Insulin-induced Early Growth Response Gene (Egr-1) Mediates a Short Term Repression of Rat Malic Enzyme Gene Transcription

Abstract

In this report we have studied insulin regulation of malic enzyme (ME) gene transcription in rat H-35 hepatoma cells and localized the insulin-responsive region of the ME promoter between positions ؊177 and ؊102. This region contains a putative insulin response element (IRE-II). When nuclear extracts from untreated or insulin-treated H-35 cells were incubated with IRE-II, transcription factors Sp1 and Sp3 were observed to bind constitutively to this element, whereas insulin induces the quick and transient binding of an insulin response factor. This induction requires de novo protein synthesis. Competition and supershift assays demonstrated that the insulin response factor is the immediate-early gene Egr-1. In vitro assays revealed that Egr-1 displaces Sp1 from its binding site in IRE-II. Insulin induces Egr-1 mRNA, with a time course pattern that corresponds perfectly to the Egr-1 binding to IRE-II. This induction depends on the activation of mitogen-activated protein (MAP) kinase, and it is phosphatidylinositol 3-kinaseindependent, as demonstrated with specific inhibitors for both pathways. By cotransfecting the wild-type or a dominant negative Ras, an upstream regulator of MAP kinase, we show that Ras inhibits ME promoter activity. Furthermore, overexpression of Egr-1 in H-35 cells represses the ME gene promoter in a dose-dependent manner. These results suggest that insulin induces a quick, transient, and Ras/MAP kinase-dependent activation of Egr-1 which leads to a transient repression of ME gene transcription. On a late phase, insulin would activate a different, Egr-1-independent pathway, which would result in activation of the ME gene.

Development of a Long-Acting Insulin Analog Using Albumin Fusion Technology

“…Treatment of the SREBP-SEAP reporter with either Albulin or insulin indicates that both Albulin and insulin can induce binding of SREBPs to their cognate sequence with approximately similar efficiency ( Table 1). We also tested the effect of Albulin and insulin on the transcription of two other key enzymes involved in hepatic fatty acid metabolism: malic enzyme and FAS (28). Treatment of FAS and malic enzyme reporters with Albulin or insulin strongly stimulated the secretion of SEAP in a dose-dependent and comparable manner.…”

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

“…Consistent with its role as an immediate early gene, Egr-1 mRNA levels rise and peak early after stimulation with insulin then decline quickly, with a corresponding pattern of protein expression (Figure 1). This time course of insulin induced increase in Egr-1 mRNA levels has been observed in many tissues (Barroso and Santisteban, 1999;Tan et al, 2003;Silverman and Collins, 1999;Leung-Theung-Long et al, 2005;Garnett et al, 2005) as has its dependence on the MAP kinase pathway (Leung-Theung-Long et al, 2005;Garnett et al, 2005;Keeton et al, 2003). The GnRH mRNA level peak after one hour of insulin treatment corresponds with the time of maximal Egr-1 protein levels, suggesting that higher protein levels, rather than protein activation, promote binding of Egr-1 to the GnRH promoter.…”

“…A variety of factors can stimulate Egr-1 gene expression, including cellular injury, radiation, and growth factors (Gashler and Sukhatme, 1995). Insulin regulates Egr-1 gene expression in a variety of tissues including hepatic, bone, pancreatic beta cells and vascular endothelial tissue (Barroso and Santisteban, 1999;Tan et al, 2003;Silverman and Collins, 1999;Leung-Theung-Long et al, 2005;Garnett et al, 2005). The induction of Egr-1 gene expression by insulin is primarily mediated by the MAP kinase pathway.…”

Regulation of apoA1 gene expression with acidosis: requirement for a transcriptional repressor

“…Egr-1 expression and activity can be induced by mitogenic signals (Sukhatme et al 1988, Lim et al 1987, atherosclerosis (McCaffrey et al 2000) and cellular stress (Santiago et al 1999, Yan et al 1999. Egr-1 acts as a transcriptional repressor of the murine deaminase promoter (Ackerman et al 1991) and the rat malic enzyme promoter (Barroso & Santisteban 1999). Of interest, Yamaji et al (1994) have previously shown that Egr-1 expression is induced by acidosis in the MCT kidney epithelial cell line as early as 15 min after the change in media pH.…”