short-chain fatty acids (SCFAs), acetate, propionate, and butyrate are produced by bacterial fermentation of dietary fiber in the colonic lumen. In the normal human large intestine, SCFA concentrations range from 70 to 100 mM; these SCFAs account for ∼80% of the anions present in the lumen (5). The relative ratio of acetate:propionate:butyrate is considered to remain stable at 60:25:15 (11). Butyrate has received the most attention due to its multiple beneficial roles in health and disease states. Butyrate is considered the preferred energy source for colonocytes and helps maintain the epithelial integrity (5, 17). Numerous beneficial roles of butyrate within and outside the intestine have been reported. In the intestine, butyrate has multiple effects (5), which include 1) increases in NaCl absorption via activating Na⁺-H⁺ and Cl⁻-HCO₃⁻ exchangers (6, 9); 2) influences on cell proliferation and differentiation (8, 17); 3) plays an anti-inflammatory role via modulating the release of prostaglandin E2, cytokines, and chemokines from immune cells (10); 4) alters gut barrier function by inducing mucin synthesis and antimicrobial peptide production, and by decreasing intestinal tight junction permeability via AMP-activated protein kinase (5); 5) affects colonic motility by modulating acetylation in the myenteric plexus and via release of 5-HT (13, 27); and 6) helps prevent and inhibit colonic carcinogenesis. Extra-intestinal beneficial effects of butyrate include 1) increasing fetal hemoglobin production; 2) lowering serum cholesterol levels; 3) stimulating neurogenesis in brain after ischemic injury; and 4) providing positive effects in the treatment of obesity, insulin resistance, cystic fibrosis, urea cycle enzyme deficiency, and sickle cell disease (5). The role of butyrate in the inhibition and prevention of colon cancer is likely one of its most important beneficial effects.

Butyrate and colon cancer.

Butyrate has been shown to act as both preventive and inhibitory in carcinogenesis of the colon. The chemopreventive effect is mediated by upregulation of detoxifying enzymes for xenobiotics and oxidants, an effect that derives from rather complex actions of butyrate on cell proliferation and differentiation, termed the “butyrate paradox” (5, 8). This term derives from the ability of butyrate to inhibit cell proliferation and induce apoptosis in colon cancer cell lines, whereas under normal conditions butyrate induces cell proliferation in colonic crypts (1). The mechanisms of the effects of butyrate on colon cancer mainly include its absorption into colonocytes followed by its multiple effects on cell proliferation/differentiation via its inhibition of histone deacetylases (HDACs) (Fig. 1). In human colonic cell lines, butyrate increases p21 gene expression, thereby inducing cell cycle arrest via inhibition of HDACs (7). However, a recent report showed that this effect of butyrate on p21 gene expression occurs by two mechanisms: HDAC inhibition and decreased expression of the miR-106b gene family (19). Additional effects of butyrate on cell apoptosis involve effects on Bcl2 family proteins, e.g., upregulation of (pro-apoptotic) BAK and downregulation of (anti-apoptotic) BclxL (24, 25). Another anticancer effect of butyrate is its effects on canonical Wnt signaling pathway, which is constitutively activated in most colonic tumors (5). Butyrate may also induce autophagy in colonic epithelial cells (28). Indirect effects of butyrate, e.g., upregulating MDR1 expression or conversion of estrone to 17β-estradiol may also underlie the decreased incidence of colon cancer (2, 22). Two very recent studies, however, showed that the anticancer effects of butyrate involved its interactions with cell surface G-protein-coupled receptors: GPR109a and GPR43 (29, 30), effects that appear to be independent of its inhibition of HDACs (30). Thus the anticarcinogenic effects of butyrate are rather complex and could involve participation of SCFA receptors as well as the uptake of butyrate into the colonocytes and subsequent effects on HDACs (Fig. 1).

Mechanisms of butyrate uptake and efflux from the colonocytes.

It was assumed for a long time that non-ionic diffusion of protonated SCFAs was the major mechanism of SCFA absorption in the intestine (9). However, other studies showed involvement of carrier-mediated processes, e.g., SCFA⁻/HCO₃⁻ or a SCFA⁻/Cl⁻ exchangers (9, 12). It is now accepted that monocarboxylate transporter 1 (MCT1) plays a major role in carrier-mediated SCFA transport in colonocytes (3, 12) (Fig. 1). However, its localization in polarized colonocytes has been controversial. Although many studies indicate it localizes to the apical membrane, its basolateral localization has also been reported (15, 20). SLC5A8 (SMCT1), a sodium-dependent monocarboxylate transporter also localizes to the apical membranes of colonocytes (4, 14). However, its functionality in the human colon has not yet been demonstrated.

