Identification of the Regulatory Region of the L-Type Pyruvate Kinase Gene in Mouse Liver by Hydrodynamics-Based Gene Transfection

Abstract

Expression of L-type pyruvate kinase (L-PK) is upregulated in the liver by dietary carbohydrate. Previously, 3 carbohydrate/insulin response elements were identified in the 5'-flanking region of the L-PK gene up to bp -170. Studies of the 5'-flanking region beyond bp -183 in transgenic mice suggested that other regulatory elements may be present upstream of bp -183, but the positions of these elements were uncertain. In the present study, the existence of regulatory regions of the L-PK gene responding to stimulation by feeding was examined using in vivo hydrodynamics-based gene transfection (HT) in mouse liver. The firefly-luciferase (FL) gene, fused with various lengths of the 5'-flanking region of the L-PK gene, was introduced into mouse liver by HT. The mice had free access to a high-carbohydrate diet. In liver homogenate, luciferase activity of pL-PK(-1467)-FL (which included the 5'-flanking region from bp -1467 to +17), was markedly stimulated by feeding. 5'-Deletion up to bp -1065 caused only minor changes in luciferase activity, but further deletion up to bp -690 and bp -203 caused significant, gradual decreases in activity. Further analyses utilizing 5'-deletion mutants indicated the existence of positive regulatory regions that respond to stimulation by feeding between bp -1065 and -945, and between -300 and -203 on the L-PK gene. These results suggest that unidentified cis-acting DNA elements exist in the upstream region of the L-PK gene, and that HT is a useful approach for detecting regulatory regions of genes expressed in the liver.