In Vivo Assessment of Acetic, Propionic and Butyric Acid Production Following Colonic Fermentation of Non-Digestible Carbohydrates From Two 13C-Labelled Barley Meals

Abstract

Extrinsic afferent neurons play an important role in the regulation of gut function. These neurons exhibit extensive plasticity in phenotype and cell number in response to a variety of conditions. We have previously reported that TNBS colitis increased the mRNA and protein levels of calcitonin gene-related peptide (CGRP) in rat dorsal root ganglia (DRG). The promoter of CGRP contains a binding site for cAMP response element binding (CREB) protein. The activation of CREB was regulated by its phosphorylation on Ser133, for which Ca2+-regulated pathways may have a role. Thus the AIMS of this study are to examine 1) if phospho (p)-CREB was regulated by colitis in DRG; 2) if p-CREB was co-localized with CGRP; and 3) the association of p-CREB with Ca2+/calmodulin-dependent protein kinase (CaMK) II in the DRG during colitis. METHODS: Colonic inflammation was induced in rats by intracolonic instillation of TNBS (1.5 mL/kg of 60 mg/mL solution in 50 % EtOH). Control animals received 50 % EtOH. Rats were sacrificed by intracardiac perfusion with 4% paraformaldehyde on day 7 or 21 following the induction of colitis. The lumbar L1-L2 DRG sections (20 μm thickness) underwent single or double immunostaining for p-CREB, CaMKII, or/and CGRP. RESULTS: In all animals examined, p-CREB immunoreactivity was expressed in the nucleus of small diameter DRG neurons (10-20 μm). Following TNBS treatment, the number of p-CREB immunoreactive neurons in L1-L2 DRG was significantly increased (p<0.05) at day 7 (29 cells per 105 μm2 DRG section) and day 21 (22 cells per 105 μm2 DRG section) when compared to control (11 cells per 105 μm2 DRG section). The number of CGRP immunoreactive neurons was also increased by 2-fold (control: 7 cells; TNBS 7 days: 13 cells; TNBS 21 days: 16 cells per 105 μm2 DRG section, p<0.05). Colocalization studies showed that the number of DRG neurons co-expressing CGRP and pCREB was increased by 2-2.5 fold in the lumbar L1-L2 DRG at 7 and 21 days following colitis induction. The neurons that contained CGRP also expressed p-CREB, however, only 50 % of p-CREB immunoreactive cells expressed CGRP. Double immunostaining also showed co-localization of p-CREB with CaMKII in the DRG during colitis. CONCLUSIONS: TNBS colitis increased the expression level of CGRP in lumbar L1-L2 DRG at day 7 and day 21, and also increased the phosphorylation level of CREB in these DRG neurons. The colocalization of p-CREB with CGRP and with CaMKII in the DRG suggested a correlation of CaMKII/p-CREB activation and the CGRP expression in DRG during colitis.