THE PYRUVATE-KINASE GENE AS A MODEL FOR STUDIES OF GLUCOSE-DEPENDENT REGULATION OF GENE-EXPRESSION IN THE ENDOCRINE PANCREATIC BETA-CELL TYPE

The insulinoma beta-cell line INS-1 expresses the L-type pyruvate kina se gene at high level and responds to a rise in extracellular glucose by strong induction of gene expression. Following the addition of gluc ose to the culture medium in the 3.5-33 mM concentration range, the ce llular level of L-type pyruvate kinase mRNA increases within 2 h and r eaches a maximum 15-fold above basal in 8-12 h. By run-on nuclear assa y, the relative transcription rate of the pyruvate kinase gene is show n to increase 4-fold at maximal stimulation, suggesting that both tran scriptional and post-transcriptional effects contribute to mRNA accumu lation. The glucose effect is totally suppressed by the hexokinase inh ibitor mannoheptulose, indicating a requirement for glucose phosphoryl ation. The mRNA induction is not inhibited in glutamine-free culture m edium or by azaserine, suggesting that the hexosamine biosynthetic pat hway is not involved. Moreover, metabolism along the glycolytic pathwa y does not appear to be an absolute requisite, since 2-deoxyglucose pa rtly mimics the inductive effect of glucose. The glucose effect on the pyruvate kinase gene is reversibly antagonized by agents increasing i ntracellular cAMP. In addition, the effect is highly specific to the p yruvate kinase gene. Neither proinsulin I mRNA nor glucokinase mRNA ar e increased in glucose-stimulated INS-1 cells. Short term transfection with CAT plasmids driven by the pyruvate kinase L promoter reveals sp ecific glucose-inducible reporter activity with the 183-base pair prom oter region upstream of the cap site. Within this region, the previous ly described L4 cis-acting element is crucial for glucose responsivene ss, as demonstrated by the fact that a plasmid with a mutation in this element does not elicit glucose-inducible CAT activity. Induction of L-type pyruvate kinase mRNA occurs in the islets of rats subjected to fasting and carbohydrate refeeding. In conclusion, the L-type pyruvate kinase gene provides an interesting model of glucose-regulated gene i n the endocrine beta-cell type.