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Cholesterol and chronic activation of hypoxia-inducible factor-1 (HIF-1) have been separately implicated in the pathogenesis and progression of liver diseases. In AML12 hepatocytes increased HIF-1α protein accumulation was evident after 2 h of incubation with cholesterol, whereas enhanced HIF-1 transcriptional activity was observed after 6 h. Investigations into the molecular mechanism have shown that cholesterol inhibited HIF-1α degradation. Mitochondrial dysfunction and enhanced mitochondrial reactive oxygen species (ROS) generation were observed in 2-h cholesterol-treated cells along with augmented nitric oxide (NO) levels. Further analysis indicated that HIF-1α stabilization at later time (6h), but not after 2h, of incubation with cholesterol was dependent on NO production. To elucidate the role of mitochondrial dysfunction in HIF-1α stabilization, mitochondrial DNA-depleted hepatocytes were prepared. In these cells the ability of cholesterol to activate the HIF-1 pathway was abolished. Similarly, catalase overexpression also attenuated cholesterol-induced HIF-1α accumulation. These results demonstrate that cholesterol promotes HIF-1 activation in a ROS- and NO-dependent manner. Chronic liver activation of HIF-1 by cholesterol may mediate its deleterious effects in the liver.

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.

Green tea polyphenol extracts (GTE) are sold commercially as cholesterol-reducing agents and for weight reduction however, they have also been reported to be hepatotoxic. Our results from both in vitro and in vivo studies indicate that green tea polyphenols can potentiate liver injury after and during treatment with high cholesterol diet to induce experimental NASH. Eight week old male C57BL mice (n=32) were fed for 6 weeks with one of the following diets: Control diet (Con); Con +1% w/w polyphenols from green tea extract (Con+GTE); High cholesterol diet, Con+ 1% cholesterol+ 0.5% cholate w/w (HCD); HCD + 1 % polyphenols w/w (HCD+GTE). Hepatic steatosis, oxidative and inflammatory markers and bile acid synthesis pathways were measured. GTE enhanced hepatic steatosis but only in animals exposed to the high cholesterol diet. In HCD treated animals GTE elevated blood levels of liver enzymes SGOT, SGPT, and bile acids. Inflammatory and oxidative markers in the liver were also significantly increased including liver mRNA expression of TNF-alpha, IL-6, SAA1, SAA2, iNOS and levels of 4-hydroxynonenal protein adducts. The mechanism of action for GTE/cholesterol hepatotoxicity was found to be related to alteration in bile acids synthesis pathways. This study shows that liver injury in the presence of lipids could be the reason for weigh loss by high dose of polyphenols.