The Robert H. Smith Faculty of Agriculture, Food and Environment, Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, Israel.
Free Radic Biol Med. 2012 May 1;52(9):1531-42. doi: 10.1016/j.freeradbiomed.2012.02.014. Epub 2012 Feb 16.
Steatosis increases the sensitivity of hepatocytes to hypoxic injury. Thus, this study was designed to elucidate the role of hypoxia-inducible factor-1α (HIF1α) 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 HIF1α activation by hypoxia was observed. The capacity of these cells to express HIF1α-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 HIF1α significantly increased cell viability as well as ATP and GLUT1 mRNA levels in steatotic hepatocytes under hypoxia. Hypoxia significantly enhanced HIF1α 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-κB (NF-κB) DNA binding. Treatment with N-acetyl-l-cysteine, a reducing agent, improved NF-κB DNA-binding capacity and restored HIF1α induction. Conversely, overexpression of an NF-κB super-suppressor in control hepatocytes (IκBαΔN-transfected cells) resulted in complete inhibition of HIF1α expression, confirming that indeed NF-κB regulates HIF1α expression in hypoxic hepatocytes. In conclusion, hypoxia in combination with hepatic steatosis was shown to promote augmented oxidative stress, leading to NF-κB inactivation and impaired HIF1α induction and thereby increased susceptibility to hypoxic injury.
脂肪变性增加了肝细胞对缺氧损伤的敏感性。因此，本研究旨在阐明缺氧诱导因子-1α（HIF1α）在缺氧时脂肪变性肝细胞中的作用。AML12 肝细胞和分离的大鼠肝细胞用油酸和棕榈酸（2:1，1mM）的游离脂肪酸混合物处理 18 小时，导致肝细胞内脂肪堆积。然后将细胞暴露于缺氧（1%氧气，6-24 小时）。缺氧后，细胞内脂肪堆积进一步增加。在脂肪变性的肝细胞中，缺氧引起的 HIF1α激活减少。这些细胞表达负责利用营养物质产生能量的 HIF1α依赖性基因的能力也受损。这导致脂肪变性肝细胞的细胞内 ATP 水平显著降低，细胞死亡增加，与对照肝细胞相比。相比之下，在缺氧条件下，过表达组成性激活的 HIF1α可显著增加脂肪变性肝细胞的细胞活力以及 ATP 和 GLUT1mRNA 水平。缺氧显著增加了对照肝细胞中的 HIF1αmRNA 水平，但在脂肪变性肝细胞中则没有。同时，与对照细胞相比，在缺氧条件下，脂肪变性的肝细胞中发现氧化应激增加。这包括更高的活性氧生成、更低的细胞和核 GSH 水平以及更高的 4-羟壬烯醛蛋白加合物积累。缺氧介导的氧化应激伴随着基础核因子-κB（NF-κB）DNA 结合的失活。用还原剂 N-乙酰-L-半胱氨酸处理可改善 NF-κB DNA 结合能力并恢复 HIF1α诱导。相反，在对照肝细胞（IκBαΔN 转染细胞）中转染 NF-κB 超级抑制剂可完全抑制 HIF1α的表达，证实 NF-κB 确实调节缺氧肝细胞中的 HIF1α表达。总之，本研究表明，缺氧与肝脂肪变性相结合会促进氧化应激增加，导致 NF-κB 失活和 HIF1α诱导受损，从而增加对缺氧损伤的敏感性。
