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Clinical Nutrition
journal homepage: http://www.elsevier.com/locate/clnu
Narrative Review
Butyrate’s role in human health and the current progress towards its clinical application to treat gastrointestinal disease
Kendra Hodgkinson a, Faiha El Abbar a, Peter Dobranowski a, Juliana Manoogian a, James Butcher a, Daniel Figeys a, b, David Mack c, d, Alain Stintzi a, 
a Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
b School of Pharmaceutical Sciences, University of Ottawa, Ottawa, ON K1H 8M5, Canada
c Department of Paediatrics, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8L1, Canada
d Children’s Hospital of Eastern Ontario Inflammatory Bowel Disease Centre and Research Institute, Ottawa, ON K1H 8L1, Canada

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Article history:
Received 30 June 2022
Accepted 29 October 2022

Keywords:
Butyrate
Inflammatory bowel disease
Resistant starch
Gastrointestinal health
Microbiome
Colon

Butyrate is a key energy source for colonocytes and is produced by the gut microbiota through fermentation of dietary fiber. Butyrate is a histone deacetylase inhibitor and also signals through three G- protein coupled receptors. It is clear that butyrate has an important role in gastrointestinal health and that butyrate levels can impact both host and microbial functions that are intimately coupled with each other. Maintaining optimal butyrate levels improves gastrointestinal health in animal models by supporting colonocyte function, decreasing inflammation, maintaining the gut barrier, and promoting a healthy microbiome. Butyrate has also shown protective actions in the context of intestinal diseases such as inflammatory bowel disease, graft-versus-host disease of the gastrointestinal tract, and colon cancer, whereas lower levels of butyrate and/or the microbes which are responsible for producing this metabolite are associated with disease and poorer health outcomes. However, clinical efforts to increase butyrate levels in humans and reverse these negative outcomes have generated mixed results. This article discusses our current understanding of the molecular mechanisms of butyrate action with a focus on the gastrointestinal system, the links between host and microbial factors, and the efforts that are currently underway to apply the knowledge gained from the bench to bedside.
© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

https://doi.org/10.1016/j.clnu.2022.10.024
0261-5614/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.		Introduction
The microbiome plays a critical role in maintaining health, in part by helping to metabolize dietary components. One key metabolite generated by the microbiome is butyrate, which is generated by the breakdown of nondigestible carbohydrates such as cellulose, inulin, and resistant starch in the lumen of the large intestine [1]. Studies using germ-free animals suggest that butyrate is essentially absent in their gastrointestinal tracts [2,3]. This coupled with the negligible levels of butyrate in our diet suggests that humans are dependant on the action of gut microbes to produce this key metabolite. Short-chain fatty acids (SCFAs), including butyrate, are synthesized by gut bacteria through several pathways [4,5]. Butyrogenic bacteria convert dietary carbohydrates into butyrate via intermediates such as pyruvate, acetyl-CoA, and crotonoyl-CoA. Bacterial species such as Faecali- bacterium prausnitzii, Roseburia intestinalis, and Agathobacter rectale perform this simple conversion in monoculture [6]. Less common pathways involve transforming and denitrifying L- glutamate (Intestimonas, [7]) and L-lysine (Flavonifractor plautii, [6]) into crotonoyl-CoA. Crotonoyl-CoA is hydrogenated into butyryl-CoA, then transformed into butyrate directly via butyryl- CoA transferase or indirectly via phosphotransbutyrylase and butyrate kinase [5]. Butyrogenic bacteria may outsource initial dietary carbohydrate breakdown to other bacteria and cross-feed upon their fermentation by-products, such as acetate (R. intestinalis and F. prausnitzii, [8]), lactate (Anaerobutyricum soehngenii, [6]), and succinate (Clostridoides difficile, [9]) to produce butyrate.
Butyrate is not the sole SCFA in the gut, with the most abundant SCFAs in the gut being acetate, butyrate and propionate, which together constitute over 90% of the SCFAs produced

Abbreviations	HIF1A	Hypoxia inducible factor 1 subunit alpha	
		HSCT	Hematopoietic stem cell transplantation	
5-ASA	5-Amino salicylic acid	IBD	Inflammatory bowel disease	
AHR	Aryl hydrocarbon receptor	IFNG	Interferon gamma	
AMP	Antimicrobial peptide	IGF1	Insulin-like growth factor 1	
AMPK	AMP-activated protein kinase	IL	Interleukin	
BMP	Bone morphogenetic protein	iNOS	Inducible nitric oxide synthase	
BSG	Basigin (Ok blood group)	MAPK	Mitogen-activated protein kinase	
CD	Crohn’s disease	MUC	Mucin	
CRC	Colorectal cancer	NFKB1	Nuclear factor kappa B subunit 1	
CYP	Cytochrome P450 family	NO	Nitric oxide	
DEFA8	Defensin alpha 8	PHD	Prolyl hydroxylases	
DEFB1	Defensin beta 1	PPARg	Peroxisome proliferator activated receptor gamma	
DEFB4A	Defensin beta 4A; previously defensin beta 2	REG3G	Regenerating family member 3 gamma	
DSS	Dextran sulfate sodium	SCFA	Short chain fatty acid	
FFAR	Free fatty acid receptor	SLC12A2	Solute carrier family 12 member 2	
FMT	Fecal microbiota transplant	SRC	SRC proto-oncogene, non-receptor tyrosine kinase	
GPCR	G-protein coupled receptor	STAT	Signal transducer and activator of transcription	
GVHD	Graft-versus-host disease	TCA	Tricarboxylic acid	
H2S	Hydrogen sulfide	TNF	Tumor necrosis factor	
HAT	Histone acetyltransferase	TUB	TUB bipartite transcription factor	
HDAC	Histone deacetylase	UC	Ulcerative colitis	

