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Endocrinology. 2001 Mar;142(3):1041-9.

Physiological concentrations of insulin promote binding of nuclear proteins to the insulin-like growth factor I gene.

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Emory University School of Medicine, Atlanta, Georgia 30322, USA.


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 B(1), which persisted despite the presence of antibodies against Sp1 and Sp3. B(1) 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 B(1) 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 B(1) 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.

[Indexed for MEDLINE]

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