Hypoxia-inducible factor 2α mediates nonesterified fatty acids and hypoxia-induced lipid accumulation in bovine hepatocytes.

Ketosis is a metabolic disorder frequently occurring in the perinatal period, characterized by elevated circulating concentrations of nonesterified fatty acids (NEFA) due to negative energy balance, resulting in fatty liver in dairy cows. However, the mechanism of hepatic steatosis induced by high concentrations of NEFA in ketosis remains unclear. Hypoxia-inducible factor 2α (HIF-2α), which mediates adaptation to hypoxic stress, plays a critical role in regulating lipid metabolism. In this study, we investigate whether HIF-2α is involved in NEFA-driven hepatic lipid accumulation in dairy cows with ketosis. Liver and blood samples were collected from 10 healthy cows (blood β-hydroxybutyrate (BHB) concentration <1.2 mM) and 10 ketotic cows (blood BHB concentration >3.0 mM with clinical symptoms) with similar lactation numbers (median = 3, range = 2 to 4) at 3 to 9 d in milk (median = 6). In cows with ketosis, serum concentrations of NEFA and β-hydroxybutyrate (BHB) were greater but serum concentrations of glucose were lower. Moreover, hepatic triglyceride content increased significantly. In the liver of ketotic cows, which was accompanied by upregulated HIF-2α expression. To determine the potential association among hypoxia, HIF-2α, and the formation of hepatocellular steatosis in vitro, we isolated hepatocytes from healthy calves for the following experiments. First, hepatocytes were treated with 0, 0.6, 1.2, or 2.4 mM NEFA (52.7 mM stock NEFA solution was diluted in RPMI-1640 basic medium supplemented with 2% fatty acid-free BSA to achieve the specified concentrations) for 18 h, showing that HIF-2α expression and cellular hypoxia occurred in a dose-dependent manner. Next, hepatocytes were infected with HIF-2α (encoded by EPAS1) small interfering RNA (Si-HIF-2α) for 48 h and then treated with 1.2 mM NEFA for 18 h. Results indicated that silencing HIF-2α decreased NEFA-induced lipid accumulation in bovine hepatocytes. Subsequently, hepatocytes treated with or without NEFA were placed in an AnaeroPack System, mimicking a hypoxic condition, for 0, 12, 18, or 24 h. Results showed that hypoxia could induce and further exacerbate lipid accumulation in bovine hepatocytes. Meanwhile, normal or NEFA-treated hepatocytes were cocultured with or without PT2385, a specific HIF-2α inhibitor, showing that hypoxia promoted steatosis through HIF-2α. Activating transcription factor 4 (ATF4) is an endoplasmic reticulum (ER) stress and hypoxia-inducible transcription factor. Here, bovine hepatocytes were treated with NEFA or hypoxia following transfecting ATF4 small interfering RNA (Si-ATF4), which demonstrated that ATF4 knockdown alleviated the extent of lipid accumulation in bovine hepatocytes. Besides, we found that ATF4 expression was correlated with HIF-2α levels in both liver tissue and cultured hepatocyte models. Moreover, overexpression of ATF4 weakened the beneficial effects of HIF-2α inhibition. Overall, these data suggest that NEFA-induced hepatic hypoxia significantly contributes to the progression of hepatic steatosis which in turn, intensifies hypoxia and leads to a self-perpetuating cycle of reciprocal causation, further exacerbating hepatic lipid deposition. Additionally, accumulated HIF-2α plays a critical role in this complex-origin steatosis, potentially through ATF4.