【摘要】 为了解葡萄糖和胰岛素调控小鼠ob基因表达调控的机制 ,应用荧光素酶报告基因、凝胶迁移率变动分析 (gelmobilityshiftassays,GMSA)、DNaseI足迹分析和突变分析技术对小鼠ob基因启动子中葡萄糖和胰岛素反应元件 (GLRE和IRE)进行了鉴定。转染及报告基因活性分析结果显示 ,在小鼠ob基因启动子 - 1719～ - 145 2bp之间存在负调控元件、GLRE和IRE。GMSA结果显示葡萄糖和胰岛素抑制转录因子与顺式元件结合。DNaseI足迹分析和突变分析显示此结合区为AGCAAAA ,位于ob基因启动子的 - 16 98～ - 16 92bp。以上结果说明 ,小鼠ob基因启动子 - 1719～ - 145 2bp之间存在小鼠ob基因表达的负调控元件 ,葡萄糖和胰岛素通过同一个途径 ,即通过抑制某转录因子与此顺式元件的结合促进小鼠ob基因的表达 ,位于小鼠ob基因启动子 - 16 98～- 16 92bp的顺式作用元件AGCAAAA为一新的GLRE和IRE。更多还原

【Abstract】 Glucose and insulin stimulate leptin gene expression in vitro and in vivo . To identify cis-elements that are responsible for the glucose and insul in effects, mouse 3T3-L1 adipocytes were transiently transfected with reporter co n structs with serial deletions in mouse ob gene promoter. The cis-elemen ts were i dentified with gel mobility shift assays (GMSA), DNase I footprint assays and PC R mediated site-directed mutation assays. Transient transfections detected a ne g ative cis-acting element, a glucose-responsive element (GLRE), and an insu lin-re sponsive element (IRE) in the region from -1 719 bp to -1 452 bp of mouse ob g ene. This region does not contain any known GLRE or IRE. GMSA identified a DNA b inding protein which specifically binds a native probe prepared from mouse ob ge ne promoter (-1 719 bp to -1 452 bp), and the binding was repressed by gluc ose or insulin. DNase I footprint assays and PCR mediated site-directed mutations assa y s identified that the binding motif AGC, spanning -1 698 bp to -1 692 bp o f the mouse ob gene promoter, was responsible for the effects of glucose and ins ulin on ob gene expression. These studies suggest that a negative cis-a cting ele ment is located between -1 719 bp and -1 452 bp of the mouse ob gene promo ter, and glucose and insulin simulate mouse ob gene expression by repressing the bin ding of a transcription factor to this element. This element, AGC, spanning -1 698 bp to -1 692 bp is a novel GLRE and IRE.更多还原