Diminution of the O2 responsiveness of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene in rat hepatocytes by long-term culture at venous PO2.
The glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene within two hours is modulated by O2 in rat hepatocytes. It was the aim of the present study to test if this short-term modulation by O2 of the glucagon induction might be influenced by long-term culture of hepatocytes for 24 hours under different O2 tensions prior to glucagon induction. Cells were precultured for 24 hours at arterial O2 (16% O2) or venous O2 (8% O2), then induced within two to four hours with 1 nM glucagon each at arterial or venous O2. In arterial O2 precultured cells PCK mRNA and activity were induced to 100% at arterial O2 and to about 60% at venous O2. In venous O2 precultured cells PCK mRNA and activity were induced only to about 70% at arterial O2 and to about 60% at venous O2. Transfected PCK promoter (-2500)-CAT constructs were activated by glucagon with the same long-term modulatory effects of oxygen as the endogenous PCK gene. Gel mobility shift assays with nuclear extracts prepared from hepatocytes and a PCK promoter fragment ranging from -149 to -42 bp revealed one complex with a higher DNA binding activity when extracts of cells precultured for 24 hours under venous O2 as compared to arterial O2 were used. Therefore, the short-term modulation by O2 of PCK gene activation by glucagon was widely lost during preculture at low O2. This diminution of O2 sensitivity of PCK induction may be due to a nuclear protein or proteins which are induced by perivenous O2 tensions and bind to the PCK promoter.
Kietzmann
T
T
Institut für Biochemie und Molekulare Zellbiologíe, Göttingen, Germany. tkietzm@gwdg.de
Bratke
J
J
Jungermann
K
K
D000818:Animals; D001483:Base Sequence; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005934:Glucagon / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation
D012333:RNA, Messenger; D014157:Transcription Factors; D004247:DNA; D005934:Glucagon; D011493:Protein Kinase C; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D010100:Oxygen