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. 2010 Oct;299(4):G928-34.
doi: 10.1152/ajpgi.00279.2010. Epub 2010 Jul 29.

The probiotic Lactobacillus plantarum counteracts TNF-{alpha}-induced downregulation of SMCT1 expression and function

Alip Borthakur  1 , Arivarasu N Anbazhagan, Anoop Kumar, Geetu Raheja, Varsha Singh, Krishnamurthy Ramaswamy, Pradeep K Dudeja
Affiliations

The probiotic Lactobacillus plantarum counteracts TNF-{alpha}-induced downregulation of SMCT1 expression and function

Alip Borthakur et al. Am J Physiol Gastrointest Liver Physiol. 2010 Oct.

Abstract

The major short-chain fatty acid (SCFA) butyrate is produced in the colonic lumen by bacterial fermentation of dietary fiber. Butyrate serves as primary fuel for the colonocytes and also ameliorates mucosal inflammation. Disturbed energy homeostasis seen in inflamed mucosa of inflammatory bowel disease patients has been attributed to impaired absorption of butyrate. Since sodium-coupled monocarboxylate transporter 1 (SMCT1, SLC5A8) has recently been shown to play a role in Na(+)-coupled transport of monocarboxylates, including SCFA, such as luminal butyrate, we examined the effects of proinflammatory TNF-α on SMCT1 expression and function and potential anti-inflammatory role of probiotic Lactobacillus species in counteracting the TNF-α effects. Rat intestinal epithelial cell (IEC)-6 or human intestinal Caco-2 cells were treated with TNF-α in the presence or absence of Lactobacilli culture supernatants (CS). TNF-α treatments for 24 h dose-dependently inhibited SMCT1-mediated, Na(+)-dependent butyrate uptake and SMCT1 mRNA expression in IEC-6 cells and SMCT1 promoter activity in Caco-2 cells. CS of L. plantarum (LP) stimulated Na(+)-dependent butyrate uptake (2.5-fold, P < 0.05), SMCT1 mRNA expression, and promoter activity. Furthermore, preincubating the cells with LP-CS followed by coincubation with TNF-α significantly attenuated the inhibitory effects of TNF-α on SMCT1 function, expression, and promoter activity. In vivo, oral administration of live LP enhanced SMCT1 mRNA expression in the colonic and ileal tissues of C57BL/6 mice after 24 h. Efficacy of LP or their secreted soluble factors to stimulate SMCT1 expression and function and to counteract the inhibitory effects of TNF-α on butyrate absorption could have potential therapeutic value.

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Figures

Fig. 1.
Fig. 1.
Expression of sodium-coupled monocarboxylate transporter 1 (SMCT1) in human and mouse intestine. A: SMCT1 mRNA levels in human intestinal epithelial cells (IECs). SMCT1 protein levels in brush border (BB) and basolateral (BL) membranes in human jejunum (Jej), ileum (Ile), proximal colon (PC), and distal colon (DC; B) and in mouse Ile, PC, and DC (C) are shown. Immunoblots for protein are representatives of three experiments.
Fig. 2.
Fig. 2.
Measurement of SMCT1 function in IEC-6 cells. Na+-dependent 14C-butyrate uptake was measured in postconfluent IEC-6 monolayers as a function of time. Values represent difference between 14C-butyrate uptake in presence and absence of sodium in the uptake buffer. Values are means ± SE; n = 4.
Fig. 3.
Fig. 3.
TNF-α inhibits SMCT1 function and expression in IEC-6 cells. The cells grown on transwell inserts were treated basolaterally with indicated concentrations of TNF-α for 24 h. A: Na+-dependent 14C-butyrate uptake was measured in control or TNF-α-treated cells. B: total RNA was isolated from control or TNF-α-treated cells, and SMCT1 mRNA was measured by real-time quantitative PCR. Values are means ± SE; n = 3. *Different from control, P < 0.001.
Fig. 4.
Fig. 4.
Long-term treatment with Lactobacillus culture supernatant (CS) stimulates SMCT1 function. Postconfluent IEC-6 cells were treated with CS (diluted 1:50 in DMEM) of L. acidophilus (LA), L. rhamnosus (LR), L. plantarum (LP), and L. casei (LC) for 24 h, and Na+-dependent 14C-butyrate uptake was measured. Values are means ± SE; n = 3. *Different from control, P < 0.001.
Fig. 5.
Fig. 5.
LP-CS enhances SMCT1 mRNA and protein expression in IEC-6 cells. Cells treated with LP-CS for 24 h were used for RNA isolation and lysate preparation. SMCT1 mRNA (A) and protein levels (B) in control and LP-CS-treated cells are shown. Values are means ± SE; n = 3. *Different from control, P < 0.001.
Fig. 6.
Fig. 6.
LP-CS stimulates SMCT1 promoter activity and attenuates TNF-α inhibition of SMCT1 function and promoter activity. A: postconfluent IEC-6 monolayers were pretreated with LP-CS from the apical surface for 8 h, which was continued for another 16 h, with and without TNF-α (10 ng/ml) added basolaterally. Na+-dependent 14C-butyrate uptake was then measured. B: Caco-2 cells were transfected with the SMCT1 promoter construct in pGL2. Twenty-four hours after transfection, cells were pretreated with LP-CS from the apical surface for 8 h, which was continued for another 16 h, with and without TNF-α (10 ng/ml) added basolaterally. Promoter activity was measured as luciferase units and normalized with the corresponding β-galactosidase units for transfection efficiency. Values are means ± SE; n = 4. *Different from control, P < 0.001.
Fig. 7.
Fig. 7.
Oral administration of live LP increases SMCT1 mRNA and protein in mouse ileum and colon. C57BL/6 mice were gavaged with live LP in sterile PBS containing 1 × 10−9 colony-forming units/ml of the bacteria. After 24 h, mice were killed, intestine removed, and mucosa was scraped from ileum and colon for RNA isolation and preparation of cell lysate. SMCT1 mRNA was measured by real-time PCR using gene specific primers (A) and SMCT1 protein levels by immunoblotting (B).
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