HNF4 Regulates Fatty Acid Oxidation and is Required for Renewal of Intestinal Stem Cells in Mice

Abstract Background & Aims Functions of intestinal stem cells (ISCs) are regulated by diet and metabolic pathways. Hepatocyte nuclear factor 4 (HNF4) are transcription factors that bind fatty acids. We investigated how HNF4 transcription factors regulate metabolism and their functions in ISCs in mice Methods We performed studies with Villin-CreERT2, Lgr5-EGFP-IRES-CreERT2, Hnf4af/f, and Hnf4gCrispr/Crispr mice. Villin-CreERT2; Lgr5-EGFP-IRES-CreERT2; Hnf4af/f; Hnf4gCrispr/Crispr mice are hereafter referred to Hnf4agDKO. Mice were given tamoxifen to induce Cre recombinase. Mice transgenic with only Cre alleles (Villin-CreERT2, Lgr5-EGFP-IRES-CreERT2, Hnf4a+/+, and Hnf4g+/+) or mice given vehicle were used as controls. Crypt and villus cells were isolated, incubated with fluorescently labeled fatty acid or glucose analog, and analyzed by confocal microscopy. Fatty acid oxidation activity and tricarboxylic acid (TCA) metabolites were measured in cells collected from the proximal half of the small intestine of Hnf4agDKO and control mice. We performed chromatin immunoprecipitation and gene expression profiling analyses to identify genes regulated by HNF4 factors. We established organoids from duodenal crypts, incubated them with labeled palmitate or acetate, and measured production of TCA metabolites or fatty acids. Acetate, a precursor of acetyl-CoA (a product of fatty acid beta-oxidation [FAO]), or dichloroacetate, a compound that promotes pyruvate oxidation and generation of mitochondrial acetyl-CoA, were used for metabolic intervention Results Crypt cells rapidly absorbed labeled fatty acid, and mRNA levels of Lgr5+ stem cell markers (Lgr5, Olfm4, Smoc2, Msi1, and Ascl2) were downregulated in organoids incubated with etomoxir, an inhibitor of FAO, indicating that FAO was required for renewal of ISCs. HNF4A and HNF4G were expressed in ISCs and throughout the intestinal epithelium. Single knockout of either HNF4A or HNF4G did not affect maintenance of ISCs, but double-knockout of HNF4A and HNF4G resulted in ISC loss; stem cells failed to renew. FAO supports ISC renewal, and HNF4 transcription factors directly activate FAO genes, including Acsl5 and Acsf2 (encode regulators of acyl-CoA synthesis), Slc27a2 (encodes a fatty acid transporter), Fabp2 (encodes fatty acid binding protein), and Hadh (encodes hydroxyacyl-CoA dehydrogenase). In the intestinal epithelium of Hnf4agDKO mice, expression levels of FAO genes, FAO activity, and metabolites of TCA were all significantly decreased, but fatty acid synthesis transcripts were increased, compared with control mice. The contribution of labeled palmitate or acetate to the TCA cycle was reduced in organoids derived from Hnf4agDKO mice, compared with control mice. Incubation of organoids derived from double-knockout mice with acetate or dichloroacetate restored stem cells Conclusions In mice, the transcription factors HNF4A and HNF4G regulate expression of genes required for fatty acid oxidation and are required for renewal of intestinal stem cells.