The gut microbiota is essential for human health. Microbial supply of short-chain fatty acids (SCFAs), particularly butyrate, is a well-established contributor to gut homeostasis and disease resistance. Reaching millimolar luminal concentrations, butyrate is sequestered and utilized in the colon as the favored energy source for intestinal epithelia. Given the steep oxygen gradient across the anoxic lumen and the highly oxygenated lamina propria, the colon provides a particularly interesting environment to study oxygen sensing. Previous studies have shown that the transcription factor hypoxia-inducible factor (HIF) is stabilized in healthy colonic epithelia. Here we show that butyrate directly inhibits HIF prolyl hydroxylases (PHDs) to stabilize HIF. We find that butyrate stabilizes HIF in vitro despite eliminating β-oxidation and resultant oxygen consumption. Using recombinant PHD protein in combination with a novel NMR approach and enzymatic biochemical assays, we identify butyrate to bind and function as a unique, noncompetitive inhibitor of PHDs relative to other SCFAs. Butyrate inhibited PHD with a noncompetitive Ki of 5.3 ± 0.5 mM, a physiologically relevant concentration. We also confirm that microbiota-derived butyrate is necessary to stabilize HIF in mice colonic tissue through antibiotic-induced butyrate depletion and reconstitution experiments. Our results suggest that the co-evolution of mammals and mutualistic microbiota has selected for butyrate to impact a critical gene regulation pathway that can be extended beyond the mammalian gut. As PHDs are a major target for drug development in the stabilization of HIF, butyrate holds great potential as a well-tolerated endogenous inhibitor with far-reaching therapeutic impact. 1R21AI171827-01

The intestinal mucosa is a dynamic surface that facilitates interactions between the host and an outside world that includes trillions of microbes, collectively termed the microbiota. This fine balance is regulated by an energetically demanding physical and biochemical barrier that is formed by the intestinal epithelial cells. In addition, this homeostasis exists at an interface between the anaerobic colonic lumen and a highly oxygenated, vascularized lamina propria. The resultant oxygen gradient within the intestine establishes “physiologic hypoxia” as a central metabolic feature of the mucosa. Although oxygen is vital for energy production to meet cellular metabolism needs, the availability of oxygen has far-reaching influences beyond just energy provision. Recent studies have shown that the intestinal mucosa has purposefully adapted to use differential oxygen levels largely through the presence of short-chain fatty acids (SCFAs), particularly butyrate (BA). Intestinal epithelial cells use butyrate for a multitude of functions that promote mucosal homeostasis. In this review, we explore how the physiologic hypoxia profile interfaces with SCFAs to benefit host mucosal tissues.

Urticarial vasculitis is a rare autoimmune disorder characterized by persistent edematous papules and plaques on the skin that last longer than 24 hours, often accompanied by systemic symptoms such as joint pain and fever. Unlike common urticaria, this condition involves inflammation of small blood vessels, leading to more severe and long-lasting skin lesions with a tendency to leave a bruiselike appearance. Diagnosis is challenging and may require a skin biopsy. Associated with underlying autoimmune diseases, treatment involves managing symptoms with medications such as antihistamines and corticosteroids, addressing the immune system's dysfunction, and treating any concurrent autoimmune conditions.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Active episodes of inflammatory bowel disease (IBD), which include ulcerative colitis and Crohn's disease, coincide with profound shifts in the composition of the microbiota and host metabolic energy demand. Intestinal epithelial cells (IEC) that line the small intestine and colon serve as an initial point for contact for the microbiota and play a central role in innate immunity. In the 1980s, Roediger et al proposed the hypothesis that IBD represented a disease of diminished mucosal nutrition and energy deficiency ("starved gut") that strongly coincided with the degree of inflammation. These studies informed the scientific community about the important contribution of microbial-derived metabolites, particularly short-chain fatty acids (SCFA) such as butyrate, to overall energy homeostasis. Decades later, it is appreciated that disease-associated shifts in the microbiota, termed dysbiosis, places inordinate demands on energy acquisition within the mucosa, particularly during active inflammation. Here, we review the topic of tissue energetics in mucosal health and disease from the original perspective of that proposed by the starved gut hypothesis.

