Physiological concentrations of insulin promote binding of nuclear proteins to the insulin-like growth factor I gene.
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.
Kaytor
E N
EN
Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Zhu
J L
JL
Pao
C I
CI
Phillips
L S
LS
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D009994:Osmolar Concentration; D051381:Rats; D012016:Reference Values; D014020:Tissue Extracts / Q000378:metabolism
D002352:Carrier Proteins; D007328:Insulin; D009687:Nuclear Proteins; D014020:Tissue Extracts; D007334:Insulin-Like Growth Factor I