Oxidative stress impairs HIF1α activation: a novel mechanism for increased vulnerability of steatotic hepatocytes to hypoxic stress

Steatosis increases the sensitivity of hepatocytes to hypoxic injury. Thus, this study was designed to elucidate the role of hypoxia-inducible factor-1 alpha (HIF1 alpha) in steatotic hepatocytes during hypoxia. AML12 hepatocytes and isolated rat hepatocytes were treated with a free fatty acid mixture of oleate and palmitate (2:1,1 mM) for 18 h, which generated intrahepatocyte fat accumulation. The cells were then exposed to hypoxia (1% oxygen, 6-24 h). After hypoxia, a further increase in cellular fat accumulation was seen. In steatotic hepatocytes, a decreased HIFI a activation by hypoxia was observed. The capacity of these cells to express HIFI alpha-dependent genes responsible for the utilization of nutrients for energy was also impaired. This resulted in significantly lower intracellular ATP levels and greater cell death in steatotic hepatocytes compared with control hepatocytes. In contrast, overexpression of constitutively active HIFI a significantly increased cell viability as well as ATP and GLUT1 mRNA levels in steatotic hepatocytes under hypoxia. Hypoxia significantly enhanced HIFI a mRNA levels in control but not in steatotic hepatocytes. Concomitantly, an increase in oxidative stress was found in steatotic hepatocytes under hypoxic conditions compared with control cells. This included higher reactive oxygen species generation, lower cellular and nuclear GSH levels, and higher accumulation of 4-hydroxynonenal protein adducts. Hypoxia-mediated oxidative stress was accompanied by inactivation of basal nuclear factor-kappa B (NF-kappa B) DNA binding. Treatment with N-acetyl-L-cysteine, a reducing agent, improved NF-kappa B DNA-binding capacity and restored HIF1 alpha induction. Conversely, overexpression of an NF-kappa B super-suppressor in control hepatocytes (I kappa B alpha Delta N-transfected cells) resulted in complete inhibition of HIF1 alpha expression, confirming that indeed NF-kappa B regulates HIF1 alpha expression in hypoxic hepatocytes. In conclusion, hypoxia in combination with hepatic steatosis was shown to promote augmented oxidative stress, leading to NF-kappa B inactivation and impaired HIF1 alpha induction and thereby increased susceptibility to hypoxic injury. (c) 2012 Elsevier Inc. All rights reserved.