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Physiological Concentrations of Insulin Promote Binding of Nuclear Proteins to the Insulin-Like Growth Factor I Gene¹

Emory University School of Medicine, Atlanta, Georgia 30322

Address all correspondence and requests for reprints to: Lawrence S. Phillips, M.D., Division of Endocrinology, Emory University School of Medicine, 1639 Pierce Drive, 1301 WMRB, Atlanta, Georgia 30322. E-mail: medlsp{at}emory.edu

Limitations in understanding the mechanism of transcriptional regulation by insulin are due in part to lack of models in which there is insulin-responsive binding of nuclear factors to critical promoter regions. The insulin-like growth factor I (IGF-I) gene responds to diabetes status via a footprinted sequence, region V, which contains an AT-rich element and a GC-rich site. We tested the hypothesis that insulin regulates nuclear factor binding to the AT-rich site. Gel shift analysis with liver nuclear extracts and a region V probe showed binding of Sp1, Sp3, and B₁, which persisted despite the presence of antibodies against Sp1 and Sp3. B₁ was detected by a probe mutated in the GC-rich site (VmSp1), but not by a probe mutated at the AT-rich site (VmAT). We then asked whether B₁ was responsive to insulin. For both region V and VmSp1 probes, nuclear extracts from normal rat hepatocytes, H4IIE cells, and CHO-IR cells exposed to 10^-6 M insulin exhibited an increase in binding, designated insulin-responsive binding protein (IRBP); IRBP comigrated with B₁ from liver extracts. IRBP binding to region V was competed by VmSp1, but not by VmAT, indicating specific interactions with the AT-rich sequence; insulin response elements from other genes also failed to compete. After addition of insulin, IRBP began to increase by 1 h and rose further at 24 h, suggesting involvement of both posttranslational and transcriptional mechanisms. IRBP responded to as little as 10^-10 M insulin, indicating physiological relevance. Induction of IRBP was blunted by the phosphatidylinositol 3'-kinase inhibitor LY294002, whereas other signal transduction inhibitors had little effect. IRBP interacts with an important sequence in the IGF-I gene and may participate in the metabolic regulation of IGF-I expression. As most insulin-responsive genes do not exhibit insulin-responsive nuclear factor binding, further studies of IRBP may also contribute to understanding of the mechanism of insulin action on gene transcription.

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