Inflammatory bowel disease (IBD) including its major clinical forms ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic, recurrent disease characterized by intestinal inflammation whose etiology is still poorly understood. Nevertheless, the pathogenesis of IBD is multifactorial and results from genetic predisposition, immunological status, and environmental factors, including intestinal microbiota disbalance (Graham and Xavier, 2020). Gut microbial dysbiosis was detected both in patients with UC and CD and animal models of IBD (Ni et al., 2017; Facchin et al., 2020) and was associated with a reduced number of short-chain fatty acid (SCFA)-producing bacteria and reduced butyrate concentration (Machiels et al., 2014; Laserna-Mendieta et al., 2018).

SCFAs including butyrate, propionate, and acetate, are produced by the fermentation of nondigestible carbohydrates (dietary fibers) by the gut microbiota and play an important role in maintaining intestinal barrier functions and immune homeostasis. SCFAs, particularly butyrate, have been shown to regulate the proliferation and differentiation of intestinal epithelial cells and their expression of mucins, antimicrobial peptides, and tight junctions proteins; furthermore, butyrate exerts anti-inflammatory effects in immune cells (Gonçalves et al., 2018; Recharla et al., 2023). SCFAs interact with target epithelial or immune cells either via cell-surface G-protein-coupled receptors or by entering cells and controlling gene expression by the direct inhibition of histone deacetylases (Dalile et al., 2019). Previous studies have established that butyrate and other SCFAs can enter enterocytes via passive nonionic diffusion or carrier-mediated transport, facilitating the transport of SCFAs derived from gut microbiota. This transport is mediated by the electroneutral H⁺-dependent monocarboxylate transporters MCT1, MCT4, and MCT5 and the electrogenic, Na⁺-dependent monocarboxylate transporter SMCT1, which are encoded by the Slc16a1, Slc16a3, Slc16a4, and Slc5a8 genes, respectively (Gill et al., 2005; Cresci et al., 2010; Al-Mosauwi et al., 2016). Moreover, the efflux of butyrate has been demonstrated through the ABCG2 efflux pump, expressed in the apical membrane of intestinal epithelial cells (Gonçalves et al., 2011). In addition to their interactions with transporters, SCFAs act as ligands capable of activating cell signaling pathways in enterocytes via at least five distinct membrane receptors: GPR109A/HCAR2 (hydrocarboxylic acid receptor 2), FFAR2 and FFAR3 (free fatty acid receptor 2 and 3), OLFR78 (olfactory receptor 78), and OLFR558/OR51E1 (olfactory receptor 558) (Priori et al., 2015; Priyadarshini et al., 2018; Halperin Kuhns et al., 2019; Nishida et al., 2021).

The findings that microbiota-derived butyrate plays an important role in maintaining intestinal barrier integrity, gut homeostasis, and reducing gut inflammation (Gonçalves et al., 2018) led to many studies investigating the therapeutic implications of butyrate treatment for IBD (Recharla et al., 2023). In animal models of colitis (DSS-, TNBS- or IL10^−/−-colitis), oral butyrate supplementation attenuated the disease activity index, inflammation and mucosal lesions (Vieira et al., 2012; Ji et al., 2016; Lee et al., 2017; Chen et al., 2018), although some studies failed to show benefits following butyrate treatment (Lee et al., 2022). Moreover, butyrate failed to protect against TNBS-colitis in Hcar2 ^−/− mice (Chen et al., 2018), and Ffar2 ^−/− and Ffar3 ^−/− mice were found to be more susceptible to TNBS-colitis (Kim et al., 2013). Similarly, the beneficial effect of dietary fibers in DSS colitis was diminished in Hcar2 ^−/− , Ffar2 ^−/− , and Slc5a8 ^−/− mice (Gurav et al., 2015; Macia et al., 2015). However, the mechanisms through which oral butyrate operates remain incompletely understood, particularly whether it can directly affect the gut mucosa or if its effects are mediated indirectly through its influence on the gut microbial community (Dou et al., 2020; Facchin et al., 2020; Lee et al., 2022). Therefore, this study aimed to compare the effects of butyrate on SCFA receptors and transporters in the presence and absence of gut microbiota and intestinal inflammation.

The mechanism by which SCFAs, particularly butyrate, promote immunity and improve IBD treatment efficacy is not completely understood, even though it is known that SCFAs act at the cell surface as endogenous ligands for some G protein-coupled receptors and intracellularly as inhibitors of histone deacetylases (Gonçalves et al., 2018; Dalile et al., 2019). Previous studies have shown that the administration of sodium butyrate in drinking water ameliorates inflammation and epithelial barrier dysfunction (Vieira et al., 2012; Ji et al., 2016; Lee et al., 2017; Chen et al., 2018; Jourova et al., 2022); nevertheless, orally administered butyrate is thought to be absorbed and utilized before reaching the colon (Daniel et al., 1989; Wang et al., 2023). Here, we demonstrate that a relatively low concentration of sodium butyrate in a drinking solution modulated the expression of the SCFA carriers Slc16a1, Slc16a3, Slc16a4, and Slc5a8 and the efflux pump Abcg2. The observed effect of butyrate on the expression of SCFA transporters does not seem to reflect direct substrate-induced regulation via butyrate. First, the effect of oral butyrate differed between SPF and GF mice. Second, we found downregulated expression of SCFA solute carriers in SPF mice treated with butyrate, but butyrate stimulated the expression and function of Slc16a1 (MCT1) and Slc16a3 (MCT4) in colonic epithelial cell lines (Borthakur et al., 2008; Ziegler et al., 2016)) or in the intestinal epithelium (Dengler et al., 2015). Third, oral administration of butyrate significantly upregulated Slc16a3 expression but only in GF and not SPF mice.

