To address this question, we used a Western diet (WD) containing less fat than routinely used in a HFD (35% kcal from fat for WD vs . 60% kcal from fat for HFD) that has been designed to represent the typical Western dietary practices in all aspects, including caloric value and macro‐ and micronutrient composition (Hintze et al ., 2012 ). With this diet, our goal was to assess enteric neuronal damage and determine how LPS and saturated FFA contribute to this disorder. Therefore, the present study aimed to: (i) evaluate the impact of WD consumption on myenteric nitrergic neurons and motility; (ii) characterize the importance of gut microbiota dysbiosis, TLR4 signalling, and plasma LPS and FFA levels in the development of WD‐induced neuropathy; and (iii) evaluate the synergic effects of LPS and palmitate in vitro on enteric NOS neuronal cell survival.

Obesity and high‐fat diet (HFD) consumption are associated with enteric neuronal dysfunction and gastrointestinal (GI) motility disorders (Taba Taba Vakili et al., 2015 ; Mushref & Srinivasan, 2013 ). Myenteric neurons that play a major role in the regulation of motor functions appear to be particularly sensitive to HFD consumption (Stenkamp‐Strahm et al., 2013 ; Reichardt et al., 2013 ; Rivera et al., 2014 ; Beraldi et al., 2014 ; Stenkamp‐Strahm et al., 2015 ; Soares et al., 2015 ). We previously have shown that HFD‐fed mice (60% kcal from fat) exhibited a reduced number of colonic myenteric neurons expressing neuronal nitric oxide synthase (nNOS), which in turn was associated with delayed colonic transit (Nezami et al ., 2014 ). We recently demonstrated the important role of HFD‐induced gut microbiota dysbiosis in driving enteric neurodegeneration via Toll‐like receptor 4 (TLR4) in mice exhibiting increased plasma lipopolysaccharide (LPS) concentrations (Anitha et al ., 2016 ). High levels of LPS induce proinflammatory cytokines release and apoptosis in cultured myenteric neurons (Arciszewski et al ., 2005 ; Voss & Ekbald, 2014 ; Coquenlorge et al ., 2014 ). This neurodegenerative process could be also mediated by saturated free fatty acids (FFA) such as palmitate (C16:0) that are abundant in HFD (Voss et al ., 2013 ). Because endotoxaemia and hyperlipidaemia have been described in HFD‐fed mice (Sumiyoshi et al ., 2006 ), both LPS and palmitate may simultaneously be present at the level of the myenteric plexus. Both LPS and palmitate can activate TLR4 expressed by enteric neurons (Barajon et al ., 2009 ). However, the relative roles of LPS and palmitate in HFD‐induced enteric nitrergic neuronal loss have not yet been defined.

Statistical analyses were performed using Student's t or Mann–Whitney tests with Prism software (GraphPad Inc, La Jolla, CA, USA) as Pearson's correlations. Data numbers are reported as appropriate. For clarity, only RD and WD animals fed for similar durations were compared together. Results from parametric data are presented as the mean ± SEM. P < 0.05 was considered statistically significant ( * P < 0.05, ** P < 0.01, *** P < 0.001).

Western blot analysis was performed as described previously (Anitha et al ., 2016 ). Lysates obtained from remaining cells after 24 h of treatment were used to probe for nNOS and PGP9.5 sing nNOS (BD Biosciences, San Jose, CA, USA) and PGP9.5 specific antibodies (Abcam). Β‐actin (Cell Signaling) was used as a loading control. Data from three or more experiments were used for the statistical analysis.

IM‐FEN cells were cultured with modified N2 medium (33 °C, 5% СO 2 ) as described previously (Anitha et al ., 2008 ). Differentiation was induced by changing medium to Neurobasal‐A Medium (Gibco, Thermo Fisher, CA, USA) supplemented with 1% FBS, 2 mm l‐glutamine, 100 mg ml –1 streptomycin (all from Sigma‐Aldrich), B27 (0.1x; Gibco) and glial cell‐derived neurotrophic factor (GDNF) (10 ng ml –1 ; Shenandoah Biotechnology Inc., Warwick, PA, USA) for 5 days at 39 °C before treatment. Palmitate (100 mm stock solution in isopropanol), LPS (in ng ml –1 , 1 ng ≈ 10 EU) and N G ‐nitro‐l‐arginine methyl ester (l‐NAME; NOS inhibitor) were provided by Sigma‐Aldrich and added to the medium for an additional 24 h. Cell survival was then investigated using the MTS [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium] assay (Promega, Madison, WI, USA) as reported previously (Nezami et al ., 2014 ). Data from five or more experiments were used in the statistical analysis.