Microbiota-derived short chain fatty acids, particularly butyrate (BA), show multiple beneficial influences on health. In the colon, BA ranges from 10-20 mM and up to 99% is utilized as a metabolic fuel by the mucosa. BA plays a key role in epithelial barrier regulation, reduces inflammation, and regulates cell growth and differentiation. There are multiple mechanisms by which BA contributes to gut health, many due to its regulatory capacity for gene expression. Our group reported (PMID: 34190032) a direct influence of BA on the stabilization of the transcription factor hypoxia-inducible factor (HIF). It is known that HIF stabilization is essential for appropriate mucosal barrier regulation and the coordination of regenerative capacity in the intestine. However, BA is constantly metabolized limiting its HIF stabilization effect. This observation led to the design and investigation of BA-mimicking compounds that stabilize HIF, but may not be involved in metabolism. We hypothesize that structural modifications of BA yield analogues that stabilize HIF with better efficacy and a longer biological half-life. A library of BA derivatives was screened in search of a non-endogenous analogue with higher potency and/or longer half-life for HIF stabilization. In vitro screenings at physiologically-relevant concentrations (5 mM) were performed using intestinal epithelial cell lines (T84 and CaCO2) in normoxia to determine HIF-1a protein abundance. Various analogues stabilized HIF, with 4-mercapto-butyrate (MBA) being the most promising candidate. The best analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CAIX by q-PCR. MBA exhibited higher induction of these targets compared to BA. Time-course studies revealed that MBA exhibits significantly longer half-life as observed in the stabilization of HIF and induction of gene targets for up to 72 h compared to BA (24 h). Loss of gene induction was observed in MBA treated cells expressing lentiviral shRNA against HIF1b (HIF1b KD), supporting a HIF-dependent transcription. Furthermore, it’s been widely reported that BA enhances epithelial barrier, primarily through HIF coordinated barrier protection. Among several other BA derivatives, MBA enhanced and prolonged epithelial barrier function in intestinal epithelial cells. In vivo studies in C57BL/6 mice explored the induction of HIF targets in tissue and specifically erythropoietin (EPO) in circulation following administration of BA and MBA at similar doses. These studies revealed that induction of EPO with a single dose of MBA exceeded that of native BA. In colonic and kidney tissue, GLUT1 and BNIP3 were moderately induced in vivo. Thus, a non-endogenous BA derivative (MBA) stabilizes HIF at higher potency and longer half-life than native BA and will be used as a template for the development of HIF stabilizing agents.

Microbiota-derived short-chain fatty acids, including butyrate (BA), have multiple beneficial health effects. In the colon, BA concentrations range from 10 to 20 mM and up to 95% is utilized as energy by the mucosa. BA plays a key role in epithelial-barrier regulation and anti-inflammation, and regulates cell growth and differentiation, at least in part, due to its direct influence on stabilization of the transcription factor hypoxia-inducible factor (HIF). It remains unclear whether BA is the optimal metabolite for such a response. In this study, we explored metabolite mimicry as an attractive strategy for the biological response to HIF. We discovered that 4-mercapto butyrate (MBA) stabilizes HIF more potently and has a longer biological half-life than BA in intestinal epithelial cells (IECs). We validated the MBA-mediated HIF transcriptional activity through the induction of classic HIF gene targets in IECs and enhanced epithelial barrier formation in vitro. In-vivo studies with MBA revealed systemic HIF stabilization in mice, which was more potent than its parent BA metabolite. Mechanistically, we found that MBA enhances oxygen consumption and that the sulfhydryl group is essential for HIF stabilization, but exclusively as a four-carbon SCFA. These findings reveal a combined biochemical mechanism for HIF stabilization and provide a foundation for the discovery of potent metabolite-like scaffolds.

