title: Multiple E-boxes in the distal promoter of the rat pyruvate carboxylase gene function as a glucose-responsive element.
abstract: Pyruvate carboxylase (PC) is an anaplerotic enzyme that regulates glucose-induced insulin secretion in pancreatic islets. Dysregulation of its expression is associated with type 2 diabetes. Herein we describe the molecular mechanism underlying the glucose-mediated transcriptional regulation of the PC gene. Incubation of the rat insulin cell line INS-1 832/13 with glucose resulted in a 2-fold increase in PC mRNA expression. Transient transfections of the rat PC promoter-luciferase reporter construct in the above cell line combined with mutational analysis indicated that the rat PC gene promoter contains the glucose-responsive element (GRE), comprising three canonical E-boxes (E1, E3 and E4) and one E-box-like element (E2) clustering between nucleotides -546 and -399, upstream of the transcription start site. Mutation of any of these E-boxes resulted in a marked reduction of glucose-mediated transcriptional induction of the reporter gene. Electrophoretic mobility shift assays revealed that the upstream stimulatory factors 1 and 2 (USF1 and USF2) bind to E1, the Specificity Protein-1 (Sp1) binds to E2, USF2 and the carbohydrate responsive element binding protein (ChREBP) binds to E4, while unknown factors binds to E3. High glucose promotes the recruitment of Sp1 to E2 and, USF2 and ChREBP to E4. Silencing the expression of Sp1, USF2 and ChREBP by their respective siRNAs in INS-1 832/13 cells blunted glucose-induced expression of endogenous PC. We conclude that the glucose-mediated transcriptional activation of the rat PC gene is regulated by at least these three transcription factors.
authors: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: Apilak
authors: A
authors: Wutthisathapornchai
authors: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: Tuangtong
authors: T
authors: Vongpipatana
authors: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: Sureeporn
authors: S
authors: Muangsawat
authors: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: Thirajit
authors: T
authors: Boonsaen
authors: UW Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States of America.
authors: Michael J
authors: MJ
authors: MacDonald
authors: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: Sarawut
authors: S
authors: Jitrapakdee
mesh_terms: D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011766:Pyruvate Carboxylase / Q000235:genetics*; D034622:RNA Interference; D051381:Rats; D020218:Response Elements / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051720:Upstream Stimulatory Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C442825:Mlxipl protein, rat; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; C497187:Usf2 protein, rat; D011766:Pyruvate Carboxylase; D005947:Glucose