Effect of individual SCFA on the epithelial barrier of sheep rumen under physiological and acidotic luminal pH conditions

Abstract

The objective of this study was to investigate whether individual short-chain fatty acids (SCFA) have a different potential to either regulate the formation of the ruminal epithelial barrier (REB) at physiological pH or to damage the REB at acidotic ruminal pH. Ruminal epithelia of sheep were incubated in Ussing chambers on their mucosal side in buffered solutions (pH 6.1 or 5.1) containing no SCFA (control), 30 mM of either acetate, propionate or butyrate, or 100 mM acetate. Epithelial conductance (Gt), short-circuit current (Isc), and fluorescein flux rates were measured over 7 h. Thereafter, mRNA and protein abundance, as well as localization of the tight junction proteins claudin (Cldn)-1, -4, -7, and occludin were analyzed. At pH 6.1, butyrate increased Gt and decreased Isc, with additional decreases in claudin-7 mRNA and protein abundance (each P < 0.05) and disappearance of Cldn-7 immunosignals from the stratum corneum. By contrast, the mRNA abundance of Cldn-1 and/or Cldn-4 were upregulated by 30 mM propionate, 30 mM butyrate, or 100 mM acetate (P < 0.05), however, without coordinated changes in protein abundance. At luminal pH 5.1, neither Gt, Isc, nor TJ protein abundance was altered in the absence of SCFA; only fluorescein flux rates were slightly increased (P < 0.05) and fluorescein signals were no longer restricted to the stratum corneum. The presence of acetate, propionate, or butyrate at pH 5.1 increased fluorescein flux rates and Gt, and decreased Isc (each P < 0.05). Protein abundance of Cldn-1 was decreased in all SCFA treatments but 30 mM butyrate; abundance of Cldn -4 and -7 was decreased in all SCFA treatments but 30 mM acetate; and abundance of occludin was decreased in all SCFA treatments but 30 mM propionate (each P < 0.05). Immunofluorescence staining of SCFA-treated tissues at pH 5.1 showed disappearance of Cldn-7, discontinuous pattern for Cldn-4 and blurring of occludin and Cldn-1 signals in tight junction complexes. The fluorescein dye appeared to freely diffuse into deeper cell layers. The strongest increase in Gt and consistent decreases in the abundance and immunosignals of tight junction proteins were observed with 100 mM acetate at pH 5.1. We conclude that SCFA may contribute differently to the REB formation at luminal pH 6.1 with possible detrimental effects of butyrate at 30 mM concentration. At luminal pH 5.1, all SCFA elicited REB damage with concentration appearing more critical than SCFA species.

Effects of glucagon‐like peptides 1 and 2 and epidermal growth factor on the epithelial barrier of the rumen of adult sheep

“…Alterations in J fluor and G t are commonly used to assess the permeability of the paracellular space to large ( J fluor ) and small ions ( G t ), assuming that the transcellular route has negligible ( J fluor ) or rather constant ( G t ) contribution to these functional barrier readouts. Nevertheless, we have demonstrated in previous studies that fluorescein is also able to use the transcellular route for permeation across RE, for example, after an acid challenge (Greco et al, ; Meissner et al, ). Based on the fact that G t and the protein expression of all tested TJ proteins were unchanged by GLP‐1 and GLP‐2 in the present study, we thus propose that paracellular barrier function of RE was not affected by these hormones during the 7‐hr incubation, despite the fact that J fluor was changed in the last flux period by either of them.…”

“…The procedures for protein extraction and Western blot analysis of claudin-1, -4, -7 and occludin were performed as described previously (Greco et al, 2018), except that blotting was performed at 80 V for 60 min and that antibodies for the reference gene (anti-βactin) were diluted 1 in 2000. The zero hypothesis was considered disproved with p-values < .05.…”

“…The epithelial barrier (EB) of the rumen provides effective protection against free noxious agents in the ruminal milieu (Aschenbach et al, ; Penner, Steele, Aschenbach, & McBride, ). However, it can become ineffective under pathological conditions, e.g., when luminal accumulation of short‐chain fatty acids and protons challenge key epithelial functions (Aschenbach & Gabel, ; Greco et al, ; Meissner et al, ). Different studies proposed a loss of barrier integrity and selectivity under such circumstances (Liu, Xu, Liu, Zhu, & Mao, ; Steele, AlZahal, Hook, Croom, & McBride, ).…”

“…However, it can become ineffective under pathological conditions, e.g., when luminal accumulation of short-chain fatty acids and protons challenge key epithelial functions (Aschenbach & Gabel, 2000;Greco et al, 2018;Meissner et al, 2017). Different studies proposed a loss of barrier integrity and selectivity under such circumstances (Liu, Xu, Liu, Zhu, & Mao, 2013;Steele, AlZahal, Hook, Croom, & McBride, 2009).…”

Growth of rumen papillae in weaned calves is associated with lower expression of insulin‐like growth factor‐binding proteins 2, 3, and 6

“…These data suggested that higher expression of IGFBP5 in rumen papillae is associated with the changes of the fermentation in rumen. Higher expression of IGFBP5 in rumen papillae might be necessary for adaptation to highly fermentable diet causing disruption of rumen epithelial barrier (Greco et al, 2018;Meissner et al, 2017;Steele et al, 2011). IGFBP5 induced monocyte migration in lung epithelium in mice (Yasuoka, Yamaguchi, & Feghali-Bostwick, 2009).…”