2. Effect of butyrate on colonocyte health

by the microbiome [10]. These SCFAs promote human health in a variety of ways; they provide energy for intestinal cells, help maintain the gut barrier, and activate anti-inflammation signalling cascades. Low levels of SCFA-producing microbes have been associated with diseases such as ulcerative colitis [11] and asthma [12]. In addition to the major SCFAs mentioned above, there are also less abundant SCFAs present such as the branched- chain SCFAs isobutyrate, isovalerate, and 2-methyl butyrate [10], which are produced from the fermentation of amino acids (valine, leucine, and isoleucine) rather than carbohydrates. Little is known about the effects of branched-chain SCFAs on human health, although there has been speculation that they may also play an important role in maintaining intestinal homeostasis [13]. SCFA levels are highest in the lumen of the gut and show a clear gradient within the body, with butyrate absorption primarily in the gut epithelium whereas propionate is absorbed mainly in the liver and acetate is absorbed mainly by the peripheral organs and tissues [14].
Butyrate is a particularly important SCFA because it is the main energy source for colonocytes; it also regulates histone acetylation by activating, at low concentrations, histone acetyltransferases (HATs) or inhibiting, at high concentrations, histone deacetylases (HDACs) classes I and II [15,16]. Butyrate can also directly impact gene expression by interacting with promoters, and several genes with butyrate response elements have been identified. Studies examining global changes in gene expression following butyrate treatment have found changes in various pathways including the cell cycle, differentiation, and fatty acid metabolism [17e22].
The goal of this review is to survey the current state of knowledge regarding butyrate’s role in host intestinal tissue and the gut microbiome, and how these effects translate to maintaining health. We discuss butyrate’s various roles in colonocytes, butyrate’s impact on inflammation, butyrate’s link with the microbiome, and finally its links with human gastrointestinal disease. In particular, we have endeavored to detail the impact of butyrate in vivo, describing the impact of butyrate supplementation in model organisms and studies employing butyrate supplementation to treating human disease.
2.1.		Butyrate uptake and signalling
Butyrate crosses the cell membrane of colonocytes by diffusion or through active transporters [23]. These transporters include the Hþ-coupled monocarboxylate cotransporter 1 (MCT1) and the Naþ- coupled monocarboxylate transporter [23,24]. Previous in vivo human work suggests that butyrate is readily absorbed in the small intestine [25,26]. Thus, minimal dietary butyrate reaches the colon, and colonic epithelial cells are probably wholly dependant on microbially derived butyrate.
Butyrate is critical for maintaining colonocyte health, in large part due to its role as an energy source. Butyrate produced by the gut microbiota is the primary energy source for colonocytes, yielding around 70% of the cell’s energy [27,28]. Butyrate is oxidized into CO2 in the mitochondria, generating ATP [29]. The generated energy is necessary to maintain the sodium electrochemical gradient by activating the Naþ/Kþ ATPase and consequently is essential for water and electrolyte absorption. Colonocytes isolated from germ-free mice appear to be energy deprived and present with deficits in mitochondrial respiration and increased levels of autophagy [28]. However, these defects can be rescued in vivo by colonizing the germ-free mice with conventional microbiotas or by treating germ-free colonocytes with butyrate ex vivo [28].
Butyrate signals through three G-protein coupled receptors (GPCRs; GPR43, GPR41 and GPR109A; Fig. 1) which are present on colonic epithelial, adipocyte and immune cells [30]. GPR43 (FFAR2) and GPR41 (FFAR3) are activated by all three major SCFAs: acetate, propionate, and butyrate [31,32]. GPR41 is expressed in many tissues, whereas GPR43 is highly specific to lymphatic tissues and various immune cells [31,32]. Butyrate binds GPR41 preferentially over GPR43 [23,33]. GPR109A is activated by butyrate and b- hydroxybutyrate [34], and niacin at high doses [35]. All three GPCRs are coupled with Gi/o and therefore inhibit cAMP-dependent pathways while activating other signaling pathways [36]. When activated in colonocyte and immune cells, these receptors cause changes in cytokine levels and various signaling pathways that promote an anti-inflammatory response (Fig. 1) [30].

2.2.		
Fig. 1. Butyrate signaling in the colon through G-protein coupled receptors (GPR41, GPR43, and GPR109A). Butyrate is mainly produced by the intestinal microbiota through the breakdown of dietary fibers. Butyrate signals through three G-protein coupled receptors present on colonic epithelial and immune cells: GPR43 (free fatty acid receptor 2; FFAR2), GPR41 (free fatty acid receptor 3; FFAR3), and GPR109A (hydroxycarboxylic acid receptor 2; HCAR2). Activation of these receptors in colonocyte and immune cells causes changes in cytokine levels and various signaling pathways, promoting an anti-inflammatory response.

