FUNCTIONAL SYNERGISM IN THE CARBOHYDRATE-INDUCED ACTIVATION OF LIVER-TYPE PYRUVATE-KINASE GENE-EXPRESSION

Hepatic expression of the liver-type pyruvate kinase (L-PK) gene is in duced at the transcriptional level by increased carbohydrate metabolis m in the rat. The carbohydrate response of the L-PK gene requires sequ ences from -171 to -124, which encompass adjacent major late transcrip tion factor (MLTF)-like and hepatic nuclear factor (HNF)-4 binding sit es, Neither site alone is capable of conferring a response, prompting us to explore the mechanism of synergy between the MLTF-like factor an d HNF-4. Spacing requirements between the two factor binding sites wer e tested by generating a series of mutations that altered the distance between these sites, Surprisingly, all of the constructs with spacing mutations were capable of responding to elevated glucose when introdu ced into primary hepatocytes. Thus the glucose response does not depen d on the rigid phasing of the MLTF-like and HNF-4 factors, suggesting that the factors binding to these two sites do not interact directly w ith each other. Substitution or inversion of the PK HNF-4 site abrogat ed the response to glucose and also significantly suppressed the promo ter activity under non-inducing conditions. We conclude that the MLTF like factor and HNF-4 co-operate functionally to maintain the basal ac tivity, as well as the carbohydrate responsiveness, of the L-PK gene, A mechanism other than co-operative DNA binding is responsible for the synergism.