HNF4a regulates acyl chain remodeling and ether lipid accumulation in hepatic steatosis

Hepatocyte nuclear factor 4 (HNF4) is an established transcriptional master regulator of differentiation, maintenance, and metabolism. Polymorphisms in HNF4 are linked to several diseases in humans including diabetes and nonalcoholic fatty liver disease (NAFLD). Identifying novel regulation of lipid metabolism by HNF4 would inform on NAFLD development and progression. We directly assessed HNF4 activity through chromatin immunoprecipitation (ChIP)-sequencing and integration of untargeted lipidomics. Direct regulation by HNF4 can be difficult to assess due to the role of HNF4 in liver homeostasis; to rapidly disrupt activity, mice were exposed to cold stress which induces hepatic steatosis in several hours. Cold exposure shifted HNF4 activity with differential genome occupancy of more than 50% of HNF4 binding sites. Focusing on HNF4 binding to promoter with active transcription determined that HNF4 directly regulates fatty acid desaturation, ether lipid synthesis, and peroxisomal biogenesis in response to cold exposure. Integration of lipidomics found that cold exposure increases the very long chain polyunsaturated fatty acid composition of the hepatic lipid pool, including ether lipids, in an HNF4 dependent manner. Because portions of ether lipid synthesis are in the peroxisome and peroxisomal biogenesis is directly HNF4 regulated, we analyzed peroxisomal abundance and found increases with cold exposure that are ablated with loss of HNF4. This peroxisomal regulation was independent of PPAR-- a known regulator of peroxisomes and lipid metabolism--since loss of HNF4 was not rescued by PPAR overexpression. These data determined that regulation of hepatic steatosis by HNF4 is more complex than triglyceride accumulation and includes acyl chain modifications, ether lipid synthesis, and peroxisomal oxidation.