REGULATION OF THE L-TYPE PYRUVATE KINASE GENE BY GLUCOSE AND cAMP IN ISLET BETA CELLS

Extracellular signals generated during both feeding and fasting coordinately regulate transcription of metabolic enzyme genes that control glucose metabolism in theƒnƒÒ cell. A post-prandial rise in extracellular glucose levels promotes expression of various genes including the gene encoding the glycolytic enzyme L-type pyruvate kinase (L-PK). Conversely, under conditions of fasting, a rise in hormones that stimulate increased intracellular levels of cAMP results in suppression of glucose-activated genes such as L-PK. The L-PK gene is coordinately regulated by these two opposing stimuli. Therefore, we explored the mechanism of induction and repression of the L-PK gene by glucose and cAMP, respectively, using the 832/13 rat insulinoma cell line.Glucose mediates induction of the L-PK gene by stimulating the recruitment of two primary DNA binding transcription factors, the basic helix-loop-helix/leucine zipper protein Carbohydrate Response Element Binding Protein (ChREBP) and the orphan nuclear receptor, Hepatic Nuclear Factor 4ƒÑ (HNF4ƒÑ) to their respective response elements in the proximal L-PK promoter. In addition, glucose stimulates the recruitment of the coactivator CREB binding protein (CBP) to the L-PK gene promoter. Assembly of these three factors on the L-PK gene promoter facilitates alterations in the pattern of acetylation and methylation of histones associated with the promoter and coding region, respectively. These changes in histone modifications correlate with increased occupancy of the RNA Polymerase II (Pol II) holoenzyme on the L-PK promoter. Finally, glucose promotes changes in the phosphorylation state of the carboxyl-terminal domain (CTD) of Pol II at serines 5 and 2, which are necessary for the promoter clearance and elongation phases of transcription.cAMP represses the glucose-mediated induction of the L-PK gene by inhibiting the assembly of the ChREBP, HNF4ƒÑ and CBP-containing complex on the L-PK promoter. The cAMP-dependent decrease in complex assembly on the promoter is associated with alterations in the acetylation and methylation status of histones on both the promoter and coding region. Furthermore, cAMP inhibits the glucose-mediated recruitment and phosphorylation of Pol II CTD, ultimately blocking initiation and elongation of the L-PK gene by Pol II. In summary, these studies provide a detailed insight into the mechanism of regulation of the L-PK gene by glucose and cAMP in islet ƒÒ cells