| Objective: Obesity is the pathological accumulation of fat caused by the imbalance of energy intake and consumption in the body,which leads to tissue dysfunction.Adipocyte differentiation and lipid accumulation are closely related to the occurrence and development of obesity.Obesity can cause insulin resistance,type 2 diabetes,heart disease,end-stage renal disease and even some neoplastic diseases,which seriously affects the quality of life of such people.Excessive accumulation of adipose tissue is the main cause of the above-mentioned diseases.Therefore,in-depth study of the regulatory factors that affect the excessive accumulation of adipose tissue has become the key to improving obesity.Nicotinamide N-methyltransferase(NNMT)is a methylase that can methylate nicotinamide in the body to generate N1-methylnicotinamide and finally excrete it from the body.The expression of NNMT in human adipose tissue is positively correlated with obesity and insulin resistance.Knockdown of NNMT expression in vivo can reduce lipid accumulation induced by high-fat diet in mice and improve glucose tolerance.However,the effect of NNMT on the metabolic function of adipocyte differentiation and its specific mechanism are still unclear.Autophagy is the main process used by cells to clear and degrade cellular contents.Lipid autophagy and traditional lipase-mediated lipolysis play an important role in lipid metabolism.It not only affects the lipid metabolism of fat cells,but is also closely related to a variety of metabolic diseases.The level of autophagy in the adipose tissue of obese patients and obese mice is significantly increased,and inhibition of autophagy can reduce obesity.The promotion of autophagy in the mouse model can accelerate the degradation of KLF2 and KLF3(negative regulators of adipocyte differentiation),thereby up-regulating the expression of PPAR-γ and C/EBPβ(positive regulators of adipocyte differentiation),which is conducive to fat formation.The purpose of this study is to detect NNMT and use overexpression and knockdown methods to interfere with NNMT expression on lipid accumulation,triglyceride content,and adipose differentiation-related transcription factors(PPARγ,C/EBPα,SREBP1)during the differentiation of 3T3-L1 adipocytes,lipid metabolism-related genes(FABP4,FAS,FATP1(Slc27a1),LPL),adipokines expression(ADIPOQ,LEP)and autophagy-related genes(Beclin1,ATG7,ATG12,ATG14)transcription and LC3 I and LC3 II,Beclin1 and P62 protein expression,to explore the possible mechanism of NNMT affecting lipid accumulation.Methods: 1.In order to observe the expression changes of NNMT during the differentiation process of 3T3-L1 preadipocytes,we used IBMX,dexamethasone and insulin to induce the differentiation of 3T3-L1 cells programmatically,and used q PCR to detect the expression of NNMT m RNA during cell differentiation.The expression of NNMT protein was detected by western blotting.2.Identification of the transfection efficiency of pc DNA3.1-Nnmt-3x Flag-C expression vector.3.The plasmid pc DNA3.1-NNMT-3x Flag-C and the control plasmid were transfected into 3T3-L1 cells respectively,and the cells were induced to differentiate,and then oil red O staining was used to detect the lipid accumulation and the cell triglyceride.The changes of esters were observed,and the changes in lipid accumulation of 3T3-L1 cells by over-expression of NNMT were observed.4.The q RT-PCR method was used to detect the adipose differentiation-related transcription factors(PPARγ,C/EBPα,SREBP1),lipid metabolism-related genes(FABP4,FAS,FATP1(Slc27a1),LPL),and adipokines(ADIPOQ,LEP)in 3T3-L1 cells caused by overexpression of NNMT,and the Western blot method was used to detect the changes in the expression levels of PPARγ and ADIPOQ proteins.5.Determination of the reinfection index(MOI)value of 3T3-L1 cells infected by NNMT lentivirus interference vector.6.NNMT lentiviral interference vector transfected 3T3-L1 cells to knock down the expression efficiency of NNMT.7.Oil Red O method was used to detect lipid accumulation in adipocytes,colorimetric method to detect triglyceride content,and observe the changes in lipid accumulation of3T3-L1 cells by knocking down NNMT expression.8.The q RT-PCR method was used to detect the adipose differentiation-related transcription factors(PPARγ,C/EBPα,SREBP1),lipid metabolism-related genes(FABP4,FAS,FATP1(Slc27a1),LPL),and adipokines(ADIPOQ,LEP)in 3T3-L1 cells caused by knocking down NNMT.Western blotting was used to detect changes in the expression levels of PPARγ and ADIPOQ proteins.9.q PCR was used to detect the changes in the transcription level