Butyrate affects crypt development and proliferation
The effect of butyrate on cell proliferation varies according to dose and cell type. In the small intestine, butyrate helps maintain normal growth, development, and homeostasis. In particular, increased villus height to crypt depth ratio is believed to increase nutrient absorption in part by increasing the total small intestine surface area. Oral butyrate supplementation increases the villus height to crypt depth ratio in juvenile animal models, including piglets [37], chicks [38,39], and calves [40]. Studies have shown mixed effects of butyrate on crypt depth. Butyrate decreased crypt depth (and decreased IGF1 expression) in calf ileums [40], but increased crypt depth in chick jejunums [41] and did not significantly affect crypt depth in sheep [42]. In the colon, butyrate has been shown to increase crypt depth in pigs by inhibiting apoptosis [43] but another study found no change in crypt depth despite increased proliferation [44].
Butyrate treatment of germ-free or antibiotic-treated mice rescued epithelial cell turnover in the ileum, suggesting that butyrate produced by the normal gut microbiome is vital for this process [45]. In the same study, butyrate promoted the development of intestinal enteroids in vitro.
Two studies in animal models have shown that butyrate promotes colonocyte proliferation [44,46]. However, another study found that 10% butyrylated high amylose maize starch increased apoptosis in the distal colon (especially in the base and proliferative zone of crypts) and did not alter proliferation of the colonic epithelium [47].
These contradictory effects in vivo may be related to dose and/or formulation, as there is a dose-specific effect of butyrate on colo- nocyte proliferation in vitro. At low doses (below approximately 2 mM), butyrate stimulates colonocyte proliferation [48]. At higher doses, butyrate inhibits proliferation of human colon epithelial cells in vitro [49] and ex vivo [50]. High-dose butyrate has been shown to induce cell cycle arrest and apoptosis [51]. Free sodium butyrate is also likely to have more of an impact in the small intestine as compared to the colon due to it being absorbed first at these sites [25,26].
Overall, these results suggest that a threshold level of butyrate is needed for optimal colonocyte survival and proliferation, whereas higher butyrate levels can have the opposite effect. Butyrate’s seemingly contradictory effects could be caused by its different mechanisms of action. At low doses butyrate is oxidized by the mitochondria activating HATs, while at higher doses (at concentrations saturating b-oxidation), butyrate’s HDAC inhibition activity becomes more relevant. The set of genes activated by HAT activation or HDAC inhibition have been shown to be significantly different, partially explaining these conflicting results [49].
2.3.		Effects of butyrate on blood flow, visceral sensitivity and gut motility
Butyrate stimulates colon contractions and gut motility in animal models. Using strip preparations of rat colon circular muscles, it was found that butyrate stimulates concentration-dependent phasic and tonic contractions [52,53]. Interestingly, this effect was not seen in mucosa-free preparations, suggesting that butyrate stimulates the colonic muscles indirectly via the mucosa [53]. Butyrate increased the contractile response of rat colon muscles ex vivo, and rats fed a butyrate-generating resistant starch diet had faster colonic transit times [54]. Both in vitro and in vivo, butyrate increased the proportion of choline acetyltransferase neurons in rats by activating the SRC-kinase signaling pathway [54]. Inhibiting the SRC signaling pathway or knocking down monocarboxylate transporter 2, which is expressed by enteric neurons, prevented this response to butyrate [54].
A mix of SCFAs (including butyrate) administered into the lumen of the proximal colon of rats stimulated intestinal contractions and shortened intestinal transit; these effects were mediated by an increase in serotonin release into the lumen [55]. Similarly, butyrate treatment rescued the delayed transit time seen in germ-free mice but did not rescue transit time in germ-free mice lacking tryptophan hydroxylase-1, an enzyme involved in serotonin synthesis [56]. This suggests that butyrate is important for maintaining normal colon function by increasing serotonin synthesis and release. Serotonin has also been implicated in visceral perception [57].
Butyrate also promotes healthy colon function by increasing blood flow to the mucosa, as shown by dilation of resistance arteries dissected from human colon biopsies [58] and increased blood flow in colon resection patients given butyrate enemas [59].
2.4.		Butyrate and epithelial repair
There are no direct studies of butyrate on mucosal healing in humans, although studies in IBD patients suggest that butyrate has a protective effect (see IBD section below). Animal models show that butyrate can protect from epithelial injury and improve tissue repair. Tributyrin protects against acetic acid injury in pigs [60] and butyrate has a similar effect in rats [61]. Butyrate attenuates inflammation and mucosal lesions in mouse models of colitis [62,63]. Transcriptomic analysis of colonic mucosa biopsies identified numerous genes that were differentially regulated by butyrate, including upregulation of Wnt signaling and BMP antagonists, which promote mucosal repair and stem cell renewal [22,64].
2.5.		Beneficial effects of butyrate on the gut barrier and ion transport
Butyrate strengthens the gut barrier by targeting three complementary elements: tight junctions, the mucus layer, and the production of antimicrobial peptides. Many tight junction proteins are upregulated by butyrate (e.g. TJP1, claudin 7, cadherin 1 in the rat ileum [65]; TJP1, claudin 3 and occludin in pig colons [66]). Tight junction protein 1 (TJP1; previously named ZO1) is particularly important, as it modulates tight junctions and is commonly used as a marker of intestinal permeability [67]. In contrast, claudin 2, a tight junction protein that forms gap channels and contributes to a leaky gut barrier [68], is downregulated by butyrate via HDAC inhibition, STAT3 activation, and upregulation of IL10RA (previously IL10R) [69]. In addition to altering the expression of tight junction proteins, butyrate promotes tight junction assembly by activating AMPK, reducing the permeability of colon cancer cell monolayers [70].
Some studies have shown that butyrate increases expression of MUC2, the main gut mucin, in LS174T colon cancer cells [71,72]; however, butyrate appears to decrease MUC2 expression in HT29 cells [73]. The difference could be due to cell line or dose differences as butyrate did not induce MUC2 at doses above 1 mM in LS174T cells [71]. LS174T cells treated with a MEK inhibitor did not show MUC2 upregulation by butyrate, implicating the MEK/ ERK pathway in this effect [74]. Upregulation of prostaglandin production has also been suggested to be a mechanism [72]. Butyrate at 0.5 mM activated MUC2 production from biopsies of ulcerative colitis (UC) patients [75]. Butyrate enemas also upregu- lated MUC1, MUC2, MUC3 and MUC4 in mice [76]; however, butyrate enemas (60 mL, 100 mM sodium butyrate) did not alter MUC2 in healthy volunteers or UC patients in remission [77]. The dose and frequency of administration might be key to observing a beneficial effect.
Butyrate increases Naþ and Cl^ absorption in the colon, as first shown in isolated colon mucosa from rats [78]. Butyrate promotes Naþ absorption through NHE2 and NHE3, which are expressed on the apical membranes of colon epithelial cells [79]. Butyrate also inhibits cAMP-mediated Cl^ secretion in rat colon sections and T84 cells [80] by blocking the activity of the SLC12A2 cotransporter [15].
3.		Anti-inflammatory mechanisms of butyrate within the intestinal tract
3.1.		Butyrate effects on cytokine levels, T regulatory cells and macrophages
Butyrate and other SCFAs have been shown to reduce inflammation in many model systems. GPR43, GPR41, and GPR109A are the main mediators of the effects of SCFAs on inflammation and immune cells. GPR43 and GPR41 bind acetate, propionate, and butyrate [31]. GPR109A has only been studied in the context of butyrate activation and it is unclear whether other SCFAs also bind to GPR109A [81].
GPR43-/- mice have been reported to have more severe inflammation than C57BL/6 wild-type mice in models of colitis, arthritis, and asthma [82,83]. In contrast, another study found that both GPR43-/- and GPR41-/- mice had reduced inflammatory responses to chemically-induced colitis as compared to C57BL/6 controls [84]. In addition, a study found conflicting impacts on inflammation with GPR43-/- mice being more sensitive to acute DSS-induced colitis but protected in a chronic DSS-induced colitis model as compared to C57BL/6 controls [85]. The reasons for these conflicting responses are unclear but could be related to differences in the mouse colonies, how colitis was induced, and the severity of induction.
Despite acting on shared receptors, butyrate has different effects from other SCFAs on cultured lymphocytes from rat mesenteric lymph nodes [86]. Butyrate inhibited the T helper-1 lymphocyte phenotype (IL2 and IFNG production), whereas acetate and propionate did the opposite [86]. In the same study, a combination of the three SCFAs led to an anti-inflammatory phenotype.
Butyrate decreases macrophage secretion of pro-inflammatory cytokines, such as IL17 [87], IL6, and IL12 [23,33] (Fig. 2). Butyrate also increases plasma levels of the anti-IL17 cytokines IL10 and IL12 [88] and increases levels of IL18, an anti-inflammatory cytokine, in the colonic epithelium [88,89]. Butyrate promotes differentiation of T-regulatory immune cells through GPR109A [90] and thus suppresses inflammation. Increases in IL10 are caused by increased differentiation of IL10-producing CD4þ T-cells [90].
3.2.		Butyrate inhibits NF-kB signaling
NF-kB (NFKB1) is a transcription factor that plays a central role in regulating inflammation by upregulating many genes that promote inflammation [23]. Butyrate inhibits NF-kB activity by

Fig. 2. Summary of butyrate mechanisms of action. Butyrate plays an important role in maintaining intestinal homeostasis through different mechanisms: as an energy source for colonocytes, via HDAC inhibition, and via GPR activation in colonocytes and immune cells.

