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.

Phosphorylation of endothelial NOS contributes to simvastatin protection against myocardial no-reflow and infarction in reperfused swine hearts: partially via the PKA signaling pathway

“…It has been reported that H-89 inhibits Akt, ROCK II, and 5'-AMP-activated protein kinase (AMPK) [35] and that the PKA pathway interacts with the PI3K/Akt pathway in the regulation of gene expression [36] . Previous studies have shown that the forskolin-induced stimulation of PKA can inhibit Akt activity in human embryonic kidney cells [37] , but epinephrine or forskolin-induced stimulation of PKA enhanced eNOS phosphorylation at Ser 1177 by activating the Akt pathway in aortic or coronary endothelial cells [28,38] .…”

Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis

“…However, because no AU-rich elements are present in the mRNA 3=-untranslated region of AQP8, mechanisms of upregulation other than mRNA stabilization seem to be taking place. In our laboratory, we have described that glucagon promoted a decrease in calpain-mediated AQP8 proteolysis in rat hepatocytes (41). During metabolic acidosis, the decreased expression of other proteases (e.g., cathepsin B) may contribute to the decreased rate of protein turnover (9).…”

The effects of over expressing aquaporins on the cryopreservation of hepatocytes

“…In the cases of embryos, larvae, oocyte and kidney, aquaporins were artificially expressed on the cellular membrane prior to cryopreservation. Contrastingly, in the current investigation, the fact that aquaporins can be increased in the cellular membrane by the translocation of AQP8 from the intracellular vesicles under the influence of choleretic stimuli such as dibutyryl cAMP (Bt2cAMP) [6] and glucagon [8,9,20], was utilized. As such, it was verified by the confocal immunofluorescence which showed increased AQP8 localization at the cellular boundaries on treatment with Bt 2 cAMP or glucagon.…”

“…Literature suggests that the longer the cells are treated with glucagon, the more AQP8 translocates to the cellular membrane. Results from Soria et al [20] suggested that treatment of hepatocytes with glucagon for 36 h showed an 120% increase in the quantity of AQP8 on cellular membrane as opposed to 80% increase for a 16 h treatment. On the contrary, for Bt 2 cAMP, some researchers indicate 10 min incubation is enough for effective translocation of AQP8 [11] whereas others recommend 12 h [6].…”

“…Of the 13 isoforms discovered so far, five were identified to be expressed in hepatocytes: AQP0, AQP8, AQP9, AQP11 and AQP12 [1,4,5,11,16,22]. Among these, AQP8 is localized in the plasma membrane [20], intracellular vesicles and the mitochondria. Prior experimental evidence shows that AQP8 has a tendency to translocate to the cellular membrane on the influence of choleretic stimulus [6,7].…”