Untitled

Abstract

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.

ChREBP Rather than USF2 Regulates Glucose Stimulation of Endogenous L-pyruvate Kinase Expression in Insulin-secreting Cells

“…We conclude that USF has no effect on either basal or glucose-stimulated L-PK expression in INS-1 cells. Consistently, induction of USF2 or DN-USF2 does not affect the L-PK promoter activity in INS-1 cells, which supports the study of Kaytor et al (7) but contradicts the report of Kennedy et al (5).…”

“…Microinjection of USF antibodies into the nuclei of INS-1 cells could have pleiotropic effects on the L-PK promoter luciferase activity (5), and data showing diminished luciferase activity should therefor be interpreted with caution. Indeed, this work (5) is strongly challenged by Kaytor et al (7) who suggest that adenovirus-mediated expression of DN-USF does not interfere with glucose-induced L-PK promoter activity. Unfortunately, the expression level and cellular localization of DN-USF in the latter study was not monitored (7).…”

“…Indeed, this work (5) is strongly challenged by Kaytor et al (7) who suggest that adenovirus-mediated expression of DN-USF does not interfere with glucose-induced L-PK promoter activity. Unfortunately, the expression level and cellular localization of DN-USF in the latter study was not monitored (7).…”

“…In addition, microinjection of USF2 antibodies into the nucleus of single INS-1 cells caused a drastic inhibition of glucose-stimulated L-PK promoter activity (5). Conversely, Kaytor et al (7) demonstrated that adenovirus-mediated overexpression of a dominant-negative form of USF in INS-1 cells failed to block the glucose-induced L-PK promoter activity and again negated the involvement of USF in glucose-regulation in INS-1 cells. The present study was aimed to clarify these conflicting results.…”

“…The pancreatic ␤-cell responds to increased glucose metabolism by releasing insulin, which is essential for maintaining glucose and fatty acid homeostasis (1). L-pyruvate kinase (L-PK) 1 represents a typical glucose-responsive gene in both hepatocytes and pancreatic ␤-cells (2)(3)(4)(5)(6)(7)(8)(9). However, there is controversy over the identity of the transcription factor controlling the glucose-stimulated L-PK expression.…”