c-Myc Is Required for the Glucose-mediated Induction of Metabolic Enzyme Genes

Abstract

Glucose exerts powerful effects on hepatocyte gene transcription by mechanisms that are incompletely understood. c-Myc regulates hepatic glucose metabolism by increasing glycolytic enzyme gene transcription while concomitantly decreasing gluconeogenic and ketogenic enzyme gene expression. However, the molecular mechanisms by which c-Myc exerts these effects is not known. In this study, the glucose-mediated induction of L-type pyruvate kinase and glucose-6-phosphatase mRNA levels was diminished by maneuvers involving recombinant adenoviral vectors that interfere with (i) c-Myc protein levels by antisense expression or (ii) c-Myc function through a dominant-negative Max protein. These results were obtained using both HL1C rat hepatoma cells and primary rat hepatocytes. Furthermore, a decrease in c-Myc abundance reduced glucose production in HL1C cells, presumably by decreasing glucose-6-phosphatase activity. The repression of hormone-activated phosphoenolpyruvate carboxykinase gene transcription by glucose was not affected by a reduction in c-Myc levels. The basal mRNA levels for Lpyruvate kinase and glucose-6-phosphatase were not altered to any significant degree by adenoviral treatment. Furthermore, adenoviral overexpression of the c-Myc protein induced glucose-6-phosphatase mRNA in the absence of glucose stimulation. We conclude that multiple mechanisms exist to communicate the glucose-derived signal and that c-Myc has a key role in the hepatic glucose signaling pathway.Insulin and glucose act jointly to influence glucose homeostasis by altering hepatic gene expression patterns. Insulin increases glucokinase (GK) 1 gene transcription and protein levels in hepatocytes (1). The phosphorylation of glucose by GK leads to increased glucose flux and metabolism, generating signaling metabolites that modify the gene expression profile of the liver (2). For example, the L-pyruvate kinase (L-PK) and glucose-6-phosphatase (Glc-6-Pase) genes (encoding key glycolytic and gluconeogenic enzymes, respectively) are stimulated by increases in hepatic glucose metabolism (3-5). Conversely, glucose metabolism can also negatively regulate gene transcription, exemplified by the repression of hormone-activated phosphoenolpyruvate carboxykinase (PEPCK) gene promoter activity (6, 7). Although glucose metabolism modulates the gene expression patterns of key glycolytic and gluconeogenic enzymes, the signaling mechanisms and coordinating transcription factors involved are not fully characterized.One transcription factor that is a candidate for establishing hepatic glucose-dependent gene expression patterns is the basic, helix-loop-helix leucine-zipper (bHLH-LZ) transcription factor c-Myc (8, 9). The Myc family of proteins (e.g. c-, N-, L-Myc, USF, Max, Mad, etc.) participates in control of proliferation, growth, differentiation, apoptosis, and metabolism (10, 11). c-Myc binds DNA by interacting with the E box sequence CACGTG (and other related non-canonical sites) as a heterodimer with Max, another bHLH-LZ protein. The Myc/Max heterodimer ...

Gene expression profiles of human liver cells mediated by hepatitis B virus X protein

“…All biological activities of c-Myc require its binding partner, Max [22] . c-Myc has been assigned roles in hepatocyte proliferation during liver development and regeneration, control of hepatic metabolism, and the dysregulated growth that occurs during heptocarcinogenesis [23][24][25][26] . Therefore, the enhancement of Max may be consistent with the induction of c-Myc in L-O2-X cells to promote hepatocyte growth and proliferation.…”

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

“…HL1C rat hepatoma cells [7] were maintained as previously described [8]. Cells were transduced with an adenovirus expressing glucokinase (Ad-GK [9]) (a gift from Dr. Christopher Newgard) and incubated for 24 h. The amount of Ad-GK required to facilitate a glucose response in the HL1C hepatomas was established empirically by a functional titration, wherein the amount of Ad-GK that conferred a 2 to 3 fold increase in L-PK gene expression was determined.…”

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

“…Total RNA was isolated from 832/13 cells using Tri reagent (Molecular Research Center) according to the manufacturer's instructions. The conditions for the RT and PCR have been described, and the linear response of the pixel densities from ethidium bromide-stained gels as a function of RNA input has been validated (10,39). For the real-time PCR analyses, 2.5% of the total RT reaction was used as input for PCR using SYBR green master mix (Applied Biosystems) or Bio-Rad's iTaq SYBR green supermix with carboxy-X-rhodamine.…”

“…Preparation of nuclear proteins and blot immunolabeling of these proteins has been described (10). For coimmunoprecipitation reactions, 500 g of nuclear protein were diluted to 1 g/l with lysis/wash buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1), and the immunoprecipitation reaction was carried out using the Catch and Release reversible immunoprecipitation kit following the manufacturer's recommendations (Upstate Cell Signaling Solutions).…”

“…Although many genes are regulated by glucose, the L-PK gene has become a prototypical model system for determining the molecular mechanisms of glucose signaling in both hepatocytes and ␤-cells (16,33,35,45,46,48,50,52,53). Our group recently demonstrated that c-Myc is required for glucose-regulated expression of the L-PK gene in hepatocytes (10), and the present study focuses on the role of c-Myc in the regulation of this gene in ␤-cells.…”