increasing the transcription and activity of peroxisome proliferator- activated receptor gamma (PPARg) in human colon cancer cells [91] (Fig. 3). Butyrate also suppresses nuclear translocation and DNA binding of NF-kB in colon cancer cells [92], and decreases the transcription of NF-kB and other pro-inflammatory genes in primary epithelial cells derived from human small intestine and colon [18]. In addition, butyrate inhibits NF-kB p65 signaling in mice macrophages through a GPR109A-dependent mechanism [93].
3.3.		Butyrate decreases nitric oxide (NO) production
Nitric oxide (NO) is a neurotransmitter and immunomodulator that can have pro-inflammatory or anti-inflammatory effects depending on dose and context [94]. Butyrate decreased NO synthesis in neutrophils isolated from rats and cultured in vitro [95]. Similarly, NO synthesis and neutrophil migration were inhibited in neutrophils from rats that had previously received tributyrin treatment in vivo [95]. Butyrate also decreased NO and other pro- inflammatory molecules in cultured intestinal macrophages, independent from GPCR signaling [33]. The HDAC inhibitor trichostatin A had similar effects, suggesting that butyrate downregulates NO through HDAC inhibition [33]. Treating mouse macrophages with butyrate resulted in decreased expression ofNO and IL6, which was independent of GPCR signalling, but instead seemed to be linked to HDAC inhibition [23]. Knockdown of the butyrate transporter SLC5A8 in mice increased expression of inducible nitric oxide synthase (iNOS) [96]. In a colon carcinoma cell line, butyrate reduced iNOS induction by inhibiting IFNG-induced STAT1 activation [96]. Thus, inhibition of NO by butyrate may contribute to the anti-inflammatory effects of butyrate (Fig. 3).
3.4.		Butyrate activates the aryl hydrocarbon receptor (AHR)
AHR is a ligand-dependent transcription factor, with ligand binding leading to nuclear translocation of AHR and its transcriptional control of target genes including cytochrome P450 family members (CYP1A1, CYP1A2, CYPAB1) and NAD(P)H-quinone oxidoreductase [97] (Fig. 3). AHR is activated by numerous ligands including tryptophan-derived microbial metabolites, food contaminants, and phytochemicals [98]. Butyrate was recently suggested as a direct ligand for AHR activation [99]. While recent reports have disputed whether this direct interaction exists [100], it is clear that butyrate can influence AHR function either through putative binding to the receptor or through HDAC inhibition.
AHR regulates different subsets of immune cells and plays an important role in gut homeostasis [97,101]. In addition, AHR activation maintains intestinal barrier integrity, and controls both stem cell proliferation and epithelial differentiation via inhibition of the Wnt/b-catenin signaling pathway [102]. It has also been shown that AHR activation prevents epithelial barrier dysfunction through upregulation of Notch1 signaling and by maintaining the expression of tight junctions [103]. In addition to its direct binding to AHR, butyrate is also reported to enhance the expression of AHR and its target genes through its HDAC inhibition activity [104]. AHR knockout mice exhibit increased susceptibility to DSS-induced colitis [105,106] and AHR expression is downregulated in Crohn’s disease (CD) patients especially at the inflamed site [107]. Collectively, these data suggest an important role for AHR and its activation by butyrate in promoting gut barrier integrity (Fig. 3).
4.		The interplay of butyrate and the intestinal microbiome
4.1.		Butyrate increases production of host defense peptides/ antimicrobial peptides
Antimicrobial peptides (AMPs) protect against a wide spectrum of pathogenic microbes and have been shown to be upregulated by HDAC inhibitors [108]. Butyrate upregulates the production of AMPs such as cathelicidin antimicrobial peptide (previously LL-37) [109], DEFA8, TUB (previously Rd5), REG3G [65], and b-defensins 1 and 2 (DEFB1, DEFB4A) [110] in various colon cancer cell lines. Butyrate has also been shown to increase AMP secretion in pigs, cows, and chickens [48]. Butyrate upregulates AMPs primarily through HDAC inhibition, but also through activation of the MAPK pathway by GPCRs [48] (Fig. 2).
Butyrate treatment in mice promoted the antimicrobial activity of intestinal macrophages in an ex vivo assay [111]. Macrophages differentiated in vitro in the presence of butyrate showed enhanced antimicrobial activity, altered metabolism, and increased AMP production; these effects were mediated by inhibition of HDAC3 [111].

4.2.		
Fig. 3. Microbial-derived butyrate modulates intestinal epithelial immunometabolic circuitry to maintain gut homeostasis. In the intestinal lumen, communities of anaerobic commensal bacteria transform dietary fibres into butyrate. Within epithelial cells, butyrate influences aryl hydrocarbon receptor (AHR), Wnt/b-catenin, peroxisome proliferator- activated receptor gamma (PPARg), Notch, and hypoxia inducible factor 1 (HIF1) signalling pathways. Butyrate promotes epithelial barrier integrity, drives intracellular and luminal hypoxia, and negatively regulates stem cell proliferation to contribute to gut homeostasis. Panel (a) represents the role of butyrate in a healthy state, with the effects of perturbed butyrate production and metabolism associated with a diseased state shown in panel (b).

