Localization of the carbohydrate response element of the rat L-type pyruvate kinase gene.
Pyruvate kinase is a major regulatory enzyme of glycolysis. Transcription of the L-type pyruvate kinase (L-PK) gene in rat liver is induced by feeding a carbohydrate-rich diet. To investigate the regulatory DNA sequences required for this response, primary hepatocytes were transfected with plasmids containing the 5'-flanking sequence of the rat L-PK gene fused to the chloramphenicol acetyltransferase (CAT) gene. Sequences from -4300 to +12 of the L-PK gene directed an increase in CAT activity when hepatocytes were switched from media containing 10 mM lactate to 25 mM glucose. Average induction was 17-fold (n = 13; S.E. = 2.9). Addition of fructose to the media also induced CAT activity. Carbohydrate regulation of the L-PK promoter was retained with 5'-deletions to -197, but constructs deleted to -96 were completely unresponsive. The 101-base pair fragment from -197 to -96 of the L-PK gene can confer carbohydrate regulation when fused in either orientation to the heterologous thymidine kinase promoter, thus defining a carbohydrate response element in this region. Expression of the transfected gene was regulated by insulin and glucagon in a pattern similar to that seen for the endogenous L-PK gene, suggesting that control of L-PK promoter activity was responsible for carbohydrate-mediated changes in L-PK mRNA production.
Thompson
K S
KS
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Towle
H C
HC
D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology*; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005934:Glucagon / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D011770:Pyruvate Kinase / Q000235:genetics*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection
D002241:Carbohydrates; D007328:Insulin; D005934:Glucagon; D011770:Pyruvate Kinase; D005947:Glucose