Insulin responsiveness of the glucagon gene conferred by interactions between proximal promoter and more distal enhancer-like elements involving the paired-domain transcription factor Pax6

Regulation of gene transcription is an important aspect of insulin's action . However, the mechanisms involved are poorly understood. Insulin inhibits glucagon gene transcription, and insulin deficiency is associated with hype rglucagonemia that contributes to hyperglycemia in diabetes mellitus. Trans fecting glucagon-reporter fusion genes into a glucagon-producing pancreatic islet cell line, a 5'-, 3'-, and internal deletion analysis, and oligonucl eotide cassette insertions failed in the present study to identify a single insulin-responsive element in the glucagon gene. They rather indicate that insulin responsiveness depends on the presence of both proximal promoter e lements and more distal enhancer-like elements. When the paired domain tran scription factor Pax6 binding sites within the proximal promoter element G1 and the enhancer-like element G3 were mutated into GAL4 binding sites, the expression of GAL4-Pax6 and GAL4-VP16 restored basal activity, whereas onl y GAL4-Pax6 restored also insulin responsiveness. Likewise, GAL4-CBP activi ty was inhibited by insulin within the glucagon promoter context. The resul ts suggest that insulin responsiveness is conferred to the glucagon gene by the synergistic interaction of proximal promoter and more distal enhancer- like elements, with Pax6 and its potential coactivator the CREB-binding pro tein being critical components. These data thereby support concepts of insu lin-responsive element-independent mechanisms of insulin action to inhibit gene transcription.