Butyrate increases epithelial O2 consumption promoting a hypoxic environment critical for intestinal homeostasis
Intracellular butyrate is transported into the mitochondria by carnitine-dependent and independent mechanisms [112]. In the mitochondria, butyrate undergoes b-oxidation and enters the TCA cycle as acetyl-CoA. In addition to fueling the cell, downstream oxidative phosphorylation depletes intracellular O2 [113]. Resultantly, a steep O2 gradient forms at the intestinal epithelial interface that contributes to homeostasis in two ways (Fig. 3).
First, the O2 gradient renders intracellular conditions hypoxic (<5% O2 tension), stabilizing hypoxia inducible factor 1 subunit alpha (HIF1A) by inhibiting O2-dependent prolyl hydroxylases (PHDs). Consequently, HIF1A and HIF1B form a heterodimer transcription factor that upregulates epithelial barrier integrity components (mucin, trefoil factors, and tight junction proteins; reviewed in [114]) and is demonstrably protective against colitis in mice [115e118]. Kelly et al. showed that mitochondrial respiration inhibition, antibiotic treatment, and germ-free conditions abrogate hypoxia and HIF1 function, which can be restored with tributyrin [113]. Interestingly, butyrate also noncompetitively inhibits PHDs [119], thereby regulating HIF1 activity independently of the O2 gradient. Hence, butyrate metabolism contributes to maintaining epithelial homeostasis via HIF1A (Fig. 3).
Second, the O2 gradient renders the lumen hypoxic and this is potentially important for microbiome homeostasis, but the evidence for this is inconsistent. In the “oxygen hypothesis” (introduced by [120] and developed further by [121]), lumen hypoxia favors butyrogenic obligate anaerobes and suppresses dysbiotic facultative anaerobes. Recent work demonstrated the concept that antioxidants can restore butyrate production by Clostridia under normoxic conditions [122]. Further, depleting Clostridia with streptomycin drives colonocyte oxygenation in C57BL/6 mice, permitting an expansion of pathogens [122]. Without antibiotics, infection with Salmonella Typhimurium in CBA mice also leads to an expansion of Gammaproteobacteria that significantly negatively correlates with Clostridia [122]. The murine pathogen Citrobacter rodentium promotes lumen oxygenation and Enterobacteriaceae outgrowth in mice, while paradoxically enriching Clostridia [123]. While hyperbaric oxygen treatment depletes luminal Anaerostipes in C57BL/6 mice, it restricts Enterobacteriaceae growth [124]. Moreover, intermittent hypoxia significantly reduces putatively butyrogenic Firmicutes genera and enriches Proteobacteria genera in C57BL/6 mice [125], although this model is potentially confounded bya compensatory accumulation of luminal CO2 [126]. Hence, butyrate’s role in maintaining microbiome homeostasis can only be inferred at present (Fig. 3). In vitro cell culture coupled to an oxygen gradient [127] presents an ideal platform to disentangle gut microbiome responses to varying oxygen conditions.
4.3.		Butyrate selectively modulates gut bacteria
Microbiome changes have been observed in healthy volunteers after ingestion of butyrylated high amylose maize starch [128]. The butyrylated maize starch increased the relative quantities of Para- bacteroides distasonis and F. prausnitzii in stool samples compared to baseline levels [128]. Treatments that increase butyrate have also been shown to increase species richness and/or diversity in animal models. For example, sorghum bran increased butyrate and increased the richness and diversity of microbial species in a DSS rat model [129]. Rats fed a resistant starch diet that increased butyrate levels had increased culturable Lactobacilli and Bifidobacteria with decreased Enterobacteria [130]. While SCFAs can inhibit gut pathogen growth and activity by acidifying the cell cytoplasm [131,132], recent studies have elucidated butyrate’s unique cytotoxic effect on certain microbes.
Butyrate was shown to selectively inhibit the common gut bacteria Bacteroides in a strain-specific manner, modulated by the presence of different glycans [133]. Butyrate-sensitive Bacteroides (e.g. Bacteroides vulgatus) express higher acyl-CoA transferase activity, causing an accumulation of butyryl-CoA and downstream cell membrane damage and cell death. Conversely, butyrateresistant Bacteroides (e.g. Bacteroides thetaiotaomicron) express higher acyl-CoA thioesterase activity, sequestering butyryl-CoA and conferring a fitness advantage in butyrate-rich environments. These dynamics are worth closer examination in light of butyrate’s role in health and disease.
B.		vulgatus has been shown to cooperate in inhibiting butyrate production in bioreactor experiments [134], ostensibly an attempt to attenuate hostile microenvironment conditions. Interestingly, B. vulgatus proteolytic activity has also been mechanistically linked to the onset of UC [135,136], suggesting contributory roles in perturbing gut homeostasis. Conversely, B. thetaiotaomicron competes with B. vulgatus for resources in the gut [137], co-responds to the presence of specific dietary fibers along with butyrate producers [138], and also expresses immunoregulatory outer membrane vesicles [139]. These findings indicate that B. thetaiotaomicron contributes to maintaining gut homeostasis. Interspecies Bacteroides competition may, at least in part, hinge upon butyrate, offering a vignette to showcase butyrate’s role in health and disease by directly modulating the microbiome.
4.4.		Butyrate activates PPARg, limiting enterobacteriaceae expansion
PPARg (PPAR-gamma) is a multi-domain transcription factor that binds lipophilic ligands such as butyrate and regulates gene transcription through transactivation and transrepression [140,141]. Butyrate activates PPARg signaling in colonocytes, which drives mitochondrial b-oxidation (and thus lumen hypoxia, described above), and inhibits epithelial expression of NOS2, a gene coding for inducible nitric oxide synthase (iNOS) [142]. Since iNOS contributes to the epithelial production of nitric oxide (NO) in the gut, its downregulation results in a decrease in nitrate levels and the maintenance of an anaerobic environment in the colonic lumen (Fig. 3). In contrast, butyrate depletion increases the synthesis of iNOS, thus increasing nitrate levels (Fig. 3). Luminal NO renders a fitness advantage to certain facultative anaerobes, such as Entero- bacteriaceae [143]. Interestingly, bacteria capable of using NO as an electron acceptor are often able to ferment protein into hydrogen sulfide (H2S) [144]. While the involvement of H2S in intestinal inflammation is unsettled [145], one study found that bacteria known to ferment sulfur-containing amino acids to H2S were associated with severe CD [146], while another study found that H2S can reduce disulfide bonds in the mucus barrier to thiols, thus acting as a mucolytic agent and further exposing the epithelium to gut microbes [147]. Host enzymes required for H2S detoxification are inhibited by NO [148]. Additionally, acyl-CoA dehydrogenase, the enzyme responsible for oxidizing butyrate by colonocytes, is inhibited by H2S [149,150]. Therefore, butyrate acting via PPARg prevents microbiome destabilization by opportunistic, disease- associated bacteria (Fig. 3).
Obligate anaerobes are thought to be a marker of gut microbiome homeostasis while an environment dominated by facultative anaerobes such as Enterobacteriaceae is a hallmark of a dysbiotic microbiome [151]. Obligate anaerobes rely on fermentation for energy and expansion while facultative anaerobes can use oxygen or nitrate as an electron acceptor for aerobic or anaerobic respiration, respectively. The increase in electron acceptor levels due to decreased butyrate allows for the expansion of facultative anaerobes and gives them an advantage over commensal obligate anaerobes (Fig. 3). As obligate anaerobes produce butyrate through the fermentation of complex carbohydrates, their depletion maintains the unbalanced microbial environment. In this way, butyrate production may contribute to the maintenance of gut microbiota homeostasis.
5.		Butyrate and intestinal disease
5.1.		Butyrate and inflammatory bowel disease (IBD)
Both UC and CD patient microbiotas present with altered compositions as compared to healthy controls (discussed in more detail below) and also have decreased levels of butyrate and other SCFAs [152]. This meta-analysis also found that butyrate levels increased in UC patients during remission. Interestingly, as discussed above,
butyrate levels impact the activity of AHR, and profiling intestinal tissue from IBD patients suggests that these patients have reduced AHR when compared to controls. Indeed, AHR signaling increases IL22 expression and provides protection against induced colitis in mice [107], suggesting a potential mechanism by which increased butyrate could improve disease. In addition, TNF (previously TNFa) treatment reduced butyrate oxidation in intestinal epithelium organoids from IBD patients [153]. This suggests that IBD patients could be suffering from a butyrate “double hit”: both reduced butyrate levels from lower levels of butyrate-producing microbes and a reduction in the ability to utilize butyrate itself. This “double hit” hypothesis is further supported by the decreased expression of two butyrate receptors, BSG and MCT1, in CD colonocytes, suggesting a decreased capacity to take up butyrate [154].
It is thus not surprising that many, but not all, butyrate treatments and interventions that increase butyrate have led to clinical improvements in several IBD patient populations (Table 1). Enemas are a reliable way to deliver butyrate to the colon, but mixed outcomes have been observed. Butyrate enemas can improve symptoms and inflammation associated with UC [155e159] and reduced NF-kB activation in lamina propria macrophages from UC patients [160]. More recently, high-dose butyrate enemas (600 mM, twice daily) improved endoscopic scores and prevented mucosal atrophy in a small group of patients following diverting ileo/colostomy (performed for IBD, colorectal cancer, or diverticulitis) relative to control enemas [22]. Due to the patient cohort being studied, this report was able to avoid the cofounding effect that diet could have in these patients (diverting ileo/colostomy prevents food from reaching this section of the gastrointestinal tract). However, studies in other settings have found no clinical effect of butyrate enemas in UC patients relative to saline enema controls [77,161,162]. These differences between studies could be due to more rigorous controls, the lower doses used, individual variation, or different disease conditions.

