We have cloned 5 kb of genomic DNA encompassing 1.72 kb of 5'-regulatory sequence and exons 1-c and 2 of the rat SREBP-1c gene. A 1.5-kb segment upstream from the transcription start site was ligated ahead of the luciferase reporter gene and tested for promoter activity by transient transfection assays in primary rat hepatocytes. We discovered that insulin strongly activated the full-length promoter, regardless of whether 5 or 20 mM glucose was in the culture medium during treatment. Stimulation by insulin was blocked by dibutyryl-cAMP and by polyunsaturated fatty acids, such as alpha-linolenic acid, gamma-linolenic acid, or eicosapentaenoic acid; palmitic or oleic acids, however, had no inhibitory effect. A truncated promoter containing 149 bp of 5' flanking DNA, including proximal NF-Y, E-box, SRE, and Sp1 sites, retained most of the response. This is the first report that insulin, cAMP, and polyunsaturated fatty acids modulate the proximal SREBP-1c promoter in rat hepatocytes mirroring physiological regulation of SREBP-1c in vivo.
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