energy metabolism.However,the receptor of irisin on human adipocytes have not been elucidated,and the signal pathways involved in the regulation of adipocyte beiging are still unclear.This study aims to elucidate the receptors and signal pathways involved in irisin-induced adipocyte beiging,so as to provide research directions and theoretical basis for the prevention and treatment of obesity.Methods:(1)CRISPR-Cas9 lentiviral vector was used to construct integrin αV/β1/β5/β6 knockout 3T3-L1 adipocytes.Firstly,the effect of lentiviral infection with Cas9 empty vector on adipogenic differentiation was evaluated,including oil red O staining to observe lipid droplet and quantitative real-time PCR(qRT-PCR)to detect the genes expression(Adipoq,Fabp4,Pparγ,Retn).Lenti-Cas9-puro was transfected into 3T3-L1 cells,puromycin screening was performed to obtain 3T3-L1-Cas9 cells.Flag protein expression was detected by western blot(WB).Design and synthesize integrin αV/β1/β5/β6-specific gene knockout sgRNA.The 3T3-L1-Cas9 cells were infected with Lenti-sgRNA-EGFP to obtain integrinαV/β1/β5/β6-KO cells,and the sgRNA activity and knockout efficiency were verified.(2)Constructing integrin αV and β1/β5/β6 double subunit knockout 3T3-L1 cell with CRISPR-Cas9 lentiviral vector.The preadipocytes were adipogenic differentiated into mature adipocytes.Effect of irisin on the differentiation of gene knockout adipocytes was evaluated.Using oil red O staining to assess lipid droplet morphology and qRT-PCR to detect the expression of Adipoq,Fabp4,Pparγ,Retn and Ucp1.(3)The high-resolution liquid chromatography-tandem mass spectrometry(LC-MS/MS)was applied to detect differentially expressed proteins and phosphorylated peptides.Statistical methods such as volcano plot and principal component analysis(PCA)were performed to analyze the intra-group and inter-group variations of differential proteins and phosphorylated peptides.The variability of proteins and phosphorylated peptides was analyzed using gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)annotations.Identification of phosphorylation events and signal pathways mediating irisin-induced adipocyte beiging from KEGG and GO enrichment data.(4)The PI3K-AKT signal pathway derived from phosphorylated proteomics data was selected for functional analysis and pathway validation.Using human subcutaneous adipocytes to assess the effect of irisin on UCP1 protein levels and phosphorylation levels of PI3K and AKT proteins levels.The adipocytes were pretreated with integrin αVβ5 inhibitor(Cyclo RGDyK)before irisin intervention,and the effects of the UCP1 expression and PI3K-AKT phosphorylation protein levels were analyzed to further validate the receptors and post-receptor signal pathways mediating the beiging effect of irisin.Results:(1)3T3-L1 preadipocytes were adipogenic differentiated into mature adipocytes.The oil red O staining showed that the size of lipid droplets of 3T3-L1 adipocytes transfected with Cas9 empty vector(3T3-L1-Cas9-NC)were not significantly different from the control group.The PCR analysis data showed that the lipogenic genes were significantly upregulated in the 3T3-L1-Cas9-NC group after differentiation(P<0.0001),confirming that transfection of Cas9 empty vector did not affect adipogenic differentiation.Adipocytes were infected with Lenti-Cas9-puro and were screened by puromycin.Compared with the control group,3T3-L1-Cas9 cells were in good status and flag protein were detected by WB,which proved that the Cas9 stable strain was successfully constructed.Sequentially infected with Lenti-sgRNA-EGFP carrying integrin αV/β1/β5/β6 target gene knockout sequences,the number of fluorescence-positive cells in each group reached more than 80%as observed by fluorescence microscopy,which indicated that the infection was effective.The application of knockout and mutation detection kits showed that cleavage bands appeared at the expected positions in each group,and WB detected a decrease in the expression of the corresponding knockout target proteins(integrin αV,integrin β1,integrin β5 and integrin(36).Combining the results of the two assays to screen out efficient knockout sgRNAs for subsequent experiments.(2)Integrin αV and β1/β5/β6 double subunit knockout 3T3-L1 cell lines were constructed with CRISPR-Cas9 lentiviral vectors.The oil red O staining showed that the lipid droplet morphology was changed in all three groups,but the lipid droplet size and number of adipocytes in integrin αVβ5-KO group were significantly downregulated compared to the integrin-NC group.The PCR showed that irisin promoted Ucpl gene expression in 3T3-L1 adipocytes,but the expression levels of adipogenic differentiation genes such as Adipoq,Fabp4,Pparγ,Retn and Ucpl in the integrin αVβ5-KO group were significantly decreased(P<0.05).Adipocytes in the integrin αVβ5-KO group were eventually selected for proteomic analysis in order to search for post-receptor signal pathways of irisin effects.(3)LC-MS/MS data revealed significant changes in the number and gene composition of proteins and phosphorylated peptides,as many as 4443 proteins were involved in this process,with 2595 of these proteins undergoing phosphorylation events within the integrin αVβ5-KO and integrin-NC groups after irisin intervention.The volcano plot showed differentially expressed proteins,of which 90 were significantly up-regulated and 180 were down-regulated,while among the differentially expressed phosphorylated peptides,34 were significantly up-regulated and 159 were down-regulated.The PCA results suggested excellent repeatability within the data of integrin αVβ5-KO or integrin-NC groups,but higher variability between the groups.Phosphorylated protein GO enrichment showed significant enrichment in inactivation of MAPK activity,negative regulation of cell-cell adhesion and insulin receptor signal pathway.KEGG enrichment showed that integrin αVβ5 knockout mainly caused significant changes in insulin signal pathway,regulation of lipolysis in adipocytes and PI3K-AKT signal pathway.(4)PI3K-AKT signal pathway was selected for functional validation with human adipocytes.WB showed that 40 nM irisin could significantly increase UCP1 protein levels(P<0.001)and phosphorylation protein levels of PI3K and AKT in human subcutaneous mature adipocytes(P<0.05),but the pre-treatment of 10 μM cyclo RGDyK reversed these changes(P<0.01).Conclusion:In this study,the integrin double subunit gene knockout 3T3-L1 adipocytes were successfully constructed using CRISPR-Cas9 lentiviral vector.Based on the combined LC-MS/MS proteomics and phosphorylated proteomics analysis data,combining the results of functional validation and signal pathway validation of human adipocytes,this study first confirmed that irisin promotes beiging of human adipocytes via integrin αVβ5,and the PI3K-AKT signal pathway is the relevant mechanism,which could provide a new drug target for intervention in obesity and metabolic diseases through promoting adipose tissue browning. |
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