In this report we characterized a genetic model of VHL-associated hepatocellular steatosis and identified HIF-2 as a central regulator of hepatic lipid metabolism. We found that activation of HIF-2 resulted in suppression of fatty acid β-oxidation and lipid synthesis as well as an increase in lipid storage capability. We propose that, in this model, HIF-2 controls at least three components of hepatic lipid metabolism: synthesis, oxidation, and storage.
Cells adapt metabolically to hypoxia by switching from aerobic to anaerobic metabolism in order to generate ATP in an oxygen-independent manner. This process, known as the Pasteur effect, is the result of increased glucose uptake and glycolysis, as well as decreased mitochondrial oxidative phosphorylation (44
). HIF-1 plays a central role in this process as it regulates the switch from pyruvate catabolism and oxidative phosphorylation to glycolysis in both hypoxic and normoxic (e.g., VHL-deficient) cells (Fig. ). HIF-1 directly stimulates glycolysis by activating the expression of glucose transporters and glycolytic enzymes (12
) and inhibits mitochondrial oxidative phosphorylation by blocking pyruvate entry and conversion to acetyl-CoA in the mitochondria through activation of pyruvate dehydrogenase kinase, a negative regulator of pyruvate dehydrogenase activity (33
). Recent studies have shown that HIF-1 can also indirectly repress mitochondrial metabolism and biogenesis by antagonizing c-Myc activity (55
). While these findings explain the effects of low oxygen on glucose and pyruvate metabolism, the regulation of lipid metabolism under hypoxic conditions is not well understood. The identification of underlying molecular mechanisms would be particularly relevant for tissues such as liver, lung, and heart, where fatty acids are the preferred carbon source for the generation of ATP. In these tissues, hypoxia results in suppressed fatty acid oxidation and neutral lipid accumulation (5
). In this report, we have generated a model of constitutive HIF activation in the liver and demonstrate that HIF-2, but not HIF-1, suppresses fatty acid oxidation and promotes lipid accumulation (Fig. ). These findings indicate that HIF-2 is the predominant HIF for the regulation of lipid metabolism.
FIG. 7. Model depicting the roles of HIF-1 and HIF-2 in the regulation of hepatic glucose and lipid metabolism. Cells adapt metabolically to hypoxia by switching from aerobic to anaerobic metabolism in order to generate ATP in an oxygen-independent manner. While (more ...)
Our findings demonstrate that activation of HIF-2 alters hepatic lipid metabolism and results in the development of severe fatty liver disease in mice. The phenotype in our model resembles that found in patients with nonalcoholic fatty liver disease (NAFLD), which raises the possibility that HIF-2 may contribute to the development of this disease. NAFLD is characterized by fat accumulation in hepatocytes and can be associated with inflammation (nonalcoholic steatohepatitis) or fibrosis (2
). Nonalcoholic steatohepatitis may progress to cirrhosis in up to 20% of patients and represents a leading cause of cryptogenic cirrhosis. Insulin resistance, oxidative injury, iron, and antioxidant deficiencies among other factors have been implicated in the pathogenesis of NAFLD, which is still poorly understood. Chronic sleep apnea has been associated with fatty livers, suggesting that liver hypoxia, which activates HIF-2, may be a contributory factor (49
). Furthermore, it has been suggested that hypoxia contributes to the development of steatosis in patients with alcohol-induced liver damage and heart failure (17
). In addition to NAFLD, our findings have relevance for patients with VHL disease. Similar to the genetic model utilized in this study, patients with germ line mutations in the VHL tumor suppressor develop tumors that accumulate lipids. Most notably, VHL-associated renal cell carcinomas are distinguished histologically from other types of renal cancer by the presence of a “clear” cytoplasm, which results from the washout of lipids during tissue processing. Furthermore, lipid accumulation is a characteristic of stromal cells, which are the neoplastic components of VHL-associated hemangioblastomas (20
). Thus, our studies identify HIF-2 as a molecular mediator of the clear-cell phenotype that is a morphological hallmark of VHL-associated neoplasms.
Excess lipid accumulation in the liver and the development of NAFLD can result from increased lipid synthesis or from a decrease in the ability to oxidize fatty acids (2
). With regard to VHL-associated heptocellular steatosis, we established that key lipogenic enzymes, such as FASN and ACC, were significantly downregulated in a HIF-2-dependent manner. In addition, we also observed a HIF-2-dependent suppression of FASN expression in hypoxic HepG2 cells (see Fig. S3 in the supplemental material). These findings suggest that HIF-2-mediated lipid accumulation was not a result of increased lipid synthesis. Instead, using polarographic measurements of oxygen consumption, we found a functional decrease in the ability of pVHL-deficient hepatocytes to oxidize fatty acids. Furthermore, gene expression analysis of pVHL-deficient mouse livers suggested a functional interaction between HIF and nuclear hormone receptor signaling through PPARα, a central regulator of fatty acid oxidation and gluconeogenesis (22
). The importance of PPARα in maintaining hepatic energy balance is illustrated by the findings that PPARα-deficient mice develop steatohepatitis and hypoglycemia (18
). We found that a number of PPARα targets, including those which control β-oxidation (Acsl1
), were decreased by HIF-2 in pVHL-deficient mouse livers and renal cell carcinoma cells (see Fig. S3 in the supplemental material). Whether HIF-2 has a direct or indirect role in suppressing PPARα transcriptional activity requires further investigation. HIF-1 has been shown to directly inhibit PPARα expression in intestinal and oral epithelial cells (31
); however, we did not observe a significant decrease in PPARα protein levels in pVHL-deficient mouse livers (data not shown). PPARα activity may also be indirectly suppressed through a decrease in lipogenesis. Using a mouse model of liver-specific Fas knockdown, Chakravarthy et al. demonstrated that de novo lipid synthesis is required to activate PPARα and stimulate β-oxidation in the liver (3
). This raises the possibility that HIF-2 may regulate β-oxidation indirectly through its suppressive effects on lipogenesis. Future studies are needed to further explore the role of HIF-2 in the regulation of hepatic PPARα signaling.
In addition to suppressed fatty acid oxidation in pVHL-deficient livers, we also observed increased expression of the lipid droplet binding protein ADFP, which may have contributed to the development of steatosis. ADFP is a member of the PAT family of lipid droplet binding proteins, which include perilipin A, TIP47, and Pmap (25
), and plays an active role in regulating lipid stores in a variety of cell types. Overexpression of Adfp increased both the number and size of lipid droplets in murine fibroblasts, while inhibition of Adfp prevented the development of steatosis in leptin-deficient and diet-induced fatty liver models (4
). We found that ADFP was induced by HIF-2 in hypoxic human hepatoma cells, indicating direct regulation by HIF-2, which is consistent with previous reports on pVHL-deficient renal cell carcinoma cells (12
). While perilipin A, TIP47, and Pmap were also increased in fatty livers of Vhlh-deficient mice, ADFP was the only PAT family member that was hypoxia inducible, suggesting that ADFP may be an early and initiating event in the pathogenesis of HIF-2-mediated steatosis. Efforts are under way in our laboratory to examine the roles of ADFP and other PAT family members in the development of HIF-2-mediated steatosis.
In summary, we have shown that constitutive HIF-2 activation in hepatocytes results in the development of severe hepatic steatosis, which is associated with suppression of lipid synthesis and fatty acid β-oxidation, and an increase in lipid storage capacity. These findings demonstrate that efficient control of HIF-2 signaling is necessary to maintain normal lipid homeostasis in the liver and may have implications for the development of new therapeutic strategies aimed at the treatment of fatty liver diseases.