Chronic elevation in glucose has pleiotropic effects on the pancreatic ␤-cell including a high rate of insulin secretion at low glucose, ␤-cell hypertrophy, and hyperplasia. These actions of glucose are expected to be associated with the modulation of the expression of a number of glucose-regulated genes that need to be identified. To further investigate the molecular mechanisms implicated in these adaptation processes to hyperglycemia, we have studied the regulation of genes encoding key glycolytic enzymes in the glucose-responsive ␤-cell line INS-1. Glucose (from 5 to 25 mM) induced phosphofructokinase-1 (PFK-1) isoform C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4-fold), and L-pyruvate kinase (L-PK) (7-fold) mRNAs. In contrast the expression level of the glucokinase (Gk) and 6-phosphofructo-2-kinase transcripts remained unchanged. Following a 3-day exposure to elevated glucose, a similar induction was observed at the protein level for PFK-1 (isoforms C, M, and L), GAPDH, and L-PK, whereas M-PK expression only increased slightly. The study of the mechanism of GAPDH induction indicated that glucose increased the transcriptional rate of the GAPDH gene but that both transcriptional and post transcriptional effects contributed to GAPDH mRNA accumulation. 2-Deoxyglucose did not mimic the inductive effect of glucose, suggesting that increased glucose metabolism is involved in GAPDH gene induction. These changes in glycolytic enzyme expression were associated with a 2-3-fold increase in insulin secretion at low (2-5 mM) glucose. The metabolic activity of the cells was also elevated, as indicated by the reduction of the artificial electron acceptor 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. A marked deposition of glycogen, which was readily mobilized upon lowering of the ambient glucose, and increased DNA replication were also observed in cells exposed to elevated glucose. The results suggest that a coordinated induction of key glycolytic enzymes as well as massive glycogen deposition are implicated in the adaptation process of the ␤-cell to hyperglycemia to allow for chronically elevated glucose metabolism, which, in this particular fuel-sensitive cell, is linked to metabolic coupling factor production and cell activation.The adaptation of the pancreatic ␤-cell to chronic hyperglycemia is characterized by an augmented secretory function, hypertrophy, and hyperplasia (1, 2). These pleiotropic actions promoted by elevated glucose are expected to be associated with the induction of a number of glucose-regulated genes that must be identified. Elevated circulating glucose has gained recognition over the last few years as a factor contributing to ␤-cell dysfunction and the subsequent development of type 2 diabetes (2-4). However, the link between sustained hyperglycemia and long-term alterations in ␤-cell function is not well understood. In animal models, prolonged hyperglycemia causes both an impaired glucose-induced insulin secretion and the development of peripheral insulin resistance (5, 6). These a...