A novel nutrient sensing mechanism underlies substrate-induced regulation of monocarboxylate transporter-1

Abstract

Monocarboxylate transporter isoform-1 (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFAs) in the colon. Butyrate, a major SCFA, serves as the primary energy source for the colonic mucosa, maintains epithelial integrity, and ameliorates intestinal inflammation. Previous studies have shown substrate (butyrate)-induced upregulation of MCT1 expression and function via transcriptional mechanisms. The present studies provide evidence that short-term MCT1 regulation by substrates could be mediated via a novel nutrient sensing mechanism. Short-term regulation of MCT1 by butyrate was examined in vitro in human intestinal C2BBe1 and rat intestinal IEC-6 cells and ex vivo in rat intestinal mucosa. Effects of pectin feeding on MCT1, in vivo, were determined in rat model. Butyrate treatment (30-120 min) of C2BBe1 cells increased MCT1 function {p-(chloromercuri) benzene sulfonate (PCMBS)-sensitive [(14)C]butyrate uptake} in a pertussis toxin-sensitive manner. The effects were associated with decreased intracellular cAMP levels, increased V(max) of butyrate uptake, and GPR109A-dependent increase in apical membrane MCT1 level. Nicotinic acid, an agonist for the SCFA receptor GPR109A, also increased MCT1 function and decreased intracellular cAMP. Pectin feeding increased apical membrane MCT1 levels and nicotinate-induced transepithelial butyrate flux in rat colon. Our data provide strong evidence for substrate-induced enhancement of MCT1 surface expression and function via a novel nutrient sensing mechanism involving GPR109A as a SCFA sensor.

Fig. 1.

Butyrate (But)-induced enhancement of monocarboxylate transporter-1 (MCT1) function is pertussis toxin (PTX)-sensitive and associated with decreased intracellular cAMP levels. {MCT1 function (pCMBS-sensitive [¹⁴C]butyrate uptake)} was calculated as nanomols butyrate per milligram protein per 5 min and results are expressed as % of control]. Time-course of butyrate (10 mM) effects on MCT1 function in C2BBe1 cells (14-day postplating) (n = 5, P < 0.05 vs. control) (A) and in IEC-6 cells (10-day postplating) (n = 3, *P < 0.05) (B). Time-course of butyrate (10 mM) effects on intracellular cAMP levels (n = 3, *P < 0.05) (C); forskolin (10 μM) and dibutyryl cAMP (50 μM) effects on MCT1 function (n = 3, *P < 0.001) (D); PTX (1 μg/ml)-sensitivity of butyrate (10 mM) induction of MCT1 function (n = 3, *P < 0.05) (E). pCMBS, p-chloromercuribenzene sulfonate.

Fig. 2.

Nicotinate receptor GPR109A as potential mediator of butyrate effects on MCT1 function. GPR109A protein expression in IEC-6 and C2BBe1 cells (75 μg protein in cell lysate was loaded, probed with anti-human GPR109A antibody) (A); time-course of 100 μM nicotinate effects on MCT1 function (B); and time-course of nicotinate effects on intracellular cAMP (n = 3, *P < 0.05) (C).

Fig. 3.

Butyrate increases cell surface MCT1 levels in C2BBe1 cells in a GPR109A-dependent manner: A: cell monolayers (14-day postplating) were treated with 10 mM butyrate for the indicated time periods, and apical membrane MCT1 levels were measured by cell surface biotinylation; top: the band intensities of apical vs. total MCT1 in different groups; bottom: densitometric analysis of band intensities (AU, arbitrary units of band intensity). B: cells were transfected with control-scrambled siRNA or GPR109A siRNA as described in materials and methods. Forty-eight hours after transfection, cells were either used to measure GPR109 levels by immunoblotting with anti-GPR109A antibody (B) or to measure surface MCT1 by cell surface biotinylation (C). C, bottom: densitometric analysis of MCT1 band intensities. D: plasma membrane MCT1-green fluorescent protein (GFP) levels in response to butyrate treatments were measured in C2BBE cells transiently transfected with MCT-GFP fusion constructs and probed with anti-GFP antibodies in immunofluorescence studies. GFP, green fluorescent protein; WGA, wheat germ agglutinin. *P < 0.05.

Fig. 4.

Effects of pectin diet vs. fiber-free diet on MCT1 function (transepithelial butyrate flux) in native colonic mucosa of rats. A: pCMBS-sensitive J_m-s transepithelial flux of [¹⁴C]butyrate in the colonic mucosa of rats fed pectin or fiber-free diet for indicated periods. B: after mounting the colonic mucosa on the Ussing chamber, nicotinate (100 μM) was applied to the mucosal side of the compartment of for 60 min, followed by measurement of pCMBS-sensitive J_m-s transepithelial flux of [¹⁴C]butyrate. *P < 0.05.

Fig. 5.

Pectin feeding increases MCT1 protein expression as well as its apical membrane levels in the colonic mucosa compared with control rats fed fiber-free diet. MCT1 (red), actin (green), nuclei (blue); scale bar = 20 μm.