title: Glucagon induces the gene expression of aquaporin-8 but not that of aquaporin-9 water channels in the rat hepatocyte.
abstract: Glucagon stimulates the vesicle trafficking of aquaporin-8 (AQP8) water channels to the rat hepatocyte canalicular membranes, a process thought to be relevant to glucagon-induced bile secretion. In this study, we investigated whether glucagon is able to modulate the gene expression of hepatocyte AQP8. Glucagon was administered to rats at 0.2 mg/100 g body wt ip in 2, 3, or 6 equally spaced doses for 8, 16, and 36 h, respectively. Immunoblotting analysis showed that hepatic 34-kDa AQP8 was significantly increased by 79 and 107% at 16 and 36 h, respectively. Hepatic AQP9 protein expression remained unaltered. AQP8 mRNA expression, assessed by real-time PCR, was not modified over time, suggesting a posttranscriptional mechanism of AQP8 protein increase. Glucagon effects on AQP8 were directly studied in primary cultured rat hepatocytes. Immunoblotting and confocal immunofluorescence microscopy confirmed the specific glucagon-induced AQP8 upregulation. The RNA polymerase II inhibitor actinomycin D was unable to prevent glucagon effect, providing additional support to the nontranscriptional upregulation of AQP8. Cycloheximide also showed no effect, suggesting that glucagon-induced AQP8 expression does not depend on protein synthesis but rather on protein degradation. Inhibitory experiments suggest that a reduced calpain-mediated AQP8 proteolysis could be involved. The action of glucagon on hepatocyte AQP8 was mimicked by dibutyryl cAMP and suppressed by PKA or phosphatidylinositol-3-kinase (PI3K) inhibitors. In conclusion, our data suggest that glucagon induces the gene expression of rat hepatocyte AQP8 by reducing its degradation, a process that involves cAMP-PKA and PI3K signal pathways.
authors.affiliation: Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Santa Fe, Argentina.
authors.forename: Leandro R
authors.initials: LR
authors.lastname: Soria
authors.forename: Sergio A
authors.initials: SA
authors.lastname: Gradilone
authors.forename: M Cecilia
authors.initials: MC
authors.lastname: Larocca
authors.forename: Raúl A
authors.initials: RA
authors.lastname: Marinelli
mesh_terms: D000818:Animals; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005934:Glucagon / Q000008:administration & dosage / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007274:Injections, Intraperitoneal; D008297:Male; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011480:Protease Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D020346:Aquaporins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D005934:Glucagon; D019384:Nucleic Acid Synthesis Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011480:Protease Inhibitors; D047428:Protein Kinase Inhibitors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C491637:Aqp9 protein, rat; C108330:aquaporin 8