74 Fecal Metabolites Associated With Nitrergic Myenteric Neuronal Impairment and Gastrointestinal Motility Disorders in Western Diet-Fed Mice

Abstract

Introduction: Western-diet (WD) is associated with reduced stool frequency in humans. High-fat fed mice exhibit delayed gastrointestinal (GI) transit associated with reduced number of nitrergic myenteric neurons. Mechanisms of these alterations are still unknown. Our aim was to investigate if WD feeding leads to modifications of fecal metabolites associated with the development of enteric neurodegeneration and the role played by the gut microbiota in such phenomenon. Methods: C57Bl/6 mice were fed a WD (34.5% kcal from fat, representative of the typical American diet fat calorie intake, n=5) or a Regular diet (RD, 16.9% kcal from fat, n=5). 6 weeks post feeding, fecal metabolites were analyzed by high-resolution mass spectrometry (LTQ-FT). 12 weeks post feeding, nitrergic myenteric neurons were quantified in the proximal colon by NADPH-diaphorase staining and GI and colonic transit were measured. Citrulline was measured by ELISA in germ-free (GF) mice stools fed a WD or a RD for 6 weeks (n=3 per group). Data are presentedas mean ± SEM and are significant to p < 0.05. Results: After 12 weeks of feeding, WD mice exhibited a reduced stool frequency in comparison to RD mice (1.5±0.5 and 4.4±0.3 stools/min, respectively) and this was associated with a delayed intestinal transit time (281.5±57.5 and 152.8±15.7 min). In addition, WD mice had a longer colonic transit time (as measured by bead expulsion time: WD: 127.5±52.5 min; RD: 13.2±2.0 min) associated with reduced number of nitrergic myenteric neurons in the proximal colon (113.0±5.6 and 64.2±7.4 neurons/field). Together, these data demonstrate an altered colonic motor function after 12 weeks of WD. We quantified 3730 metabolites in the feces but only 185 were significantly higher in WD mice compared to RD mice. In addition to palmitate, we found increased concentrations of citrulline (associated with enhanced nitric oxide synthase activity), 3-hydroxykynurenine (reflecting neuroinflammation) and bacterial muramic acid [Figure 1]. In order to understand the possible role of the gut microbiota in these changes, we measured fecal citrulline in GF mice fed a WD or a RD for 6 weeks and found similar concentrations (3.0±0.5 and 2.6±0.3 ng/mg). Conclusion: Our results show that long-term WD consumption is associated with nitrergic myenteric neuronal loss contributing to delayed GI transit. These alterations were preceded by gut dysbiosis and elevated fecal citrulline, potentially reflecting higher plasma concentrations that lead to oxidative stress and apoptosis in NO producing neurons. Importantly, fecal citrulline was not affected by a WD feeding under germfree conditions, suggesting that the gut microbiota is required for such effects. Fecal citrulline may be considered as a marker of myenteric neuronal impairments responsible for the motor alterations observed during WD feeding.