Delivery Mode	Format (dose)/Length	Design	Groups (n)	Effects and comments	Ref.	
Enema	60 mL, 60 mM acetate, 30 mM propionate
40 mM butyrate, daily for 14 d	Double-blind	DC (13)	^ No improvement in endoscopic and histologic scores [159]	
Enema	100 mL, acetate 80 mM, propionate 30 mM and
40 mM SB, 2/d for 6 wks	DB-RCT	UC (40)	^ Minor differences between groups but this could be [156] due to increased number of moderate patients in the study arm	
Enema	60 mL, 60 mM acetate, 30 mM propionate
40 mM butyrate, pH 7.0, 2/d for 6 wks	Open Label	UC (10)	^ Therapy refractive patients; 5/10 showed [155] improvement at end of trial.	
Enema	100 mL, 100 mM SB, 40 mM NaCl pH 7.0; 2/day for 2 wks	Single blind with crossover	UC (10)	^ Improved inflammation scores and reduced stool [158] frequency	
Enema	60 mL, 80 mM SB pH 7.0; daily for 6 wks	Open Label	UC (10)	^ Therapy refractive patients; 6/10 showed [157] improvement at end of trial.	
Enema	60 mL, 80 mM SB pH 7.0; daily for 6 weeks	RCT	UC (38)	^ No significant difference between placebo and [161] treatment groups	
Enema	60 mL, 100 mM SB, pH 5.5; 2/day for 4e8 wks	DB-RCT	UC (47)	^ No difference between disease activities at end of trial [162]
^ Fewer colonic segments affected after butyrate as compared with placebo
^ Trial also included an arm with all three SCFA’s in enema; no difference seen	
Enema	60 mL, 100 mM SB, pH 5.5; 2/d for8 wks	RCT	UC (11)	^ Reduction in NF-kB positive macrophages	[160]
^ Reduction in Disease Activity Index	
Enema
Enema	60 mL, 100 mM SB, 40 mM NaCl; daily for 20 d
60 mL, 50 or 100 mM SB, pH 7.0; daily for 7 d for each SB conc.	DB-RCT with crossover
DB-RCT with crossover	UC (35)
IBS (11)	^ NCT00696098	[198]
^ Minor impact on IL10/IL12 ratio
^ Decreased perception of pain, urge and discomfort [20]	
Enema	30 mL, 600 mM SB, 2/d, 30 d	DB-RCT	Mixed (17)	^ Patients with enterostomies for diverticular disease, [22] cancer or IBD
^ Placebo group enemas contained 3 mM SB to add odour to enema
^ Reduced mucosal atrophy in SB group	
Oral	Zacol NMX Tablet; 300 mg butyrate & 250 mg inulin; 3/d	Open, Multi-center	UC (216)	^ Combination therapy with 3 ^ 800 mg 5-ASA/day [197]
^ Reduced disease activity	
Oral	Encapsulated, 5/d; 800 mg SB/capsule	DB-RCT	UC (25)	^ Combination therapy with 2.4 g 5-ASA/day	[199]
^ No difference in outcomes between treatment and placebo
^ Better improvement as compared to baseline for treatment group	
Oral	Encapsulated, 3/day; 500 mg SB/capsule; 60 d	DB-RCT	CD (19)
UC (30)	^ Stimulated growth of butyrogenic microbes	[164]
^ No change in disease activity was observed
^ Improved quality of life in UC patients	
Oral	Encapsulated; 2/d; 500 mg SB/capsule; 12 mo	Observational, singlecenter, prospective cohort	UC (39)	^ Reduced fecal calprotectin	[200]
^ Maintained clinical remission
^ Higher rate of clinical and biochemical improvements
^ Improved quality of life	
Oral	Encapsulated; 2/d, 150 mg SB/capsule, 12 wks	DB-RCT	IBS (66)	^ Reduction in pain during defecation and in urgency [178]
^ No difference in abdominal pain and flatulence	

Table 1
Effects of butyrate interventions. (DB-RCT ¼ Double blind randomized, placebo controlled trial; SB ¼ sodium butyrate, DC ¼ diverting colitis).

