An indirect role for upstream stimulatory factor in glucose-mediated induction of pyruvate kinase and S14 gene expression.

Transcription of the L-type pyruvate kinase (L-PK) and S14 genes is induced in hepatocytes in response to increased glucose metabolism. The regulatory sequences of these genes responsible for induction by glucose have been mapped to related E-box containing motifs in the promoters. Similarly, L-PK promoter activity is stimulated in a differentiated pancreatic beta-cell line, INS-1, in response to elevated glucose. By mutational analysis, we demonstrate that the sequence requirements for glucose induction in the INS-1 cell are identical to those observed in the hepatocyte, suggesting that the same transcriptional factor(s) is responsible for regulation of L-PK expression in the two cell types. One nuclear factor that binds to the glucose regulatory sequences of both of these genes is the Upstream Stimulatory Factor (USF), a ubiquitous E-box binding protein. Mice deleted for the USF2 gene display a severely delayed response to carbohydrate feeding (Vallet et al. [26]). This observation, however, does not differentiate between a direct and an indirect role for USF in the process. To gain further insight into the possible involvement of USF in glucose signaling, we have used a recombinant adenoviral construct that expresses a dominant negative form of USF. This dominant negative can dimerize with endogenous USF and is shown to inhibit DNA binding of USF in hepatocytes and INS-1 cells. However, expression of the dominant negative USF did not block the ability of glucose to stimulate L-PK or S14 gene expression in hepatocytes or L-PK promoter activity in INS-1 cells. We conclude that USF does not act by binding to the glucose regulatory sequences of the S14 or L-PK genes and the role of USF in the process of glucose induction is indirect.