ABSTRACT
Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that ß2â1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether ß2â1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 ß2â1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by ß2â1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain ß2â1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain ß2â1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with ß2â1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that ß2â1-fructans are specific ligands for TLR2. To conclude, ß2â1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of ß2â1-fructan-mediated health effects.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/metabolism , Colon/metabolism , Fructans/metabolism , Intestinal Mucosa/metabolism , Protective Agents/metabolism , Tight Junctions/metabolism , Toll-Like Receptor 2/agonists , Anti-Inflammatory Agents, Non-Steroidal/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Antibodies, Blocking/pharmacology , Cell Line , Colon/drug effects , Colon/immunology , Diglycerides/pharmacology , Fructans/antagonists & inhibitors , Fructans/chemistry , Gastrointestinal Agents/antagonists & inhibitors , Gastrointestinal Agents/chemistry , Gastrointestinal Agents/metabolism , Humans , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Kidney/drug effects , Kidney/immunology , Kidney/metabolism , Ligands , Membrane Transport Modulators/antagonists & inhibitors , Membrane Transport Modulators/toxicity , Molecular Structure , NF-kappa B/agonists , NF-kappa B/metabolism , Oligopeptides/pharmacology , Prebiotics/analysis , Protective Agents/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/antagonists & inhibitors , Tetradecanoylphorbol Acetate/toxicity , Tight Junctions/drug effects , Tight Junctions/immunology , Toll-Like Receptor 2/antagonists & inhibitors , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/metabolism , Transcription Factor AP-1/agonists , Transcription Factor AP-1/metabolismABSTRACT
The inhibition of fructan accumulation by phosphate was investigated in warm-grown and cold-treated barley (Hordeum vulgare) plants. Detached leaves were incubated in water or phosphate for 24 h under lighting or in darkness. Fructosyltransferase, sucrose phosphate synthase (SPS) and cytosolic fructose-1,6-bisphosphatase (FBPase) activities were subsequently analysed, as well as the content of carbohydrates, hexose-phosphates, phosphate, amino acids and protein. In warm-grown leaves, phosphate decreased fructan accumulation and total carbon in carbohydrates and did not affect protein content. Phosphate increased hexose-phosphates, phosphate and amino acids. Fructosyltransferase and FBPase activities were not affected by phosphate feeding, while SPS activity was inhibited by phosphate in incubations in both light and darkness. In cold-treated leaves, which before incubation had higher SPS activities than warm-grown leaves, phosphate had no inhibitory effect on fructan accumulation, carbohydrate content or total C in carbohydrates. The activities of SPS and FBPase were unaffected by phosphate. The results indicate that phosphate decreases fructan accumulation through an inhibition of SPS whenever this activity is not high before a rise in phosphate content.