Metabolite mimicry identifies butyrate analogs with select protective functions in the intestinal mucosa

Abstract Microbial-derived short-chain fatty acids regulate a variety of pathways in the healthy colonic mucosa. In particular, butyrate serves as the primary energy source for colonocytes and regulates gene transcription by stabilizing the transcription factor hypoxia-inducible-factors (HIF) and functioning as a histone deacetylase (HDAC) inhibitor. A limitation of butyrate as a therapeutic is its rapid metabolism in differentiated colonocytes. Furthermore, intestinal stem cells (ISCs) respond differently to butyrate, preferentially using glucose for energy procurement. To address these limitations, we explored metabolite-mimicry to discover compounds with potent or selective biological responses within the butyrate pathway(s). We discovered an analog, 3-chlorobutyrate (3-Cl BA), that significantly enhances epithelial barrier formation and wound healing in vitro. Mechanistically, we revealed that 3-Cl BA is a potent HDAC inhibitor. Furthermore, unlike butyrate, 3-Cl BA does not stabilize HIF and it is not used as metabolic fuel. In vivo studies in a DSS-colitis model revealed that contrary to butyrate, 3-Cl BA is protective. Studies in stem-like colonoids demonstrated that only butyrate inhibits ISC proliferation and differentiation. Furthermore, it was recently reported that HIF stabilization inhibits ISCs activity. Given the fact that butyrate but not 3-Cl BA stabilizes HIF, we surmised that 3-Cl BA would circumvent these detrimental functional consequences. We demonstrate here that pharmacologic HIF stabilization inhibits colonoid differentiation and that genetic loss of HIF significantly promotes ISC differentiation. This study reveals a promising butyrate analog protective in colitis and demonstrates the advantages of metabolite-mimicry to dissect selective biological functions from major metabolites in the gut. Significance statement Butyrate is a well-studied microbial short-chain fatty acid that regulates a number of mucosal pathways and is paramount in maintaining intestinal integrity. In health, it is a major source of energy for colonocytes and regulates gene transcription. The role of butyrate in disease is still controversial and not well understood. When butyrate is not metabolized or well-utilized (e.g. disease), it accumulates in intestinal stem cells leading to reduced cell proliferation and differentiation, thereby hampering intestinal barrier recovery. In this study, we describe a butyrate analog that enhances epithelial barrier formation and wound healing. Furthermore, as opposed to native butyrate, this butyrate analog is protective in a colitis mouse model and does not exhibit detrimental influences on intestinal stem cells. Competing Interest Statement The authors have declared no competing interest. Footnotes Grant and Funding Support: This work was supported by NIH grants DK50189, DK95491, DK104713, DK143211, T34GM149812, T32GM149361, DP2AI184728, by VA grants BX002182, BX006475, IK2BX006088 and by the Crohn’s and Colitis Foundation. Data availability statement All data generated in this study are included in this article and the supplementary information or are available from the corresponding author upon reasonable request. Abbreviations - HIFs - hypoxia-inducible-factors - HDAC - histone deacetylase - HDACi - histone deacetylase inhibitor - ISCs - intestinal stem cells - 3-Cl BA - 3-chlorobutyrate - DSS - dextran sulfate sodium - SCFAs - short-chain fatty acids - IBD - inflammatory bowel disease - IECs - intestinal epithelial cells - TEERs - transepithelial electrical resistance - 4-Cl BA - 4-chlorobutyrate - 3-OH - 3-hydroxybutyrate - GABA - γ-aminobutyrate - FITC-dextran - fluorescein isothiocyanate-dextran - NMR - nuclear magnetic resonance - PBS - phosphate-buffered saline - TJ - tight junctions - H3 - histone 3 - OCLN - occludin - CLDN2 - claudin-2 - CLDN4 - claudin-4 - CGN - cingulin - SYNPO - synaptopodin - DAI - disease activity index - WT - wild-type - MUC2 - mucin-2 - TFF3 - trefoil factor-3 - BCS - bovine calf serum - CCaspase3 - cleaved caspase3