title: Identification of Glucose-responsive and Insulin-responsive Elements in Promoter of Mouse ob Gene.
abstract: Glucose and insulin stimulate leptin gene expression in vitro and in vivo. To identify cis-elements that are responsible for the glucose and insulin effects, mouse 3T3-L1 adipocytes were transiently transfected with reporter constructs with serial deletions in mouse ob gene promoter. The cis-elements were identified with Gel mobility shift assays (GMSA), DNase I footprint assays and PCR mediated site-directed mutation assays. Transient transfections detected a negative cis-acting element, a glucose-responsive element (GLRE), and an insulin-responsive element (IRE) in the region from -1 719 bp to -1 452 bp of mouse ob gene. This region does not contain any known GLRE or IRE. GMSA identified a DNA binding protein which specifically binds a native probe prepared from mouse ob gene promoter (-1 719 bp/-1 452 bp), and the binding was repressed by glucose or insulin. DNase I footprint assays and PCR mediated site-directed mutations assays identified that the binding motif AGCAAAA, spanning -1 698 bp to -1 692 bp of the mouse ob gene promoter, was responsible for the effects of glucose and insulin on ob gene expression. These studies suggest that a negative cis-acting element is located between -1 719 bp and -1 452 bp of the mouse ob gene promoter, and glucose and insulin simulate mouse ob gene expression by repressing the binding of a transcription factor to this element. This element, AGCAAAA, spanning -1 698 bp to -1 692 bp is a novel GLRE and IRE.
authors.affiliation: The Laboratory of Molecular Genetics, Shanghai Medical University, Shanghai 200032, China. hysong@shmu.edu.cn
authors.forename: Fang-Nian
authors.initials: FN
authors.lastname: Wang
authors.forename: Chun-Gu
authors.initials: CG
authors.lastname: Ma
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Zhang
authors.forename: Nai-Xian
authors.initials: NX
authors.lastname: Zhang
authors.forename: You-Mei
authors.initials: YM
authors.lastname: Chen
authors.forename: Qi-Qun
authors.initials: QQ
authors.lastname: Tang
authors.forename: Hou-Yan
authors.initials: HY
authors.lastname: Song