Determining the retinoic acid response element in the liver glucokinase gene promoter using deletion and linker‐scan analysis

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Glucokinase is a key enzyme of hepatic glucose utilization when plasma glucose level is high. We have reported that retinoids in a liver derived lipophilic extract synergized with insulin to induce the expression of the glucokinase gene (Gck) in primary rat hepatocytes. Experiments were designed to determine the retinoic acid response element (RARE) in Gck promoter. We have shown that retinoic acid (RA) activated the minimal luciferase reporter construct containing 234‐bp (−237/−3, Gck‐234‐WT‐LUC) of the Gck promoter sequence both in primary rat hepatocytes and INS‐1 insulinoma cells. A series of reporter constructs containing overlapping 10‐ to 12‐bp scramble mutations covering the nucleotide sequence from −237 to −3 were made to narrow down the RARE to a 20‐bp region. Subsequently, a series of 5‐ to 6‐bp linker‐scan scramble mutations covering the entire 20‐bp region were made to narrow down the RARE to a 5‐bp region. RA induced Gck‐234‐WT‐LUC activity by 2.74±0.25‐fold in INS‐1 cells. The scramble mutation covering the sequence −177/−173 destroyed the RA‐induced luciferase activity. Moreover, single nucleotide mutations at −177, −176, and −175 significantly impaired the response of the reporter constructs to RA treatment. We conclude that the RA‐induced Gck transcription is mediated by a transcription activator associated with the RARE sequence covering −177/−175 of the liver Gck promoter.Grant Funding Source: American Heart Association