In this report, we also showed that not only the expression of SCFA transporters but also that of SCFA receptors is modulated by the gut microbiota and by oral intake of butyrate. We found that the expression of Ffar2, which is expressed both in enteroendocrine cells (EEC) and colonocytes, and Ffar3, which is expressed in EEC and enteric neurons but not colonocytes (Priyadarshini et al., 2018), strongly depends on gut microbiota but not on oral butyrate treatment. A similar effect of the microbiota on the expression of Ffar2 was observed in the mouse ileum (Yajima et al., 2016). In contrast to Ffar2 and Ffar3, the expression of Hcar2, a colonic SCFA receptor (Priyadarshini et al., 2018), depended not only on gut microbiota, as was already shown earlier by Cresci et al. (Cresci et al., 2010), but also on oral butyrate. Similar to the colonic Slc16a1 and Slc5a8 transporters, oral butyrate dampened Hcar2 expression in SPF but not GF mice. These data indicate a relationship between the regulation of Hcar2, the principal butyrate receptor in the colon, and the colonic butyrate transporters Slc16a1 and Slc5a8. A similar link between the expression of Hcar2 and Slc5a8 was shown by Cresci et al. (Cresci et al., 2010). The importance of the microbiota for the effect of oral butyrate on the gut is also supported by the results of Pyy and Muc2 expression. In both cases, the effect of butyrate was evident only in SPF but not GF animals, with downregulation in the case of Pyy expression and upregulation in the case of Muc2 expression; this effect is similar to the effect of rectal butyrate enemas on Muc2 expression in conventional mice (Gaudier et al., 2009). The lack of the inhibitory effect of butyrate on Pyy expression observed in our in vivo experiments with GF mice aligns with findings from experiments conducted using sterile mouse intestinal epithelial cell culture (Larraufie et al., 2018).

Indirect transcriptional regulation of colonic SCFA transporters and receptors may depend on the interaction of oral butyrate with the gut microbiota or with upstream parts of the gastrointestinal tract. In the case of the effect of oral butyrate in SPF mice, we cannot exclude the possibility that oral butyrate modulates the gut microbiota and microbiota-secreted soluble factors, which might control the expression of SCFA transporters and receptors. There is strong evidence that oral sodium butyrate remarkably alters the gut microbiota (Dou et al., 2020; Lee et al., 2022) and that Lactobacillus plantarum-derived soluble factors or Lactobacillus delbrueckii consumption upregulate Slc5a8 and Slc16a1 expression (Hou et al., 2022; Kim et al., 2022). Furthermore, the SMCT1, MCT1, and MCT4 transporters and the FFAR2 and FFAR3 receptors have been found in the duodenum (Akiba et al., 2015; Kaji et al., 2015), and butyrate has been shown to activate vagal afferents and release gut hormones that might modulate intestinal transport (Dalile et al., 2019).

Since butyrate has ameliorative effects in the treatment of colitis (Recharla et al., 2023), and we have shown that butyrate together with the microbiome affects the expression of transporters and receptors in the colon, we further investigated the interaction between oral treatment of butyrate and the presence/absence of microbiome in a model of acute colitis. Even though downregulation of Slc16a1 and MCT1 protein expression and upregulation of Slc16a3 and MCT4 expression were detected in biopsies of patients with IBD and in the colon of rats or mice with DSS-induced colitis (Thibault et al., 2007; Erdmann et al., 2019; Zhang et al., 2019), our results did not show any effect of DSS on SCFA transporters in SPF mice. This discrepancy may result from differences in the colitis models used including strain, time of colitis, and degree of inflammation. In this regard, the expression levels of SCFA transporters depend on the inflammatory state of the colonic mucosa (Thibault et al., 2007; Zhang et al., 2019). Moreover, DSS treatment significantly alters the structure of the intestinal microbiota (Lin et al., 2023), whose diversity and composition might differ among various locations/research groups.

In addition, we analyzed the expression of SCFA receptors in mice with colitis and demonstrated that DSS treatment leads to upregulation of Olfr78 expression regardless of the presence or absence of gut microbiota and significant downregulation of Ffar2 expression in SPF mice and upregulation in GF mice. In addition, Ffar3 expression in SPF mice was also decreased, but the decrease was not significant. In contrast, Hcar2 and Olfr558 expression was not changed in either SPF or GF mice. In this regard, acute DSS colitis in conventional C57BL/6 mice decreased the expression of Ffar2, Ffar3, and Hcar2 (Lin et al., 2023; Nan et al., 2023), although this result was not confirmed by others (Kotlo et al., 2020; Han et al., 2021). These findings indicate that Olfr78, Ffar2, and Ffar3 may play a more important role in our model of colitis/inflammation than other SCFA receptors and that the response of Ffar receptors to inflammation depends on the gut microbiota.

In conclusion, our study demonstrates that the intestinal microbiota actively modulates the response of genes encoding colonic SCFA transporters and receptors, mucin, and the gut hormone PYY to oral butyrate administration. It is noteworthy that the responses to butyrate did not differ significantly between the colons of healthy subjects and those with experimentally induced murine colitis. Our observations indicate alterations in the expression of certain SCFA receptors and the hormone PYY in colitis, while the expression of SCFA transporters remained unchanged. It is important to note that these changes occurred regardless of the presence or absence of gut microbiota. Overall, our results suggest a promising avenue for further research in this area. These findings highlight the potential indirect influence of butyrate supplementation on its anti-colitic effects.