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Objectives: The epithelial barrier of the ruminal epithelium is provided by a tight junction protein (TJ) strand network sealing the space between contiguous cells. It constitutes the intercellular fence against solutes, microorganisms and toxins. In jejunal epithelia of monogastric organisms, it has been shown that the epithelial barrier can be tightened by glucagon-like peptide 2 (GLP-2) (1). On the other hand, glucagonlike peptide 1 (GLP-1) decreases endothelial permeability in injured mesenteric endothelium (2). Against this background, we addressed the hypothesis that GLP-1 and GLP-2 may also affect the epithelial barrier of the ovine rumen.
Materials and Methods: Six adult sheep were fed on standardized 80% hay and 20% concentrate diet for at least 2 weeks prior to the experiment. Sheep were stunned and exsanguinated, and ruminal tissue was removed from the ventral ruminal sac. The epithelial layer was stripped off the tunica muscularis and tunica serosa. Epithelia were cut in ~3×3 cm pieces and mounted in Ussing chambers with an exposed area of 3.14 cm2. The incubation buffer on the mucosal side was titrated to pH 6.1 and contained short chain fatty acids (SCFA; 40 mM). The incubation buffer at the serosal side had a pH 7.4 and was SCFA-free. Epithelial conductance was continuously monitored under short-circuit conditions. One control group was incubated for 7 hours in buffer solution without hormones. Simultaneously, four treatment groups were incubated on the serosal side with incubation buffer containing either 25 nM or 250 nM of either GLP-1 or GLP-2. After 7 hours, epithelial samples were collected and stored in RNAlater. The changes in mRNA expression of the TJ proteins claudin-1, -4, -7 and occludin were measured by reverse transcription quantitative real-time PCR (RT-qPCR). Statistical analysis was performed with the software SPSS 22 using one-way ANOVA and the post-hoc Dunnett test.
Results: The electrical conductance was not different between control epithelia incubated without hormones and epithelia incubated with GLP-1 or GLP-2 at the two tested concentrations. However, the mRNA patterns of tight junction proteins were different between epithelia incubated with and without hormones. The mRNA expression of claudin-7 decreased in all epithelia incubated with GLP-1 at 25 nM (by -75 ± 7%) or 250 nM (by -82 ± 8%), as well as GLP-2 at 25 nM (by -56 ± 10%) or 250 nM (by -57 ± 9%), when compared to control expression (all P < 0.05). The mRNA expression of occludin increased only in epithelia incubated with 250 nM of GLP-2 (by +239 ± 75%; P < 0.05) but was not different to controls in the presence of 25 nM or 250 nM GLP-1 or 25 nM GLP-2. The mRNA expression of claudin-1 and claudin-4 was not different between epithelia incubated with or without hormones.
Conclusions: We conclude that both GLP-1 and GLP-2 decrease the mRNA expression of the tight junction protein claudin-7. Very high concentrations of GLP-2 additionally increase the mRNA expression of occludin. These changes may be relevant for adaptation processes of the ruminal epithelium in the longer term but may not be associated with immediate changes in epithelial barrier function.