Effects Of Insig-2 Gene On 3T3-L1 Preadipocytes Differentiation And Lipogenesis

Part 1 Effects of different concentration of glucose on the expression ofinsig-1, -2 and the differentiation in 3T3-L1 preadipocytesObjective: Insulin-induced gene 1 (insig-1), plays an important regulatory role in lipogenesis andadipocytes differentiation. The effects of insig-lon adipocytes differentiation and lipogenesis, andthe expression of insig-1, -2 during 3T3-L1 preadipocyte differentiation under differentconcentration of glucose were investigated.Methods: 3T3-L1 preadipocytes differentiation was induced with different concentration of glucose(5.5 and 25mol/L). Cell differentiation was observed by oil red O staining, RT-PCR and in situhybridization were used to detect the expressions of insig-1,-2, and fatty acid binding protein(AP₂)mRNAResults: With 3T3-L1 preadipocytes differentiation, the expression of insig-1,2 and AP₂ mRNAwas gradually increased. However, the differentiated cell extent under low glucose concentrationwas significantly lower than that under high glucose concentration. A higher expression of insig-1,-2 mRNA with a lower expression of AP₂ mRNA was observed under low glucoseconcentration (P＜0.05).Conclusion: During 3T3-L1 preadipocytes differentiation, the expression of insig was graduallyincreased. In low glucose condition, much higher expression of insig was probably related to ainhibition of cell differentiation and lipidoses Part 2 Effects of insig-2 overexpression on 3T3-L1 preadipocytesdifferentiation and the expression of related genesChapter 1Construction of eukaryotic expression vectors EGFP-C3-insig-2,pcDNA3.1(+)-Insig-2, and the subcellular localization of insig-2 in 3T3-L1preadipocytesObjective: To construct the eukaryotic expression vectors EGFP-C3-insig-2, pcDNA3.1(+)-Insig-2and to subcellularly localize insig-2 protein in 3T3-L1 preadipocytes.Method: The open reading frame (ORF) of insig-2 was obtained from 3T3-L1 pre- adipocytes byRT-PCR. The fragment encoding for insig-2 gene was inserted into pUCm-T vector, sequenced, andidentified by PCR. pUCm-T-insig-2 was digested by two restrictive enzymes and insig-2 wassubcloned into eukaryotic expressing vector EGFP-C₃ and pcDNA3.1(+)-Insig-2. The recombinedplasmid EGFP-C3-insig-2 was transfected transiently into 3T3-L1 preadipocytes withLipofeetamineTM 2000 Transfection Reagent. Green fluorescence protein expression andlocalization of the plasmid were determined by fluorescence microscopy, and the expression ofdown-stream gene AP₂ was detected by RT-PCR.Results: Eukaryotic expression vectors EGFP-C3-insig-2 and pcDNA3.1(+)-Insig-2 wasconstructed successfully. After EGFP-C3-insig-2 transfected into 3T3-L1 preadipocytes,insig-2protein was found to localize in cytoplasm other than nucleus. RT-PCR showed that the amount ofinsig-2 transcription was significantly enhanced with a lowered expression of its down-stream geneAP2.Conclusion: Overexpression of insig-2 could produce an impact on cell lipid metabolism during3T3-L1 preadipocyte differentiation. Chapter 2Effects of insig-2 stable expression on 3T3-L1 preadipocytes differentiation andlipogenesisObjective: To study the effects of insig-2 stable expression on 3T3-L1 preadipocytes differentiationand lipogenesis.Methods: The recombinant plasmid pcDNA3.1(+)-insig-2 and control plasmid pcDNA3.1(+) weretransfected into 3T3-L1 preadipocytes with LipofeetamineTM 2000 Transfection Reagent and thepositive clones were screened by G418. Cell cycles and apoptosis index in pcDNA3.1(+)-insig-2,pcDNA3.1(+) and non-transfected cells were analyzed by Flow Cytometry(FCM). Celldifferentiation and cell growth were identified using oil red O staining and cell growthcurves, respectively. Semi-quantitative RT-PCR was performed to detect the expression of insig-1,insig-2, sterol regulatory element binding proteins (SREBPs), cleavage-activating protein (SCAP),fatty acid synthetase (FAS), and fatty acid binding protein (AP2) in 3T3-L1 pre adipocytes anddifferentiation-induced adipocytes. The expression of insig-2 protein was determined byimmunohistochemistry.Results: Semi-quantitative RT-PCR and immunohistochemistry showed that the expression ofinsig-2 in 3T3-L1 preadipocytes transfected with insig-2 was significantly increased than thattransfected with blank pcDNA3.1(+) vector and none-transfected cells, suggesting thatpcDNA3.1(+)-insig-2 was transfected in 3T3-L1 preadipocytes successfully with high-efficientexpression. There was no remarkable effect on 3T3-L1 preadipocytes cell cycle and apoptosis indexwith insig-2 gene stably expression. After 6 and 12 days of induction, it was found that the maturedadipocytes in transfected pcDNA3.1(+)-insig-2 were showed to have bright red fat droplets with nosignificant changes of the cell growth curve. After transfected with pcDNA3.1(+)-insig-2, theexpression of insig-2 related genes including insig-1, FAS, AP2, and SREBP1c was down-regulated,but with up-expressions during cell differentiation. However, the expression of insig-2 increasedrapidly than that of FAS and AP2.Conclusion: Overexpression of insig-2 could inhibit 3T3-L1 preadipocytes differentiation intomature adipocytes and suppress the expression of genes related to lipogenesis. Chapter 3Effects of insig-2 stable expression on adiponectin secretion in 3T3-L1preadipocytesObjective: Adiponectin plays an important role in the regulation of glucose and lipid metabolismand energy balance. To approach the influence of overexpression of insig-2 on adiponectin secretionin 3T3-L1 adipocytes, the expression of adiponectin mRNA and the adiponectin secretion duringpreadipocytes differentiation were observed.Methods: After 24 and 72 h of transfection, semi-quantitative RT-PCR was performed to detect theexpression of adiponectin mRNA in transfected with cDNA3.1(+)-insig-2) and transfected withblank pcDNA3.1(+) vector or non-transfected cells. Adiponectin was detected by ELISA.Results: RT-PCR showed that the expression of adiponectin mRNA was decreased significantlyafter insig-2 gene stable expression in 3T3-L1 preadipocytes with a rather low level of adiponectinsecretion in the culture medium (P＜0.05). It was constently low during 0～4 days of induction, thengradually increased, but was still significant lower in transfected with cDNA3.1(+)-insig-2) cells(P＜0.05).Conclusion: Overexpression of insig-2 could suppressed the expression of adiponectin mRNA andprotein in 3T3-L1 preadipocytes.