Interestingly, two recent pilot clinical trials have examined the effect of encapsulated butyrate as adjuvant therapy in maintaining remission in CD and UC patients [163,164]. Butyrate was found to decrease fecal calprotectin, promote the growth of butyrogenic bacteria, and improve the quality of life of UC patients, but no significant improvement in disease activity was observed. Conversely, a topical butyrate adjuvant therapy improved the effects of 5-amino salicylic acid (5-ASA) in refractory UC [165]. Further studies are needed to ascertain a beneficial effect of butyrate in IBD patients. Importantly, butyrate’s absorption within the small intestine [25,26] necessitates oral butyrate supplementation in a form that allows butyrate to reach the colon for benefits to be realized.
Butyrate producers in the Firmicutes phylum, including F. prausnitzii, are often decreased in the CD microbiome [166,167], and CD patients have lower butyrate levels than healthy controls [168]. Several groups have found that Enterobacteriaceae are increased in treatment-naïve pediatric IBD patients compared to non-IBD control patients, often at the expense of butyrate producers [146,166]. As discussed above, dysregulation of PPARg by low butyrate levels may lead to increases in NO and nitrate (Fig. 3). Specifically targeting the pathways used by Enterobacteriaceae to respire via nitrate led to the specific depletion of these taxa, while butyrate producers were unaffected by this treatment [169].
A recent meta-analysis showed an overall decrease in butyrate and other SCFAs in patients with IBD, but noted inconsistent results across different studies [152]. Notably, UC patients in remission had increased butyrate levels relative to controls, whereas active UC patients had a strong decrease in butyrate [152]. It remains unclear whether these observations are a cause or an effect of the disease. Fecal microbiota transplantation (FMT) studies could help answer that question. UC patients prior to FMT had altered levels of butyrate producers (reduced bacterial groups from the Clostridium cluster XIVa and higher levels of Bacter- oidetes) relative to their FMT donors [170]. After transplantation, these differences were reduced. When responders and nonresponders were compared 12 weeks post-FMT, a poor response was associated with high levels of Proteobacteria and Bacter- oidetes and low levels of butyrate-producing bacteria from Clostridium clusters IV and XIVa [170]. This observation suggests that an increase in butyrate producers may be required to induce and maintain remission in UC patients.
5.2.		The effect of butyrate in gastrointestinal GVHD
Graft-versus-host disease (GVHD) is a common and serious complication of hematopoietic stem cell transplantation (HSCT) and has been previously linked to a loss of microbiome diversity and gut barrier integrity due to prolonged antibiotic use [171]. Indeed, an earlier study in mice had identified butyrate as a significantly reduced metabolite in the intestinal tissue of HSCT recipients and showed that administering butyrate-producing probiotics could prevent acute GVHD [172].
A recent retrospective study found that HSCT recipients who took antibiotics targeting anaerobic bacteria (piperacillin-tazo- bactam, or carbapenems) had higher acute GVHD incidence and mortality relative to patients who took antibiotics (aztreonam, cefepime, or ceftazidime) with minimal activity against anaerobes [173]. Shotgun metagenomic results suggested that these anaerobe-targeting antibiotics reduced the relative abundance of butyrate producers such as Bifidobacteriales and Clostridiales and also decreased the presence of bacterial butyrate biosynthesis genes [173]. These changes preceded the development of acute gut/ liver GVHD, suggesting a role for butyrate production in protection against GVHD. Antibiotic treatment may also impact epithelial O2 consumption, thereby altering intestinal homeostasis via HIF1a as described above [113,119].
Butyrate levels are strongly decreased in acute GVHD patients [173] and in pediatric HSCT recipients who later developed acute GVHD [174]. Romick-Rosendale et al. also found that at disease onset, acute GVHD patients had decreased relative abundances of known butyrate producers from the Lachnospiraceae and Rumi- nococcaceae families. Plasma butyrate levels were lower in two cohorts of patients who developed chronic GVHD after allogeneic HSCT, compared with the patients who did not develop this complication [175]. However, this difference was not seen in serum samples from a third patient cohort in the same study.
5.3.		Butyrate and irritable bowel syndrome (IBS)
A recent meta-analysis found that fecal butyrate levels are increased in one subset of IBS relative to healthy controls and decreased in a different subset [176]. Interestingly, a third subset of IBS patients had increased Firmicutes, but no difference in clinical phenotype. Importantly, restrictive diets correlated with reduced butyrate in IBS patients.
Interventions that increase butyrate have shown mixed effects in IBS patients. A drug that alters the microbiome to increase the relative abundance of butyrate-producing bacteria (geraniol) improved IBS symptoms [177]. Butyrate capsules reduced the frequency of one clinical symptom (pain) in IBS patients but did not alter severity or frequency of the other symptoms examined [178]. Volunteers who self-administered a butyrate enema immediately before a rectal barostat procedure reported lower scores for pain, discomfort, and urgency than volunteers receiving a saline enema [20]. Interestingly, a low-FODMAP diet that reduced butyrate producers and SCFA levels led to improved IBS symptoms relative to baseline levels [179]. As discussed above, there are links between butyrate, serotonin, and visceral perception [57]. This could be a mechanism underlying the observed effects of butyrate on visceral sensitivity.
5.4.		Butyrate and colorectal cancer
Experimental models support a protective role of butyrate in colon cancer. Butyrate inhibits the growth of stem/progenitor cells which are implicated in carcinogenesis [180]. Butyrate also has a well-known inhibitory effect on the proliferation of colon carcinoma cell lines in vitro, inducing cell cycle arrest through multiple molecular pathways [181]. Butyrate’s HDAC inhibitor activity leads to transcription of genes such as p21/Cip1, causing cell cycle arrest in the G1 phase [182]. Butyrate also reduces carcinogen-induced DNA damage and increases apoptosis through a variety of signaling mechanisms [181].
In clinical studies, the evidence is more variable. Although many studies support the use of dietary fiber to reduce the risk of colorectal cancer (CRC), butyrate’s potential link is unclear. Butyrate levels were higher in one study of CRC patients [183], and a systematic review found that SCFA levels are higher in CRC patients and individuals at higher risk of CRC [184]. However, an earlier systematic review showed that colorectal carcinogenesis is associated with decreased butyrate and microbial dysbiosis, including decreased butyrate producers (Bifidobacterium, Lactobacillus, Ruminococcus, Faecalibacterium spp., Roseburia, and Treponema) in CRC [185]. In a small study, supplementation with butyrylated resistant starch increased fecal butyrate and prevented increases in oncogenic miRNAs (and associated cell proliferation) induced by a red meat diet [186]. In a follow-up study, butyrate reduced DNA adducts and cell proliferation induced by a red meat diet, and increased butyrate-producing bacteria [187].
CRC is a key risk for UC patients [188] and a meta-analysis found that routine 5-ASA use reduced the risk of CRC in these patients [189]. Interestingly, 5-ASA has been suggested to exert an anti-CRC effect at least in part by activating PPARg [190]. As discussed above, butyrate also activates PPARg activity with concomitant effects on the microbiome. As such, it is possible that butyrate and 5-ASA could have synergistic impacts on CRC prevention in susceptible populations by driving host and microbial homeostasis.
Table 2
Currently recruiting and upcoming clinical trials examining the effects of butyrate on the gut microbiome or intestinal diseases.
Name of trial	Type	Identifier/Status	Condition	Intervention	Location	
OptiMized REsistaNt Starch in Inflammatory
Bowel Disease: The MEND Trial	DB-RCT	NCT04520594/
Recruiting	IBD	Personalized resistant starch	Ottawa, Canada	
Pilot and Feasibility Study of 2'-FL as a Dietary Supplement in IBD Patients Receiving Stable	RCT	NCT03847467/
Recruiting	IBD	2'-Fucosyllactose	Hartford, USA
Cincinnati, USA	
Maintenance Anti-TNF Therapy (PRIME)		
Resistant Starch in Pediatric Inflammatory
Bowel Disease (Crohn's Disease or Ulcerative	DB-RCT	NCT04522271/
Recruiting	IBD	Personalized resistant starch	Ottawa, Canada	
Colitis)		
Analysis of Microbiota Changes Induced by Microencapsulated Sodium Butyrate in	DB-RCT	NCT04879914/
Enrolling by invitation	IBD	Butyrate capsules	Padua, Italy	
Patients With Inflammatory Bowel Disease (IBDMicro)		
A Dietary Intervention Study on the
Microbiome in Crohn's Disease Patients	Open Label	NCT04065048/
Recruiting	IBD (CD)	Soy based diet	Cleveland, USA	
Use of a Novel Diet (UC DIET) for Treatment of Mild to Moderate Active Pediatric Ulcerative Colitis	Open Label	NCT02345733/
Recruiting	IBD (UC)	Ulcerative Colitis diet	Philadelphia, USA
Halifax, Canada
Holon, Israel	
Hydroxocobalamin Approach to Reduction of
Calprotectin With Butyrate for Ulcerative	DB-RCT	NCT04259060/Not yet recruiting	IBD (UC)	Hydroxocobalamin with butyrate	Chestnut Hill, USA	
Colitis Research		
Butyrate in Children With IBS: Double Blind
Placebo Controlled Randomized Clinical Trial	DB-RCT	NCT04566679/
Recruiting	IBS	Dibuzin supplement	Bari, Italy	
(BUZIR)		
The Efficacy of Sodium Butyrate and Probiotics in Patients With Irritable Bowel Syndrome (IBS-Plus)	DB-RCT	NCT05013060/
Recruiting	IBS	Probiotics and microcapsulated sodium butyrate	Ło^dz^, Poland	
Modulation of Intestinal Barrier Function and Inflammation Via Butyrate-promoting	DB-RCT	NCT05058131/
Recruiting	Microscopic colitis	Dietary fibre	O€ rebro, Sweden	
Dietary Fibre		
Effect of Butyrate on Inflammation and Albuminuria in Patients With Albuminuria,	DB-RCT	NCT04073927/
Recruiting	Type 1 Diabetes & Albuminuria	Sodium butyrate supplement	Gentofte, Denmark
Helsinki, Finland	
Type 1 Diabetes and Intestinal Inflammation		
Effects of Butyrate Enemas on Postoperative
Intestinal Mobility Disorders in	Open Label	NCT03660176/Not yet recruting	Hirschsprung's
Disease	Butyrate enemas	Marseille, France	
Hirschsprung's Disease		
Gut Microbiota and Behcet's Syndrome: a
Dietary Intervention Trial (MAMBA Study)	Open Label with crossover	NCT03962335/
Recruiting	Behcet Syndrome	Vegetarian diet & Mediterranean diet with butyrate	Florence, Italy	
The Effects of SCFA Supplementation in Subjects Receiving Abdominopelvic RT: A	DB-RCT	NCT04700527/Not yet recruiting	GI related Radiation Toxicity	SCFA supplements	Chapel Hill, USA	
Randomized Controlled Study		
Fiber to Reduce Colon Cancer in Alaska Native People	DB-RCT	NCT03028831/
Recruiting	Colon Cancer	Resistant starch	Anchorage, USA
Pittsburgh, USA	
Evaluation of a Supplement of Agave Tequilana
Weber Azul vs Psyllium Plantago in Patients	DB-RCT	NCT04716868/
Recruiting	Functional Constipation	Agave supplement at multiple levels	M^exico, Mexico	
With Functional Constipation (SATWAVPP)		
Effect of Prebiotics on the Gut Microbiome Profile and Beta Cell Function	Open Label	NCT04114357/
Recruiting	Type 1 Diabetes	Acetylated & butyrylated high amylose maize starch	Indianapolis, USA	
Effect of Mediterranean Diet During Pregnancy on Gut Microbiota and on the Epigenetics	Open Label	NCT03337802/
Recruiting	Food Allergy	Mediterranean diet	Naples, Italy	
Modification of Gut Microbiota in the Treatment of Insulin Resistance: a Personalized	Open Label	NCT03710850/
Recruiting	Insulin Resistance	Inulin	Prague, Czechia	
Approach		
Dietary Manipulation of the Microbiome- metabolomic Axis for Mitigating GVHD in	RCT	NCT02763033/
Recruiting	HSCT	Resistant starch (Bob's
Red Mill®)	Ann Arbor, USA	
Allo HCT Patients		
Oral Supplementation of 2'-Fucosyllactose in Allogeneic Bone Marrow Transplant	DB-RCT	NCT04263597/
Recruiting	HSCT	2'-fucosyllactose	Cincinnati, USA	
Recipients		
Effects of Resistant Starch Diet on the Gut Microbiome in Chronic Kidney Disease	Open Label	NCT03356990/
Recruiting	Chronic Kidney
Disease	Resistant Starch	Little Rock, USA	
Impact of Intestinal Microbiota on Uremic
Toxins Productions (GUTCOL)	Open Label	NCT04768309/Not yet recruiting	Chronic Kidney
Disease	Probiotics	Pierre-B^enite, France	
DASH INtervention to INvestigate the Gut (DINING)	DB-RCT	NCT04538482/Not yet recruiting	Cancer	DASH Diet	Birmingham, USA	
Luxembourgish Fiber Cohort	Open Label	NCT04352231/Not yet
recruiting	Healthy volunteers	High diber Diet	Esch-sur-Alzette, Luxembourg
Strassen, Luxembourg	
Dietary Fiber Effects on the Microbiome and Satiety (FEMS)	RCT	NCT04611217/
Recruiting	Healthy volunteers	High-fiber diet	Columbia, USA	

