c-Myc and ChREBP regulate glucose-mediated expression of the L-type pyruvate kinase gene in INS-1-derived 832/13 cells

Abstract

Increased glucose flux generates metabolic signals that control transcriptional programs through poorly understood mechanisms. Previously, we demonstrated a necessity in hepatocytes for c-Myc in the regulation of a prototypical glucose-responsive gene, L-type pyruvate kinase (L-PK) (Collier JJ, Doan TT, Daniels MC, Schurr JR, Kolls JK, Scott DK. J Biol Chem 278: 6588–6595, 2003). Pancreatic β-cells have many features in common with hepatocytes with respect to glucose-regulated gene expression, and in the present study we determined whether c-Myc was required for the L-PK glucose response in insulin-secreting (INS-1)-derived 832/13 cells. Glucose increased c-Myc abundance and association with its heterodimer partner, Max. Manipulations that prevented the formation of a functional c-Myc/Max heterodimer reduced the expression of the L-PK gene. In addition, glucose augmented the binding of carbohydrate response element binding protein (ChREBP), c-Myc, and Max to the promoter of the L-PK gene in situ. The transactivation of ChREBP, but not of c-Myc, was dependent on high glucose concentrations in the contexts of either the L-PK promoter or a heterologous promoter. The glucose-mediated transactivation of ChREBP was independent of mutations that alter phosphorylation sites thought to regulate the cellular location of ChREBP. We conclude that maximal glucose-induced expression of the L-PK gene in INS-1-derived 832/13 cells involves increased c-Myc abundance, recruitment of c-Myc, Max, and ChREBP to the promoter, and a glucose-stimulated increase in ChREBP transactivation.

The glucose-responsive transcription factor ChREBP contributes to glucose-dependent anabolic synthesis and cell proliferation

“…ChREBP has been shown to play critical roles in glycolysis and lipogenesis in hepatocytes and adipocytes (12)(13)(14)(15)(16)(17). The present findings suggest that ChREBP also plays a role in redirecting glucose metabolism for lipid biosynthesis during cell proliferation.…”

“…The basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor carbohydrate responsive element binding protein (ChREBP) is a critical mediator of glucose-dependent induction of glycolytic and lipogenic enzyme genes in metabolic tissues (12)(13)(14)(15)(16)(17). Cellular level of nutrients (such as glucose and fatty acid) regulates the level and activity of ChREBP in hepatocytes and adipocytes (18,19).…”

“…RNA biosynthesis was assayed by measuring incorporation of 14 C into RNA after culture of cells with universally labeled D-[U-14 C6] glucose (PerkinElmer). RNA was then extracted using RNeasy columns (Qiagen) and 14 C incorporation was assayed using a Beckman LS 6500 scintillation counter. 14 C cpm for each sample were normalized by the amount of RNA.…”

Detailed molecular analysis of the induction of the L-PK gene by glucose

“…RT-PCR was carried out using a SYBR green master mix (Bio-Rad, Hercules, CA) in an Applied Biosystems Prism 7300 Real-Time PCR System as previously described [6]. Fold change in mRNA expression was determined using the ΔΔcT method, with all genes normalized to cyclophilin [11].…”

Activation and repression of glucose-stimulated ChREBP requires the concerted action of multiple domains within the MondoA conserved region

“…It was further postulated that its dephosphorylation was mediated by a xylulose-5-phosphate-stimulated protein phosphatase 2A isoform in response to elevated glucose metabolism, resulting in nuclear localization and DNA binding (11,12). However, ChREBP mutations that block phosphorylation at the proposed regulatory sites still require glucose for transcriptional activity (3,13,24). Furthermore, ChREBP mutations of the NES region that result in its nuclear accumulation under basal conditions are also dependent on increased glucose for activation (5).…”