RESUMO
BACKGROUND & AIMS: Steatohepatitis drives fibrogenesis in alcohol-related liver disease. Recent studies have suggested that hepatic stellate cells (HSCs) may regulate the parenchymal cell injury and inflammation that precedes liver fibrosis, although the mechanism remains incompletely defined. Neuropilin-1 (NRP-1) and synectin are membrane proteins implicated in HSC activation. In this study, we disrupted NRP-1 and synectin as models to evaluate the role of HSC activation on the development of steatohepatitis in response to alcohol feeding in mice. METHODS: Mice with HSC-selective deletion of NRP (ColCre/Nrp1loxP) or synectin (ColCre/synectinloxP) vs. paired Nrp1loxP or synectinloxP mice were fed a control diet or the chronic/binge alcohol feeding model. Several markers of steatosis and inflammation were evaluated. RESULTS: ColCre/Nrp1loxP mice showed less fibrosis, as expected, but also less inflammation and steatosis, with lower hepatic triglyceride content. Similar results were observed in the synectin model. Hepatocytes treated with supernatant of HSCs from ColCre/Nrp1loxP mice compared to supernatant from Nrp1loxP mice were protected against ethanol-induced lipid droplet formation. An adipokine and inflammatory protein array from the supernatant of HSCs with NRP-1 knockdown showed a significant reduction in Igfbp3 (a major insulin-like growth factor-binding protein with multiple metabolic functions) and an increase in SerpinA12 (a serine-protease inhibitor) secretion compared to wild-type HSCs. Recombinant Igfbp3 induced lipid droplets, triglyceride accumulation, and lipogenic genes in hepatocytes in vitro, while SerpinA12 was protective against ethanol-induced steatosis. Finally, Igfbp3 was increased, and SerpinA12 was decreased in serum and liver tissue from patients with alcoholic hepatitis. CONCLUSION: Selective deletion of NRP-1 from HSCs attenuates alcohol-induced steatohepatitis through regulation of Igfbp3 and SerpinA12 signaling. LAY SUMMARY: Hepatic stellate cells are known for their role in fibrosis (scarring of the liver). In this study, we describe their role in the modulation of fat deposition and inflammation in the liver, which occurs secondary to alcohol damage.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fígado Gorduroso Alcoólico , Células Estreladas do Fígado/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Neuropilina-1/metabolismo , Serpinas/metabolismo , Animais , Modelos Animais de Doenças , Fígado Gorduroso Alcoólico/complicações , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/patologia , Fibrose/etiologia , Fibrose/imunologia , Inflamação/metabolismo , Camundongos , Inibidores de Serina Proteinase/metabolismo , Transdução de SinaisRESUMO
Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability. Immunoblotting analysis in subcellular fractions from isolated rat hepatocytes indicated that glucagon caused a significant, dose-dependent increase in the amount of AQP8 in plasma membranes (e.g., 102% with 1 micromol/L glucagon) and a simultaneous decrease in intracellular membranes (e.g., 38% with 1 micromol/L glucagon). Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the glucagon-induced redistribution of AQP8 from intracellular vesicles to plasma membrane. Polarized hepatocyte couplets showed that this redistribution was specifically to the canalicular domain. Glucagon also significantly increased hepatocyte membrane water permeability by about 70%, which was inhibited by the water channel blocker dimethyl sulfoxide (DMSO). The inhibitors of protein kinase A, H-89, and PKI, as well as the microtubule blocker colchicine, prevented the glucagon effect on both AQP8 redistribution to hepatocyte surface and cell membrane water permeability. In conclusion, our data suggest that glucagon induces the protein kinase A and microtubule-dependent translocation of AQP8 water channels to the hepatocyte canalicular plasma membrane, which in turn leads to an increase in membrane water permeability. These findings provide evidence supporting the molecular mechanisms of glucagon-induced hepatocyte bile secretion.