IL-38 is a recently discovered cytokine and member of the IL-1 Family. In the IL-1 Family, IL-38 is unique because the cytokine is primarily a B lymphocyte product and functions to suppress inflammation. Studies in humans with inflammatory bowel disease (IBD) suggest that IL-38 may be protective for ulcerative colitis or Crohn’s disease, and that IL-38 acts to maintain homeostasis in the intestinal tract. Here we investigated the role of endogenous IL-38 in experimental colitis in mice deficient in IL-38 by deletion of exons 1-4 in C57 BL/6 mice. Compared to WT mice, IL-38 deficient mice subjected to dextran sulfate sodium (DSS) showed greater severity of disease, more weight loss, increased intestinal permeability, and a worse histological phenotype including increased neutrophil influx in the colon. Mice lacking IL-38 exhibited elevated colonic Nlrp3 mRNA and protein levels, increased caspase-1 activation, and the concomitant increased processing of IL-1β precursor into active IL-1β. Expression of IL-1α, an exacerbator of IBD, was also upregulated. Colonic myleloperoxidase protein and Il17a, and Il17f mRNA levels were higher in the IL-38 deficient mice. Daily treatment of IL-38 deficient mice with an NLRP3 inhibitor attenuated diarrhea and weight loss during the recovery phase. These data implicate endogenous IL-38 as an anti-inflammatory cytokine that reduces DSS colitis severity. We propose that a relative deficiency of IL-38 contributes to IBD by disinhibition of the NLRP3 inflammasome.

Short chain fatty acids and specifically butyrate (BA) have been well documented to show multiple beneficial influences on health. Typically, the concentration of colonic butyrate can range from 10-20 mM and 95-99% is metabolized into energy by the mucosa. BA plays a key role in epithelial barrier regulation, ameliorates inflammation and regulates cell growth and differentiation. There are multiple mechanisms on how it plays such an important role in gut health, many related to its regulatory capacity for gene expression. Our laboratory recently reported a direct influence of BA on the stabilization of the transcription factor hypoxia-inducible factor (HIF). BA acts directly and non-competitively inhibits HIF prolyl hydroxylase 2 (PHD2), responsible for the rapid degradation of HIF in normoxia (NX). However, the efficacy of BA on HIF stabilization is limited due to its rapid metabolism as fuel. Based on these observations, we hypothesize that analogues structurally related to butyrate stabilize HIF with a longer biological half-life. A small library of BA derivatives was screened in search of a non-endogenous analogue with higher potency and/or half-life for HIF stabilization. In vitro screenings at a physiologically-relevant concentration (5 mM) were performed in intestinal epithelial cell lines T84 and CaCo2 in NX and HIF-1a ELISA was used to determine protein abundance. Several hits were observed, with 4-mercapto-butyrate (SHBA) being the most promising candidate. The best BA analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CaIX by q-PCR. SHBA exhibited higher induction of these targets compared to BA. Dose response and time-course studies were performed to compare SHBA and BA. Notably, SHBA exhibits a much longer half-life as observed in the stabilization of HIF and induction of gene targets at least up to 72 h compared to BA (24 h). In vivo studies in C57BL/6 mice explored the induction of classic HIF targets and specifically erythropoietin (EPO) in circulation following administration of BA and SHBA at similar doses. These studies revealed that induction of EPO with a single dose of SHBA exceeded that of native BA. Thus, a non-endogenous BA derivative (SHBA) stabilizes HIF at a higher potency and longer half-life than BA and will be useful as a template for the development of a HIF stabilizing agent.

