Induction of the glucokinase gene by insulin in cultured neonatal rat hepatocytes. Relationship with DNase-I hypersensitive sites and functional analysis of a putative insulin-response element.
Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1-day-old newborn rats, but not in cultured hepatocytes from 21-day-old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver-specific DNase I hypersensitive sites located in the 5' flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site-1 and hypersensitive site-2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1-day-old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase-I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site-1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase-I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin-response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene.
Parsa
R
R
Centre de Recherche sur l'Endocrinologie Moléculaire et le Développement, CNRS, Meudon, France.
Decaux
J F
JF
Bossard
P
P
Robey
B R
BR
Magnuson
M A
MA
Granner
D K
DK
Girard
J
J
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000096:biosynthesis / Q000235:genetics*; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D012045:Regulatory Sequences, Nucleic Acid; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics
D007328:Insulin; D012333:RNA, Messenger; D005941:Glucokinase; D013937:Thymidine Kinase; D003850:Deoxyribonuclease I