6.		Future directions
Research continues to unravel the mechanisms underlying butyrate’s health benefits and show its clinical benefits across various populations. However, we currently infer butyrate’s effect on host physiology from fecal concentrations, which assumes a direct correlation with luminal concentrations which is unlikely to hold across the gastrointestinal tract [191]. In addition, direct butyrate supplementation (via capsules or enemas) differs from endogenous butyrate production that is not generated in discrete, punctuated boluses, but is rather coupled to fermentation, which is diurnal and rhythmic [192,193]. Sustained-release formulations of butyrate await clinical evaluation [194]. Alternatively, boosting butyrate production via slowly fermentable prebiotics like fiber or resistant starch might promote a more sustained supply that better matches physiological patterns, and is more amenable to clinical evaluation owing to favourable safety profiles [195]. It should be noted that resistant starch-driven butyrate production is profoundly variable from person-to-person [196], and thus efforts to restore or boost butyrate production via microbial fermentation will likely require tailoring the selection of resistant starches to each individual’s microbiome. Indeed, we have initiated two clinical trials to evaluate personalized resistant starch in boosting butyrate producing bacteria as an adjuvant therapy in pediatric IBD (Table 2). There is also increasing interest in combining butyrate-boosting supplements with other co-therapies. In particular, there is a theory in IBD referred to as the “double hit” hypothesis where it is both the production and metabolism of butyrate that are perturbed in these patients. Thus, butyrate-based therapies in IBD might benefit if coupled to adjuvant therapies that also activate butyrate oxidation (e.g. PPARg activation via 5-ASA) as has been previously reported in the IBD subtype UC [197]. Finally, it remains to be shown whether the health benefits of these therapies are derived solely from the presence of butyrate or whether other SCFAs or potentially uncharacterized fermentation by-products play roles as well.
7.		Conclusion
Butyrate helps maintain intestinal homeostasis through a multifaceted approach that includes significant changes in gene expression as well as rapid signaling pathways. It supports normal intestinal function by acting as an energy source for aerobic metabolism and increasing proliferation of normal colonocytes. Butyrate also promotes blood flow and gut motility in the colon, which are important for digestion. Furthermore, butyrate supports wound healing processes that are needed to repair epithelial injury.
Butyrate also helps protect from disease and infection through several complementary processes. It strengthens the gut barrier by enhancing tight junctions, the mucus layer, and the production of antimicrobial peptides. Its anti-inflammatory actions protect from inflammatory diseases and help mediate an appropriate response to pathogens.
Although clinical studies of butyrate have shown far more mixed results than experimental models, there is some evidence to support a protective role of butyrate in several intestinal diseases, particularly IBD and GVHD. Previous studies have been limited in scope and number, limiting the conclusions that can be drawn. However, there are some promising upcoming clinical studies (Table 2) that will help clarify the role of butyrate in intestinal disease and may uncover new ways to harness its effects to treat these diseases.
Funding sources
Salary support was provided by Genome Canada and the Ontario Genomics Institute (OGI-149), CIHR, the Weston Foundation and the Government of Ontario. PD is supported by a Canada Scholarship (Crohn’s and Colitis Canada & AbbVie), a Vanier Canada Graduate Scholarship (Canadian Institutes of Health Research), and an Ontario Graduate Scholarship. DM is supported by a Clinical Research Chair in Pediatric IBD from the University of Ottawa.
Author contributions
KH: Conceptualization, Writing - Original Draft, Visualization; FE: Writing - Original Draft, Visualization; PD: Writing - Original Draft, Visualization; JM: Writing - Original Draft, Visualization; JB: Writing - Original Draft; DF: Writing - Review & Editing, Supervision; DM: Writing - Review & Editing, Supervision; AS: Conceptualization, Writing - Original Draft, Supervision.
Conflict of Interest
AS, DM and DF cofounded MedBiome, a clinical microbiomics company. All other authors have no competing interests to declare.
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