Effect Of Novel Gene JAZF1 Overexpression On Glucose And Lipid Metabolism In Adipocytes And Hepatocytes

PART ONE Construction and identification of the eukaryotic ex- pression vector of mouse JAZF1 geneObjective:TO construction and identification of the eukaryotic expression vector of mouse JAZF1 gene.Methods:The cDNA fragment of mouse JAZF1 containing two restriction sites(ECORI,SalI) was obtained from mouse brain tissue by using reversal transcript-polymerase chain reaction(RT-PCR).Then,it cloned into the pIRES2-EGFP vector by ECORI/SalI double digestion and ligation.The recombinant plasmid was identified by enzyme digest and DNA sequence analysis.Results:The recombinant plasmid pIRES2-EGFP-JAZF1 digested with ECORI/SalI or without digestion were by agarose gel electrophoresis and presented with two bands(540bp:JAZF1 cDNA product,5.3kb:pIRES2- EGFP plasmid)or bands(5.84kb:recombinant plasmid pIRES2-EGFP- JAZF1),respectively.This was a preliminary marker of successful construction.Sequencing results were ananlysis by pairs using DNAssist software,its nucleotide sequence completely matched the JAZF1gene mRNA coding CDS domain sequence(BC04877)in GeneBank,which was further comfirmed as mouse JAZF1 cDNA.Conclusion: The eukaryotic expression plasmid pIRES2-EGFP- JAZF1 has been successfully constructed.It will help futher studies on the physiological function of JAZF1 gene and its effect on glucose and lipid metabolism.PART TWO The mRNA expression of JAZF1 gene in mice and in 3T3-L1 adipocytes differentiationObjective: To observe the mRNA expression of JAZF1 gene in healthy C57BL/6J mice and in 3T3-L1 adipocytes differentiation.Methods:Culture of 3T3-L1 preadipocytes and induction of differentiation were performed in vitro.Preadipocytes and differential cells at four time points were collected.At the same time,various tissue of healthy C57BL/6J male mice were obtain such as liver,heart,brain,lung,fat, testis,pancreas,muscle,kidney.Total RNA were extracted from each tissue and cells,and reversed transcription synthesis cDNA,quantitative real-time PCR(SYBR Green I) was performed in order to measure levels of JAZF1 mRNA.Each experiment was repeated three times. Results:Expression of JAZF1 were detected in various tissues of mice at varying degrees.The relative mRNA expression levels of JAZF1 from high to low in order is testis,fat,brain,lung,heart,liver,pancreas,muscle,kind- ny.JAZF1 mRNA expression dramatically increased during the process of differentiation of 3T3-L1 cells except for cells of day5.Conclusion:JAZF1 might play a role in maintaining normal physiological function,and was expressed most highly in fat,liver.For this reason,adipocytes and hepatocytes were selected as vitro model of researching JAZF1 function.In addition,JAZF1 participates in 3T3-L1 adipocytes differentiation which indicates that it may play a role in the development of metabolic diseases such as obesity.PART THREE Effect of gene JAZF1 overexpression of glucose and lipid metabolism in adipocytes and hepatocytesObjective: To observe the effects of JAZF1(juxtaposed with another zinc finger gene 1)overexpression on glucose and lipid metabolism in 3T3-L1 adipocytes and mouse hepatocytes(IAR-20 cells,Hepa1-6 cells) .Methods:Expression vector of JAZF1 gene was transient transfected into 3T3-L1 adipocytes,hepatocytes(IAR-20,Hepa1-6).The mRNA levels of JAZF1, the nuclear orphan receptor TAK1,GLUT1 ,GLUT4,FAS, ACC, SREBP1,ATGL,HSL,and FGF21 implicated in glucose,lipid metabolism and cytokine were determined by RT-QPCR;JAZF1 protein levels were measured by western blot. Intercelluar lipid accumulation were measured by Oil Red O staining and colorimetric method in 3T3-L1 cells.The glucose uptake was determined by 2-Deoxy-D-[3H]-glucose assay in 3T3-L1 adipocytes.Results: In JAZF1-transfected adipocytes and hepatocytes(IAR-20, Hepa1-6), JAZF1 mRNA expression and protein levels were significantly higher than control cells after transfected 48h. The mRNA levels of HSL was increased obviously(3T3-L1 cells:P<0.05;hepatocytes:P<0.001)inJAZF1 transfection group compared with negative control and empty vector group, and the relative expression of SREBP1,FAS,ACC,TAK1 mRNA were decreased significantly(all P<0.001). However, the mRNA levels of GLUT1(except hepatocytes),GLUT4,ATGL were not changed (all P>0.05). In addition,the expression of PPARα,GLUT1 mRNA were significantly increased (P<0.001).Intercelluar lipid accumulation was decreased obviously(P<0.05)by Oil Red O staining and colorimetric in JAZF1-transfected 3T3-L1 adipocytes compared with negative control and empty vector group.There were no significant difference of glucose uptake in both basic state and insulin stimulated among these groups in 3T3-L1 adipocytes.(all P>0.05). Conclusion: These results show that overexpression of JAZF1 in 3T3- L1 cells and hepatocytes can reduce lipid synthesis, increase lipolysis and improve lipid accumulation in 3T3-L1 or increases basal glucose transport in hepatic cells. We speculate that JAZF1 might provide a new potential therapeutic target for obesity and diabetes.