The gut microbiota is essential for human health. Microbial supply of short-chain fatty acids (SCFAs), particularly butyrate, is a well-established contributor to gut homeostasis and disease resistance. Reaching millimolar luminal concentrations, butyrate is sequestered and utilized in the colon as the favored energy source for intestinal epithelia. Given the steep oxygen gradient across the anoxic lumen and the highly oxygenated lamina propria, the colon provides a particularly interesting environment to study oxygen sensing. Previous studies have shown that the transcription factor hypoxia-inducible factor (HIF) is stabilized in healthy colonic epithelia. Here we show that butyrate directly inhibits HIF prolyl hydroxylases (PHDs) to stabilize HIF. We find that butyrate stabilizes HIF in vitro despite eliminating β-oxidation and resultant oxygen consumption. Using recombinant PHD protein in combination with nuclear magnetic resonance and enzymatic biochemical assays, we identify butyrate to bind and function as a unique, noncompetitive inhibitor of PHDs relative to other SCFAs. Butyrate inhibited PHD with a noncompetitive Ki of 5.3 ± 0.5 mM, a physiologically relevant concentration. We also confirm that microbiota-derived butyrate is necessary to stabilize HIF in mice colonic tissue through antibiotic-induced butyrate depletion and reconstitution experiments. Our results suggest that the co-evolution of mammals and mutualistic microbiota has selected for butyrate to impact a critical gene regulation pathway that can be extended beyond the mammalian gut. As PHDs are a major target for drug development in the stabilization of HIF, butyrate holds great potential as a well-tolerated endogenous inhibitor with far-reaching therapeutic impact.

Inflammatory bowel disease (IBD) coincides with profound shifts in microbiota and host metabolic energy supply and demand. The gastrointestinal epithelium is anatomically positioned to provide a selective barrier between the anaerobic luminal microbiota and host lamina propria, with the microbiota and epithelium participating in an intricate energy exchange necessary for homeostasis. Maintenance and restoration of the barrier requires high energy flux and places significant demands on available substrates to generate ATP. It is recently appreciated that components of the microbiota contribute significantly to a multitude of biochemical pathways within and outside of the mucosa. Decades-old studies have appreciated that byproducts of the microbiota provide essential sources of energy to the intestinal epithelium, especially the colon. More recent work has unveiled the existence of numerous microbial-derived metabolites that support energy procurement within the mucosa. It is now appreciated that disease-associated shifts in the microbiota, termed dysbiosis, places significant demands on energy acquisition within the mucosa. Here, we review the topic of host- and microbial-derived components that influence tissue energetics in health and during disease.

The intestinal mucosa exists in dynamic balance with trillions of luminal microbes. Disruption of the intestinal epithelial barrier, commonly observed in mucosal inflammation and diseases such as inflammatory bowel diseases (IBDs), is often associated with dysbiosis, particularly decreases in species producing short-chain fatty acids (SCFAs), such as butyrate. It remains unclear to what extent microbiota-derived factors contribute to the overall maintenance of intestinal homeostasis. Initial studies revealed that butyrate selectively promotes epithelial barrier function and wound healing. We aimed to define the specific mechanism(s) through which butyrate contributes to these epithelial responses. Guided by an unbiased profiling approach, we identified the dominant regulation of the actin-binding protein synaptopodin (SYNPO). Extensions of this work revealed a role for SYNPO in intestinal epithelial barrier function and wound healing. SYNPO was localized to the intestinal epithelial tight junction and within F-actin stress fibers where it is critical for barrier integrity and cell motility. Butyrate, but not other SCFAs, induced SYNPO in epithelial cell lines and murine colonic enteroids through mechanisms possibly involving histone deacetylase inhibition. Moreover, depletion of the microbiota abrogated expression of SYNPO in the mouse colon, which was rescued with butyrate repletion. Studies in Synpo-deficient mice demonstrated exacerbated disease susceptibility and increased intestinal permeability in a dextran sulfate sodium colitis model. These findings establish a critical role for the microbiota and their products, specifically butyrate, in the regulated expression of SYNPO for intestinal homeostasis and reveal a direct mechanistic link between microbiota-derived butyrate and barrier restoration.

