表現外源性芳香烴基受體抑制3T3-L1脂肪細胞分化

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Title:	表現外源性芳香烴基受體抑制3T3-L1脂肪細胞分化
Other Titles:	Overexpression of aryl hydrocarbon receptor inhibits 3T3-L1 adipocyte differentiation
Authors:	蘇敬評 Su, Gin-Ping
Contributors:	陳珠亮 Chen, Chu-Liang 東海大學畜產與生物科技學系
Keywords:	芳香烴基受體;脂肪細胞分化 aryl hydrocarbon receptor;adipocyte differentiation
Date:	2006
Issue Date:	2011-04-07T05:56:40Z (UTC)
Abstract:	3T3-L1前脂肪細胞可表現芳香烴基受體 (Aryl hydrocarbon receptor; AhR)。當細胞進入分化階段時，AhR之表現快速下降。本研究在探討表現外源性AhR對脂肪細胞分化之影響。使用3T3-L1細胞株，經穩定轉染，以CMV啟動子表現AhR cDNA (pcDNA3.1/zeo+-AhR)。首先藉聚合?連鎖反應，西方點墨法，及免疫螢光染色確認此穩定轉染之細胞(AhR細胞）比對照組細胞(穩定轉染pcDNA3.1/zeo+之3T3-L1細胞）或是3T3-L1細胞，表現較高的AhR mRNA與蛋白質。細胞分別經分化試劑(dexamethasone, Dex；3-methyl-1-isobutylxanthine, IBMX；insulin）誘使分化後，oil- Red O脂質染色分析顯示，AhR細胞相較於對照組或3T3-L1細胞，其油滴堆積明顯較少；同時其甘油磷酸去氫?(GPDH）活性亦較低。相對定量反轉錄聚合?連鎖反應分析 (相對定量RT-PCR)之結果顯示，AhR細胞的分化標幟基因peroxisome proliferator-activated receptor γ (PPARγ)與leptin等mRNA表現量均較對照組或3T3-L1細胞者為低，而會隨分化而表現降低的AhR與preadipocyte factor-1 (Pref-1) mRNA，其表現在AhR細胞亦較對照組或3T3-L1細胞者為高。西方點墨法之結果也顯示，AhR細胞之PPARγ蛋白質的表現受抑制，但分化早期的標幟基因CCAAT/enhancer-binding protein β (C/EBPβ)則不受影響。細胞計數發現在分化早期，AhR細胞比3T3-L1細胞有較低的細胞數目，顯示表現外源性AhR會抑制細胞擴增。利用相對定量RT-PCR進一步檢視分化早期(0 h至24 h）調控細胞擴增之重要轉錄因子C/EBPβ mRNA的表現，發現AhR比3T3-L1細胞有較低的C/EBPβ mRNA表現量。另外，當使用個別分化試劑添加處理發現，DEX的添加即足以造成脂肪細胞分化早期AhR表現的下降與C/EBPβ的增加。綜上所述，表現外源性AhR可能透過降低PPARγ的表現抑制脂肪細胞的分化，降低GPDH活性與脂質堆積；同時於分化早期，高量的AhR亦可能降低C/EBPβ的表現而抑制細胞擴增。而分化試劑中的DEX，是造成脂肪細胞分化早期AhR表現下降與誘發C/EBPβ表現的因子。 Overexpression of aryl hydrocarbon receptor inhibits 3T3-L1 adipocyte differentiationAbstractTo investigate the roles of AhR in adipocyte differentiation, the AhR cDNA was amplified by RT-PCR and cloned into a mammalian expression vector (pcDNA3.1/zeo+) to construct pcDNA3.1/zeo+-AhR. Single clones were selected in zeocin-containing medium after 3T3-L1 preadipocytes were transfected with pcDNA3.1/zeo+ or the pcDNA3.1/zeo+-AhR plasmid to obtain the control or AhR cell line, respectively. Expression of AhR in the AhR cell line was confirmed by relative quantitative RT-PCR, immunoblotting, as well as immunofluorescent staining analysis. After treatment with differentiation-stimulating reagents (dexamethasone, DEX；3-methyl-1-isobutylxanthine, IBMX; insulin), less lipid accumulation was observed in the AhR cells by oil-Red O staining, comparing to the control cell line. Furthermore, the AhR cells have lower glycerophosphate dehydrogenase (GPDH) activity than the control cells. Relative quantitative RT-PCR analysis showed that the AhR cells accumulated lower mRNA levels of adipocyte differentiation markers, PPARγ and leptin, in terminal differentiation cells, and higher levels of AhR and Pref-1, which usually are suppressed during differentiation, than control cells. Western blot analysis also showed decreased levels of adipocyte differentiation marker, PPARγ; but no difference in the early-stage marker C/EBPβ in the AhR cells, comparing to those in control cells. Cell counting analysis showed that AhR cells accumulated less cells than the 3T3-L1 cells at early stages, suggesting inhibition of cell clonal expansion by overexpression of the heterologous AhR. C/EBPβ is an important transcription factor during adipocyte clonal expansion. Results of relative quantitative RT-PCR analysis showed that the expression of C/EBPβ mRNA at early times (0-24 h) was lowered in the AhR cells than in the 3T3-L1 cells. In addition, results of cells treated with individual differentiation inducers showed that DEX along was sufficient to lead to lowered levels of AhR and elevated levels of C/EBPβ that are commonly observed at early stages of adipocyte differentiation. Taken together, overexpression of heterologous AhR is likely to decrease GPDH activity and lipid accumulation through reducing the expression of PPARγ. Moreover, higher levels of AhR appeared to lower the expression of C/EBPβ and lead to inhibition of clonal expansion at early stages of adipocyte differentiation. And treatment of DEX along, but not IBMX or insulin, led to the expression pattern of AhR and C/EBPβ commonly observed at early stages of adipocyte differentiation.
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