Mechanism of the permissive action of dexamethasone on the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes.
Rat hepatocytes were cultured for 24 h in the presence or absence of 100 nM dexamethasone (DX). After a medium change, phosphoenolpyruvate carboxykinase (PCK) was induced by addition of glucagon at different concentrations, from physiological 0.1 nM to hyperphysiological 10 nM, again in the presence or absence of 100 nM dexamethasone. 1. With dexamethasone addition during the culture and induction phase (DX+/+), 10 nM glucagon increased PCK mRNA abundance (Northern blot analysis) and activity (in vitro translation) synchronously to the same extent with maxima after 2 h and PCK enzyme activity after a time lag with a maximum after 6 h. The total detectable PCK mRNA amount was apparently also translationally active. 10 microM N6,2'-O-dibutyryladenosine 3',5'-(cyclic)phosphate (Bt2cAMP) as the second messenger had essentially the same effect as 10 nM glucagon. 2. In the absence of dexamethasone during the preculture and the induction phase (DX-/-), 10 nM glucagon and 10 microM Bt2cAMP could enhance PCK mRNA only about half-maximally. Glucagon or dexamethasone added alone in physiological concentrations of 0.1 nM and 100 nM, respectively, were unable to increase PCK mRNA. However, treatment of the cells with dexamethasone also enabled 0.1 nM glucagon to enhance PCK mRNA to a maximum after 2 h, independent of the presence of dexamethasone during the induction period (DX+/+ and DX+/- cells). Thus, dexamethasone was a permissive agent in that it shifted the sensitivity of the cells towards glucagon into the physiological concentration range. 3. In the presence of dexamethasone during the culture and induction phase (DX+/+) 0.1 nM glucagon maximally enhanced the transcription of the PCK gene (nuclear run on) fourfold after 30 min; in the absence of dexamethasone during both phases (DX-/-) glucagon was without any effect. The overall transcriptional rate was not significantly different in cells with and without dexamethasone during the culture and induction phase (DX+/+ vs. DX-/-). Thus, dexamethasone acted permissively mainly on the transcription of the PCK gene. 4. With culture in the presence of dexamethasone over decreasing periods of time, 1 nM glucagon could induce submaximal PCK mRNA amounts already after 1-3 h steroid culture. This restitution by dexamethasone of the PCK mRNA inducibility by glucagon was inhibited by cycloheximide. This suggested that ongoing protein synthesis was required for the permissive action of dexamethasone on the expression of the PCK gene. The results allow the following conclusions.(ABSTRACT TRUNCATED AT 400 WORDS)
Runge
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Institut für Biochemie, Fachbereich Medizin, Georg-August-Universität Göttingen, Federal Republic of Germany.
Schmidt
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Christ
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B
Jungermann
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D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005934:Glucagon / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008957:Models, Genetic; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic / Q000187:drug effects*
D012333:RNA, Messenger; D012313:RNA; D003907:Dexamethasone; D005934:Glucagon; D003513:Cycloheximide; D010729:Phosphoenolpyruvate Carboxykinase (GTP)