Intestinal epithelial cells (IECs) form a dynamic barrier that maintains homeostasis by separating the host immune system from an external environment of pathogenic and commensal microorganisms. Disruption of epithelial barrier in diseases such as inflammatory bowel diseases (IBD) can increase bacterial translocation and result in inappropriate immune responses. Under such conditions, rapid wound healing to restore this barrier is central to inflammation resolution. Mucosal barrier disruption is often associated with dysbiosis, particularly decreases in species producing short chain fatty acids (SCFAs). While SCFAs are an established energy source for IECs, it was hypothesized that microbial‐derived SCFAs could promote IEC barrier function through specific gene regulation. The SCFA butyrate was shown to selectively augment barrier formation, monitored by increased electrical resistance, and enhance wound healing, measured by increased scratch‐wound closure, in T84 model IEC monolayers. An unbiased single cell RNA sequencing analysis (scRNAseq) was performed to define potential mechanisms of butyrate regulation and identified a number of gene targets that could coordinate epithelial barrier function. Of particular interest was the prominent butyrate‐induced expression of synaptopodin (SYNPO), an actin‐associated protein involved in cell shape and motility previously shown to be expressed in neuronal dendrites and kidney podocytes. Validation of the scRNAseq revealed that butyrate induces IEC SYNPO mRNA and protein expression by nearly 10‐fold in IECs, and that lentiviral knockdown of SYNPO results in diminished barrier formation and wound healing not rescued with butyrate treatment. Immunofluorescence studies revealed that SYNPO distinctly localizes to the IEC tight junction (e.g. co‐localization with ZO‐1), the linkage between cells responsible for regulating paracellular flux. In vivo studies using antibiotic‐depletion of microbiota followed by butyrate add‐back in mice confirmed SYNPO induction by butyrate, and mice subjected to dextran sulfate sodium (DSS)‐induced colitis showed decreased SYNPO expression. Studies in Synpo‐deficient mice demonstrated exacerbated disease susceptibility and increased intestinal permeability in the DSS colitis model. These findings establish a critical role for the microbiota and their products, specifically butyrate, in the regulated expression of SYNPO for intestinal homeostasis and reveal a direct mechanistic link between microbiota‐derived butyrate and barrier restoration. Understanding butyrate regulation of barrier function in the intestinal mucosa may offer insight into new IBD therapeutics to promote wound healing.Support or Funding InformationThis work was supported by NIH grants DK1047893, DK50189, DK095491, DK103712, and by the VA Merit Award BX002182. R.X.W. was supported by an NIH NRSA fellowship F30DK120072 and NIH MSTP training grant T32GM008497. J.S.L. was supported by an NIH NRSA fellowship F32DK122741.

The gastrointestinal mucosa forms a dynamic physical and biochemical barrier to isolate the host immune system from potentially pathogenic microorganisms. A dysfunctional barrier allows bacterial contact with and translocation across the epithelium. Recent studies have appreciated that epithelial barrier formation and maintenance is energetically demanding, yet the colonic epithelium exists in a rather energetically‐depleted state of physiological hypoxia. Moreover, the intestinal mucosa is short lived, undergoing turnover every 3 – 5 days, incurring a substantial need for nucleotide genesis to preserve the epithelial cell population and provide ATP for energy balance. In the present work, we demonstrate that large quantities of purines are made and released by the gut microbiota and that such microbiota‐derived purines (MDP) are available to the intestinal mucosa. Through a series of experiments in which MDP production was depleted by antibiotic treatment, and purines reconstituted by supplementation and colonization with purine‐producing bacteria, the contribution of MDP to colonic proliferation and energetics during DSS‐induced colitis was delineated. Through HPLC‐based metabolite analyses, exogenously supplied purines showed incorporation into the murine colonic purine metabolite pool, were utilized for nucleotide genesis, and promoted energy balance. Immunofluorescent and metabolite analyses revealed that DSS‐insulted colon tissue lacking MDP substrates were proliferatively stunted, with notable energetic and endoplasmic reticulum (ER) stress, to the detriment of mucus barrier integrity. Upon purine reconstitution, the energetic state of the tissue was improved and ER stress alleviated, with concomitant reclamation of proliferative capacity and mucus barrier sterility. Together, this work establishes MDP as a critical substrate for colonic tissue metabolism and facilitates mucosal homeostasis and inflammatory resolution.Support or Funding InformationThis work was supported by NIH grants F32DK122741, DK1047893, DK50189, DK095491, DK103712 and by the Veterans Administration Merit Award BX002182.