Very little information is available with respect to the efflux of the absorbed SCFAs from the colonocytes, although basolateral MCTs (e.g., MCT1, MCT4, or MCT5) may play a role in this process (Fig. 1) (15). In other cell types (e.g., hepatocytes), butyrate efflux has been linked to the uptake of extracellular sulfate-conjugated steroids via organic anion transporter-7 (OATP-7) (26). A suppression in the expression of both MCT1 and SMCT1 appears to be an adaptive mechanism of colon cancer cells to avert the anticancer effect of butyrate (14, 21). Interestingly, Glut1 expression is upregulated in colon cancer, indicating that, during malignancy, colonic tissue switches from utilizing butyrate to glucose for energy needs (21). It is important to note that colonic cancer cells and normal cells also express other mechanisms that may lower butyrate levels in the cells: multi-drug resistance (MDR)-related transporters [e.g., MDR1, MRPs or BCRP (members of the ABC superfamily of transporters)] that can reduce the intracellular availability of anticancer agents and other xenobiotics. Interestingly, butyrate can upregulate the expression of MDR1 expression (22). However, whether any of these transporters play a role in transporting butyrate has not been investigated.

A novel role of BCRP in butyrate efflux from colonocytes.

In this issue of the American Journal of Physiology–Cell Physiology, Gonçalves et al. (16) demonstrate that butyrate is a substrate for BCRP but not for MDR1 or MRPs. The authors use pharmacological approaches with known inhibitors of these proteins to study the uptake or efflux of butyrate in two intestinal cell lines and one breast cancer cell line in which butyrate serves as a substrate of BCRP and can be pumped out of the cell via this protein. The authors utilized a transformed intestinal cell line (Caco2) and a non-transformed rat intestinal cell line (IEC-6) to examine the potential role of these transporters in both normal and transformed cells. Interestingly, authors showed that IEC-6 cells, but not Caco2 cells, have BCRP-mediated efflux of butyrate. The absence of an efflux process for butyrate through BCRP was shown to result from the absence of BCRP expression in Caco2 cells. However, in butyrate-treated (48 h) Caco2 cells, BCRP expression was increased and resulted in the transport of butyrate. The authors also demonstrated that this role of BCRP in butyrate efflux was not cell line-specific: MDA-MB-231, a breast cancer line known to express high levels of BCRP, also showed a role for BCRP in efflux of butyrate. The functionality of BCRP in butyrate transport was further confirmed by examining the effects of butyrate on cell proliferation in IEC-6 cells: butyrate combined with a BCRP inhibitor potentiated the inhibitory effect of butyrate on cell proliferation. The authors also showed a role for butyrate in inhibiting the efflux of [³H]folate, a known substrate for BCRP, in IEC-6 cells. Gonçalves et al. (16) speculate that, since butyrate is a substrate for BCRP, it may compete with chemotherapeutic drugs (e.g., 5-fluorouracil, methotrexate, etc.) for exit from the cells and thus might increase their intracellular concentration and enhance their therapeutic effect. These studies, therefore, provide a new facet to consider in understanding the mechanisms that underlie beneficial effects of SCFAs as well as in designing butyrate-based therapeutic modalities.

This exciting report also raises many questions. For example: 1) What is the physiological significance of BCRP in butyrate efflux in view of multiple beneficial effects of butyrate in healthy subjects? This argument should also consider the fact that BCRP expression is highest in the human duodenum and lowest in the colon (18); however, butyrate is produced mainly in the colon. 2) Does BCRP function as an efflux protein for other SCFAs (e.g., acetate or propionate), which also have beneficial effects? 3) BCRP expression is suppressed in cancer cells (23), consistent with the low expression seen in Caco-2 cells; however, the present study shows upregulation of BCRP in Caco2 cells by butyrate. Thus it is not clear whether the findings of Gonçalves et al. (16) will occur under in vivo conditions where high concentrations of luminal butyrate exist. 4) Will BCRP-knockout (KO) mice be more susceptible to butyrate for its therapeutic potential for colon cancer or other diseases? 5) Which signaling pathways regulate BCRP function and its expression in the colon? 6) Since both the MCT1 and SMCT1 are downregulated in colon cancer, hence limiting the availability of butyrate inside the cells (14, 21), what role will butyrate play by itself or by enhancing the therapeutic potential of other chemotherapeutic drugs through inhibition of their efflux from the cells by BCRP, as discussed above? The answers to many of these questions in future studies utilizing BCRP KO mice as well as molecular approaches utilizing siRNA for BCRP or its overexpression should be important to further establish this novel role of BCRP in butyrate transport and its implications in health and disease.

GRANTS

R. K. Gill's work is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-74458, and P. K. Dudeja's laboratory is supported by the department of Veterans Affairs and the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54016, DK-81858, and P01 DK-067887.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).