The effect of oral butyrate on colonic short-chain fatty acid transporters and receptors depends on microbial status

Abstract

Butyrate, a metabolite produced by gut bacteria, has demonstrated beneficial effects in the colon and has been used to treat inflammatory bowel diseases. However, the mechanism by which butyrate operates remains incompletely understood. Given that oral butyrate can exert either a direct impact on the gut mucosa or an indirect influence through its interaction with the gut microbiome, this study aimed to investigate three key aspects: (1) whether oral intake of butyrate modulates the expression of genes encoding short-chain fatty acid (SCFA) transporters (Slc16a1, Slc16a3, Slc16a4, Slc5a8, Abcg2) and receptors (Hcar2, Ffar2, Ffar3, Olfr78, Olfr558) in the colon, (2) the potential involvement of gut microbiota in this modulation, and (3) the impact of oral butyrate on the expression of colonic SCFA transporters and receptors during colonic inflammation. Specific pathogen-free (SPF) and germ-free (GF) mice with or without DSS-induced inflammation were provided with either water or a 0.5% sodium butyrate solution. The findings revealed that butyrate decreased the expression of Slc16a1, Slc5a8, and Hcar2 in SPF but not in GF mice, while it increased the expression of Slc16a3 in GF and the efflux pump Abcg2 in both GF and SPF animals. Moreover, the presence of microbiota was associated with the upregulation of Hcar2, Ffar2, and Ffar3 expression and the downregulation of Slc16a3. Interestingly, the challenge with DSS did not alter the expression of SCFA transporters, regardless of the presence or absence of microbiota, and the effect of butyrate on the transporter expression in SPF mice remained unaffected by DSS. The expression of SCFA receptors was only partially affected by DSS. Our results indicate that (1) consuming a relatively low concentration of butyrate can influence the expression of colonic SCFA transporters and receptors, with their expression being modulated by the gut microbiota, (2) the effect of butyrate does not appear to result from direct substrate-induced regulation but rather reflects an indirect effect associated with the gut microbiome, and (3) acute colon inflammation does not lead to significant changes in the transcriptional regulation of most SCFA transporters and receptors, with the effect of butyrate in the inflamed colon remaining intact.

Keywords: butyrate; butyrate receptors; butyrate transporters; colitis; dextran sulfate; germ-free (GF); microbiota; short chain fatty acid (SCFA).

FIGURE 1

Experimental timeline. DSS, dextran sodium sulfate; BT, sodium butyrate; for more details see the text. The number of animals in each experimental group is given in parentheses (SPF/GF mice).

FIGURE 2

Time course of the effect of butyrate on the expression of colonic SCFA transporters and receptors in specific pathogen-free (SPF) and germ-free (GF) mice. The mice drank either water (CTRL group) or 0.5% sodium butyrate solution for 7 (1BT group) or 14 days (2BT group). The results are presented as the mean ± SEM (n = 4–10 per group). The data for SPF (lowercase letters) and GF (uppercase letters) were analyzed separately by one-way ANOVA followed by Tukey’s post hoc test. Values with same letters are not statistically different.

FIGURE 3

Effects of the microbiota and butyrate on the expression of genes encoding colonic SCFA transporters and receptors, gut hormone PYY, and mucin 2 in specific pathogen-free (SPF) and germ-free (GF) mice. CTRL, mice receiving standard diet and water ad libitum; 2BT, mice receiving standard diet and sodium butyrate solution (0.5%) replacing drinking water in the last 14 days before sacrifice. The results are presented as the mean ± SEM (n = 4–10 per group) and were analyzed by a two-way ANOVA followed by Tukey’s post hoc test. The results of two-way ANOVA are given in tables and the results of post hoc tests in the graphs: *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001; ns, not significant.

FIGURE 4

Dextran sodium sulfate (DSS)-induced colitis and the effect of oral butyrate (BT) on crypt length and expression of proinflammatory cytokines. (A) Hematoxylin and eosin staining of colon tissue from specific pathogen-free (SPF) and germ-free (GF) mice treated with DSS. The histopathological changes in the colonic mucosa following DSS treatment were assessed through histological scoring at the end of the experiment. These changes are illustrated using representative histological sections. (B) The length of colonic crypts and (C) mRNA expression of interleukin 1β (IL-1β) and tumor necrosis factor α (Tnfα) in SPF and GF mice treated with DSS and BT. CTRL, control, untreated mice; DSS, mice that drank 2.5% DSS for 7 days; 1BT+1BT/DSS, mice that drank 0.5% BT solution for 1 week and then the mixture of 0.5% BT and 2.5% DSS for the second week; 2BT + DSS, mice pretreated with 0.5% BT for 2 weeks before drinking 2.5% DSS the following week. The results are presented as the mean ± SEM (n = 6 per group for the measurement of crypt lengths; n = 4–10 per group for the analysis of proinflammatory cytokine expression) and were analyzed by one-way ANOVA separately for SPF and GF mice. Different from the CTRL group: *p < 0.05, **p < 0.01, ****p < 0.0001; different from the DSS group: ^x P < 0.05, ^xxxx P < 0.0001; different from the 1BT+1BT/DSS group: ⁺ p < 0.05, ⁺⁺⁺ p < 0.001. Differences between SPF and GF mice in identical experimental groups were compared using Student’s t-test: ^$ p < 0.05, ^$$ p < 0.01, ^$$$ p < 0.001, ^$$$$ p < 0.0001.

FIGURE 5

Effect of oral butyrate administration on colonic SCFA transporters and receptors, gut hormone PYY, and mucin 2 in specific pathogen-free (SPF) and germ-free (GF) mice treated with DSS. CTRL, control, untreated mice; DSS, mice that drank 2.5% DSS for 7 days; 1BT+1BT/DSS, mice that drank 0.5% BT solution for 1 week and then the mixture of 0.5% BT and 2.5% DSS for the second week; 2BT + DSS, mice pretreated with 0.5% BT for 2 weeks before drinking 2.5% DSS the following week. The results are presented as the mean ± SEM (n = 4–10 per group) and were analyzed by one-way ANOVA separately for SPF and GF mice. Different from the CTRL group: *p < 0.05, **p < 0.01, ***p < 0.001; different from the DSS group: ^x P < 0.05, ^xx P < 0.05, ^xxx P < 0.001, ^xxxx P < 0.0001; different from the 1BT+1BT/DSS group: ⁺ p < 0.05, ⁺⁺⁺⁺ p < 0.0001. Differences between SPF and GF mice in identical experimental groups were compared using Student’s t-test: ^$ p < 0.05, ^$$ p < 0.01, ^$$$ p < 0.001.