The intestinal mucosa requires high levels of nucleotides for energy procurement, proliferation, and innate immunity. This need for nucleotide substrates substantially increases during injury, infection, and wound healing. In the present studies, we profile potential sources of purine nucleotides in murine mucosal tissue. This work reveals the gut microbiota as a prominent source of exogenous purines and that such microbiota-sourced purines (MSPs) are available to the intestinal mucosa. The MSPs are utilized for nucleotide genesis and promote energy balance. Further analyses reveal that colitic tissues lacking MSPs are proliferatively stunted, with notable energetic and endoplasmic reticulum stress to the detriment of mucous barrier integrity. Purine reconstitution either directly or through colonization of germ-free/antibiotic-treated mice with MSP-sufficient E. coli alleviates such deficits, establishing MSP as a critical source of substrate for tissue metabolism, wound healing, and mucous barrier sterile integrity.

Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, small-molecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small-molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass spectrometry-based approaches, hydrogen-deuterium exchange and fast photochemical oxidation of proteins (FPOP), and corroborated the findings with our X-ray structure of the PD-L1/macrocycle complex. Although all three approaches show that the macrocycle binds directly to PD-L1 over the regions of residues 46-87 and 114-125, the two protein footprinting approaches show additional binding at the N-terminus of PD-L1, and FPOP reveals some critical binding residues. The outcomes not only show the binding regions but also demonstrate the utility of MS-based footprinting in probing protein/ligand inhibitory interactions in cancer immunotherapy.

Extracellular adenosine signaling is established as a protective component in mucosal inflammatory responses. The sources of extracellular adenosine include enzymatic processing from nucleotides, such as ATP and AMP, that can be liberated from a variety of cell types, including infiltrating leukocytes. Here we demonstrate that activated human neutrophils are a source of diadenosine triphosphate (Ap3A), providing an additional source of nucleotides during inflammation. Profiling murine enteroids and intestinal epithelial cell lines revealed that intestinal epithelia prominently express apical and lateral ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), a member of the ENPP family of enzymes that metabolize diadenosine phosphates, especially Ap3A. Extensions of these studies demonstrated that intestinal epithelia metabolize Ap3A to ADP and AMP, which are further metabolized to adenosine and made available to activate surface adenosine receptors. Using loss and gain of ENPP1 approaches, we revealed that ENPP1 coordinates epithelial barrier formation and promotes epithelial wound healing responses. These studies demonstrate the cooperative metabolism between Ap3A and ENPP1 function to provide a significant source of adenosine, subserving its role in inflammatory resolution.

Intestinal epithelial cells form a selectively permeable barrier to protect colon tissues from luminal microbiota and antigens and to mediate nutrient, fluid, and waste flux in the intestinal tract. Dysregulation of the epithelial cell barrier coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically depleting state of physiological hypoxia. However, studies that systematically examine energy flux and adenylate metabolism during intestinal epithelial barrier development and restoration after disruption are lacking. Here, to delineate barrier-related energy flux, we developed an HPLC-based profiling method to track changes in energy flux and adenylate metabolites during barrier development and restoration. Cultured epithelia exhibited pooling of phosphocreatine and maintained ATP during barrier development. EDTA-induced epithelial barrier disruption revealed that hypoxanthine levels correlated with barrier resistance. Further studies uncovered that hypoxanthine supplementation improves barrier function and wound healing and that hypoxanthine appears to do so by increasing intracellular ATP, which improved cytoskeletal G- to F-actin polymerization. Hypoxanthine supplementation increased the adenylate energy charge in the murine colon, indicating potential to regulate adenylate energy charge-mediated metabolism in intestinal epithelial cells. Moreover, experiments in a murine colitis model disclosed that hypoxanthine loss during active inflammation correlates with markers of disease severity. In summary, our results indicate that hypoxanthine modulates energy metabolism in intestinal epithelial cells and is critical for intestinal barrier function.