High Fat Induces Altered Colonic Motility and Enteric Neuronal Apoptosis

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Aims: Obesity has been associated with several gastrointestinal motility problems including constipation. Previous studies have demonstrated central neuronal loss related to high fat diet. To study the etiology of the obesity related gastrointestinal symptoms we investigated the effect of twelve weeks of high fat diet (HF, 23%) on C57/BL6 mice compared to regular diet (6% fat). In addition we assessed the in-vitro effects of fatty acids on enteric neurons. Materials and method: Eight week old C57/BL6 mice were treated for 12 weeks with HF diet and their body weight and stool pellet number/hour, stool wet weight, and dry weight and water content were recorded. Isotonic muscle recordings were performed on the proximal colons. In-vitro studies were performed on postnatal IM-PEN enteric neuronal cell line. The neuronal cells were exposed to different doses of palmitate for 24 hours and MTS assay (for cell viability) were performed. Cells were also assessed for apoptosis using western blot analysis and immunostaining for cleaved caspase-3. Autophagy was assessed by western blot analysis for LC-3 and Beclin. Results: At the end of twelve weeks mice fed with the HF diet had a significant weight gain compared to normal diet group (34.2 ± 2.3 % and 23.2 ± 2.5 %, P<0.05). Mice on HF diet had a significantly lower pellet frequency (P<0.01) and pellet water content, as well as wet and dry pellet weight, resembling a constipation phenotype (Figure 1). Colonic isotonic muscle recording showed significantly less EFS-induced contractions in HF diet fed mice compared to normal diet (P<0.05), indicating loss of cholinergic neuronal function. Enteric neurons treatedwith palmitate showed a dose dependent reduction in viability (Figure 2). Exposure of enteric neuronal cells to 0.5 and 1 mM palmitate resulted in a dose dependent increase in cleaved caspase-3, which was associated with an increase in expression of LC3B and Beclin (n=3 independent blots).In addition immunostaining for cleaved caspase-3 showed enhanced apoptosis in cells exposed to palmitate compared to vehicle (15.6 ± 1.8 % and 10.6 ± 1.4 %, respectively P<0.01). To determine the signal transduction pathway involving fatty acid induced enteric neuronal apoptosis we assessed the c-Jun, p-AKT and NF-κB pathways. Exposure to palmitate (0.5 mM) was associated with a significant decrease in c-Jun and NK-κB protein expression, while it had no effect on p-AKT expression. Conclusion: In summary our data demonstrates the effects of high fat diet on reducing colonic motility. Exposure to fatty acids induced a reduction in enteric neuronal viability and enhanced enteric neuronal apoptosis. This data suggests there is a role for fatty acid induced enteric neuronal apoptosis accompanied by activation of autophagy pathways and down-regulation of c-Jun and NK-κB in mediating high fat diet-induced colonic dysmotility.