Transcriptional Regulation of Insulin Receptor Gene Promoter in Rat Hepatocytes

Abstract

To investigate the DNA regulatory sequences −618 to −593 (initiator ATG is +1) of the insulin receptor (IR) promoter required for IR gene expression, primary cultured hepatocytes of rats were transfected with plasmids containing the 5′-flanking sequences of the rat IR gene fused to the luciferase gene. When three copies of the nucleotides −618 to −593 of the IR promoter were transfected, the reporter activity was significantly increased in the presence of glucose and more increased in the presence of glucose/insulin. The glucose/insulin stimulation was inhibited by the addition of polyunsaturated fatty acids. These results were similar to those found earlier for the transcriptions of the fatty acid synthase, FAS(−57/−35), ATP citrate-lyase, ACL(−64/−41), and leptin(−101/−83) genes, which promoter sequences have a high similarity to IR(−618/−593). However, similarly to the leptin gene, the IR gene was more responsive to glucose stimulation than the FAS and ACL genes. Mutation of either one of the Sp1 binding sites (−618/−593) did not significantly affect the transcription, whereas mutation of three or four Sp1 binding sites resulted in a loss of responsiveness to glucose/insulin. Gel mobility shift assays revealed that nuclear factor(s) from rat liver specifically formed complexes with the sequence of IR(−618/−593). By antibody supershift assays, the transcription factors Sp1and Sp3 were found to bind with the IR(−618/−593). The IR, leptin, ACL and FAS genes contain common DNA-sequences responsible for the glucose/insulin-stimulation, suggesting that these genes are similarly regulated.