Cohen, R. H. | bioRxiv Science

bioRxiv2026

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

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 statementButyrate 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/697087v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1dd0f0borg.highwire.dtl.DTLVardef@6d27forg.highwire.dtl.DTLVardef@1e5d07corg.highwire.dtl.DTLVardef@dab74f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Microbially derived butyrate plays a key role in intestinal homeostasis. It is the primary source of energy for colonocytes, contributing to a metabolic and oxygen gradient as it is metabolized by differentiated cells along the intestinal crypt. Through the regulation of transcription factors such as HIF and the inhibition of HDAC, it regulates barrier formation and wound healing promoting a strong tight junction profile. Furthermore, well oxygenated ISCs at the bottom of the crypt are unaccustomed to the effects of butyrate, including HIF stabilization (left). In disease, loss of intestinal architecture leads to a disrupted metabolic/oxygen gradient where butyrate accumulates in stem cells leading to decreased proliferation, differentiation, and increases in apoptosis. 3-Cl BA selectively acts as an HDACi and does not stabilize HIF, exhibiting no significant detrimental effects on ISCs (right). Created in BioRender. Ornelas, A. (2026) https://BioRender.com/x9cy8mw C_FIG

molecular biology↗

bioRxiv2025

Regulation of Epithelial HIF by Probiotic Escherichia coli

The gastrointestinal tract is home to trillions of microorganisms that interact with their host in profound ways, including regulation of immune, endocrine, and neurological functions. One mechanism by which these microbes interact with their eukaryotic host is through the generation of short-chain fatty acids (SCFAs), which are metabolized by the intestinal epithelium creating a state of "physiologic hypoxia". This hypoxia, in turn, results in stabilization and activation of hypoxia-inducible factor (HIF), a transcription factor family shown to support gut barrier function and homeostasis, in the intestinal epithelium. The association between HIF and intestinal homeostasis has been long understood, as both genetic and pharmacologic potentiation of the HIF signaling pathway has been shown to promote barrier function both in vitro and in vivo. Although it has been previously established that pathogenic bacteria regulate HIF stabilization and activity in the intestinal epithelium independent of SCFA metabolism, it is not clear whether this property extends to noninfectious and/or commensal bacterial species. Here, we demonstrate that nonpathogenic, commensal strains of Escherichia coli stabilize HIF in intestinal epithelial cells in vitro. Further, we show that HIF is transcriptionally active in these cells and drives a "pro-barrier" transcriptional program. This property was found to be dependent on bacterial aerobic respiration, as genetic elimination of E. coli aerobic respiration abolished HIF stabilization and the subsequent transcriptional phenotype. Finally, we observed induction of tissue hypoxia in vivo using antibiotic-treated mice colonized with wild-type, but not respiration-deficient, E. coli. These findings demonstrate a novel ability for probiotic E. coli to regulate intestinal homeostasis through activation of HIF and suggest that this mechanism might be leveraged in as a novel therapeutic to combat intestinal inflammation, such as that observed during inflammatory bowel disease (IBD).

cell biology↗