Transcriptional regulation of leptin gene promoter in rat.

To investigate the DNA regulatory sequences required for stimulation and suppression of leptin gene expression, primary cultured hepatocytes and adipocytes of rats were transfected with plasmids containing the 5'-flanking sequences of the rat leptin gene fused to the luciferase gene. When two copies of the sequences spanning nucleotides -101 to -83 of the leptin promoter were used for transfection, the reporter activity significantly increased in the presence of glucose/insulin in comparison with glucose alone. The glucose/insulin stimulation of the transcription was inhibited by addition of polyunsaturated fatty acids. These results were similar to those found earlier for the transcription of the fatty acid synthase, FAS(-57/-35) and ATP citrate-lyase, ACL(-64/-41) genes. Cotransfection studies in the cells with a Sp1 expression vector and leptin (-101/-83) constructs showed the inactivation of the leptin promoter by Sp1. Gel mobility shift assays using an end-labeled leptin (-101/-83) construct as a probe revealed that nuclear factor(s) from rat liver or adipose tissue specifically formed complexes with the sequence. The DNA-protein complexes were common to the glucose/insulin-responsive regions of the leptin, ACL and FAS genes, suggesting that these genes are coordinately regulated. In addition, by antibody supershift assays, the transcription factor Sp1 was found to bind the GC-rich region located between nucleotides -101 and -83 of the leptin gene. Mutational analysis of this region showed that the sequence of the region was critical for glucose/insulin stimulation of transcription. Thus, we postulated that the region from -101 to -83 of the leptin gene is responsible for glucose/insulin stimulation of transcription, and that Sp1 is somehow involved in this regulation.