of autophagy-related genes Beclin1,ATG7,ATG12,ATG14 on the 0th day,2nd day,4th day,and 6th day of differentiation of 3T3-L1 cells.10.3T3-L1 cells was transfected with NNMT overexpression plasmid,then was induced differentiation,and was detected the changes in autophagy level on the 6th day.The m RNA levels of Beclin1,ATG7,ATG12,and ATG14 were detected by q PCR,and the protein expression levels of LC3 I and LC3 II,Beclin1 and P62 were detected by western blotting.11.3T3-L1 cells with the lentiviral interference vector LV-NNMT-RNAi3 was transfected,then was induced differentiation,and was detected the changes in the level of autophagy on the 6th day.The m RNA levels of Beclin1,ATG7,ATG12,and ATG14 were detected by q PCR,and the protein expression levels of LC3 I and LC3 II,Beclin1 and P62 were detected by western blotting.Results: 1.During the induction and differentiation of 3T3-L1 cells(day 0,day 2,day4,day 6),Nnmt m RNA expression gradually increased(P <0.0001),and protein level also gradually increased(P <0.05 and P <0.001)),where the expression increase level was more obvious on the 4th and 6th day(P <0.0001).2.Compared with the control plasmid transfection group,the m RNA level of the pc DNA3.1-NNMT-3x Flag-C group was significantly increased(P<0.0001),and the protein expression was also significantly increased(P<0.01).3.Compared with the control group,the 3T3-L1 cells transfected with pc DNA3.1-NNMT-3x Flag-C had more obvious lipid accumulation on the 6th day of differentiation(P<0.0001),and higher triglyceride content(P<0.0001).4.Compared with the control plasmid transfection group,the m RNA levels of Pparg,Srebf1,Cebpa,Fabp4,Fasn,Slc27a1,and Lpl in the pc DNA3.1-NNMT-3x Flag-C group increased significantly,while the m RNA levels of Adipoq and Lep decreased significantly(P<0.0001).The expression of PPARγ protein was significantly increased,and the expression of ADIPOQ protein was significantly decreased(P<0.0001).5.The lentiviral vector LV-NNMT-RNAi transfects 3T3-L1 cells.When the MOI value is 100,the transfection efficiency was over 80% and the cells grew well.6.Compared with the control group,the Nnmt m RNA level of the LV-NNMT-RNAi3 group decreased most significantly(P <0.0001),and the protein expression of NNMT also decreased most significantly(P <0.001).It showed that the constructed LV-NNMT-RNAi3 lentiviral interference vector can effectively interfere with the expression of the target protein NNMT in 3T3-L1 cells,so the lentiviral interference vector LV-NNMT-RNAi3 was selected for subsequent experiments.7.Compared with the control group,the lipid accumulation of 3T3-L1 cells in the LV-NNMT-RNAi3 group was significantly reduced on the 6th day of differentiation(P<0.0001),and the triglyceride content decreased significantly(P<0.05).8.Compared with the control group,the transcription levels of Pparg,Srebf1,Cebpa,Fabp4,Fasn,Slc27a1,and Lpl in the LV-NNMT-RNAi3 group were significantly decreased,the m RNA levels of Adipoq and Lep were significantly increased(P<0.0001),and the expression of PPARγ protein was decreased significantly,the protein expression of ADIPOQ increased significantly(P<0.0001).9.In the process of induced differentiation of 3T3-L1 cells,the m RNAs of autophagy-related genes Beclin1,ATG7,ATG12,ATG14 gradually increased,and the increase in m RNA was the most obvious on the 6th day.10.Compared with the control group,Beclin1,ATG7,ATG12,ATG14 m RNA levels in the NNMT overexpression group were significantly increased(P<0.0001 and P<0.05),the protein expression levels of LC3 II/I and Beclin1 were significantly increased and the level of P62 protein decreased significantly(P<0.0001).11.Compared with the control group,the m RNA levels of Beclin1,ATG7,ATG12,and ATG14 in the NNMT knockdown group were significantly reduced(P<0.0001 and P<0.05),the protein expression levels of LC3 II/I and Beclin1 were significantly decreased,and the level of P62 protein increased significantly(P<0.0001).Conclusion: Overexpression of NNMT promoted lipid accumulation in 3T3-L1 cells by increasing adipose-differentiation-related transcription factors and enhanced the function of differentiated adipocytes by increasing lipid metabolism-related genes and reducing adipokines.Reducing expression of NNMT inhibited lipid accumulation in3T3-L1 cells by decreasing adipose-differentiation-related transcription factors and reduced the function of differentiated adipocytes by decreasing lipid metabolism-related genes and increasing adipokines.Autophagy was involved in the differentiation process of adipocytes,and NNMT affected autophagy of adipocytes. |
