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.
Highlights
Glucose homeostasis is tightly regulated by glucagon and insulin which counteract each other to maintain the concentration of plasma glucose within a narrow range
Pyruvate formed after glycolysis appears to enter beta cell mitochondria through pyruvate decarboxylation catalyzed by pyruvate dehydrogenase complex (PDH) and pyruvate carboxylation by pyruvate carboxylase (PC) with similar proportions, flux toward the latter reaction is tightly associated with the glucose concentration the cells is exposed to and correlates with the magnitude of insulin secretion [6,7]
Because the distal promoter of the rat PC gene is the only promoter that is transcriptionally active in rat pancreatic b-cells [17] and mostly active in the insulinoma cell line, INS-1 832/13 [26] we examined whether the distal promoter of the PC gene contains a glucose-responsive element (GRE) by transfecting the 1.2 kb distal promoter-luciferase chimeric reporter constructs into the INS-1 832/13, a cell line that responds to glucose more robust than the INS-1 cell line [30]
Summary
Glucose homeostasis is tightly regulated by glucagon and insulin which counteract each other to maintain the concentration of plasma glucose within a narrow range. Glucagon secreted during prolonged starvation raises the blood glucose level by stimulating glycogen breakdown and gluconeogenesis [1] while insulin secreted during fed conditions [glucose-induced insulin secretion (GSIS)] lowers blood glucose levels by stimulating glucose uptake, glycolysis and glycogen synthesis in liver and skeletal muscle [2]. This process occurs efficiently in the pancreatic b-cell due to the presence of GLUT2 and glucokinase [3], which together acts as a sensor allowing high concentrations of glucose to enter to the cell for aerobic glycolysis and oxidative phosphorylation [4]. Several studies performed in models of diabetic rats and human subjects with type 2 diabetes clearly show that downregulation of PC expression in pancreatic islets is associated with type 2 diabetic phenotypes further supporting the role of PC in GSIS [14,15,16]
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