BACKGROUND: Both endoplasmic reticulum (ER) stress and autophagy have been shown to display dual roles in cell survival in multiple cell lines. There is a reported but poorly understood link between ER stress, autophagy, and cell death. We hypothesized that autophagy plays a role in ER stress-dependent cell death in rat hepatocytes. MATERIALS AND METHODS: Primary hepatocytes isolated from both lean and obese male Zucker rats were cultured and treated with tunicamycin (TM), tauroursodeoxycholic acid, 3-methyladenine, and wortmannin for 12 h. The ER stress-associated genes glucose-regulated protein 78 and C/EBP homologous protein were examined via quantitative real time polymerase chain reaction. Immunostaining with microtubule-associated protein 1 light chain 3 as well as electron microscopy were used to evaluate autophagy activity. Trypan blue exclusion was used to determine hepatocyte cell viability. RESULTS: In both lean and steatotic hepatocytes, we found that TM induced both C/EBP homologous protein and glucose-regulated protein 78 messenger RNA expression. Cells with increased ER stress were undergoing increased autophagy and had a significant decrease in cell viability. Both tauroursodeoxycholic acid and 3-methyladenine treatments attenuated TM induced ER stress, autophagy, and cell death, whereas wortmannin treatment reduced autophagy and cell death but without changing ER stress. CONCLUSIONS: These data suggest that autophagy is a likely downstream mediator of ER stress-induced cell death in rat hepatocytes. Further exploration of the link between autophagy and ER stress in hepatocyte injury will yield important information that may be leveraged for treatment of liver injuries such as ischemia/reperfusion.
BACKGROUND: Both endoplasmic reticulum (ER) stress and autophagy have been shown to display dual roles in cell survival in multiple cell lines. There is a reported but poorly understood link between ER stress, autophagy, and cell death. We hypothesized that autophagy plays a role in ER stress-dependent cell death in rat hepatocytes. MATERIALS AND METHODS: Primary hepatocytes isolated from both lean and obese male Zucker rats were cultured and treated with tunicamycin (TM), tauroursodeoxycholic acid, 3-methyladenine, and wortmannin for 12 h. The ER stress-associated genes glucose-regulated protein 78 and C/EBP homologous protein were examined via quantitative real time polymerase chain reaction. Immunostaining with microtubule-associated protein 1 light chain 3 as well as electron microscopy were used to evaluate autophagy activity. Trypan blue exclusion was used to determine hepatocyte cell viability. RESULTS: In both lean and steatotic hepatocytes, we found that TM induced both C/EBP homologous protein and glucose-regulated protein 78 messenger RNA expression. Cells with increased ER stress were undergoing increased autophagy and had a significant decrease in cell viability. Both tauroursodeoxycholic acid and 3-methyladenine treatments attenuated TM induced ER stress, autophagy, and cell death, whereas wortmannin treatment reduced autophagy and cell death but without changing ER stress. CONCLUSIONS: These data suggest that autophagy is a likely downstream mediator of ER stress-induced cell death in rat hepatocytes. Further exploration of the link between autophagy and ER stress in hepatocyte injury will yield important information that may be leveraged for treatment of liver injuries such as ischemia/reperfusion.
Authors: Goran Petrovski; Somak Das; Bela Juhasz; Attila Kertesz; Arpad Tosaki; Dipak K Das Journal: Antioxid Redox Signal Date: 2011-01-17 Impact factor: 8.401