Proximal colon and distal ileum (1 cm above the cecum) were freshly dissected to isolate myenteric ganglia and longitudinal muscle layers. Proximal colon mucosa was used to measure FFA content. Myenteric neurons were stained for the neuronal marker peripherin (Millipore, Billerica, MA, USA) or nicotinamide adenine dinucleotide phosphate (NADPH)‐diaphorase (1 h of incubation with β‐NADPH diaphorase, 1 mg ml –1 ; nitroblue tetrazolium, 0.1 mg ml –1 ; and 0.3% Triton‐X 100 in PBS at 37 °C; all from Sigma‐Aldrich, St Louis, MO, USA) that colocalizes with nNOS (Belai et al ., 1992 ). Neuronal quantifications were performed blinded on 10 images per mouse randomly chosen with the same magnification (20×) and expressed by number of neurons/field, as described recently (Carbone et al ., 2016 ). Cryosections from 6‐week fed mice colons were stained for cleaved‐caspase3 (Cell Signaling, Beverly, MA, USA) and PGP, version 9.5 (Abcam) and five sections per mouse were used to score the cleaved‐caspase3 area within the myenteric tissue using Photoshop (Adobe Systems, San Jose, CA, USA).

Feces sampled at 6 weeks were extracted in an acetonitrile/water solution (2:1) containing a mixture of internal standards and analysed as described previously (Uppal et al ., 2015 ). Mass spectrometry was performed using a LTQ‐Velos‐Orbitrap mass spectrometer (Thermo Fisher, San Diego, CA, USA). Metabolites were characterized according their mass/charge ratio with associated retention time using bioinformatics databases (METLIN: https://metlin.scripps.edu ; HMDB: http://www.hmdb.ca ). Peaks intensities analyses and heatmap were made using MetaboAnalyst, version 3.0 ( www.metaboanalyst.ca ).

Interferon (IFN)γ, interleukin (IL)‐6, keratinocyte‐derived chemokine (KC) (or CXCL1) and MCP1 expressions were analysed by quantitative RT‐PCR as described previously (Etienne‐Mesmin et al ., 2016 ).Total RNA was isolated from mesenteric (fat pads surrounding the proximal colon) and epididymal adipose tissue using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and purified using a RNeasy mini kit (Qiagen, Hilden, Germany). Quantitative RT‐PCR was performed in a CFX96 apparatus Touch Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA, USA) using the iScriptTM One‐Step RT‐PCR Kit with SYBR Green (Bio‐Rad) and gene‐specific oligonucleotides primers: IFNγ (forward) 5′‐ AGCAAGGCGAAAAAGGATGC‐3′ and (reverse) 5′‐TCATTGAATGCTTGGCGCTG‐3′; IL‐6 (forward) 5′‐GTGGCTAAGGACCAAGACCA‐3′ and (reverse) 5′‐GGTTTGCCGAGTAGACCTCA‐3′; KC (forward) 5′‐TTGTGCGAAAAGAAGTGCAG‐3′ and (reverse) 5′‐TACAAACACAGCCTCCCACA‐3′; MCP1 (forward) 5′‐GCTGGAGCATCCACGTGTT‐3′ and (reverse) 5′‐TGGGATCATCTTGCTGGTGAA‐3′. Results were normalized to 36B4 (housekeeping gene).

Six‐week‐old male C57Bl/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and placed after acclimation on WD (34.5% calories from fat: TD.140304; Harlan Laboratories, Madison, WI, USA) or regular diet (RD) (16.9% calories from fat: TD.140305) for 3, 6, 9 and 12 weeks. Age‐matched TLR4 −/− mice on a BL/10 background, also purchased from Jackson Laboratories, were fed RD or WD for 12 weeks. Female Germ‐Free (GF) wild‐type (WT) C57BL/6 mice maintained under GF conditions in the Georgia State University gnobiotic facility using Park Bioservices isolators were fed for 6 weeks. The WD was designed as described by Hintze et al . ( 2012 ), RD is the control of WD. Diet compositions provided by the manufacturer (Table 1 ) suggest that the relative palmitate content in WD and RD is ∼4:1. Mice were monitored for body weight (BW) and stool indices throughout the experiments. Blood glucose was measured in 6 h fasted RD and WD mice fed for 12 weeks with a Performa Accu‐Check glucose meter (Roche, Basel, Switzerland). Mice were killed after 4 h of fasting.

A , incubation for 24 h of an enteric neuronal cell line with palmitate (from 20 to 500 μ m ) induced cell loss. Results are expressed as the percentage increase in cell viability over vehicle‐treated cells. B , incubations with LPS (from 0 to 2 ng ml –1 ). C , co‐incubations of low LPS (0.5 ng ml –1 ) with palmitate 20 or 30 μ m for 24 h in presence or absence of the NOS inhibitor l ‐NAME (200 μ m ). Western blot analyses and representative gels showing nNOS ( D ) and PGP9.5 ( E ) expression after incubation for 24 h with palmitate 20 or 30 μ m , +/– LPS 0.5 ng ml –1 and + l ‐NAME (200 μ m ).

Having noted in vivo changes in myenteric neurons, we cultured enteric neurons in vitro in the presence of palmitate and LPS aiming to clarify the mechanisms leading to neurodegeneration. Incubation for 24 h with palmitate ≥ 30 μm induced enteric neuronal loss (Fig. 7 A ). By contrast, LPS (0.5–2 ng ml –1 ) alone had no effect on neuronal survival (Fig. 7 B ). When incubated with palmitate at 20 and 30 μm, LPS 0.5 ng ml –1 exacerbated the neuronal loss induced by palmitate alone compared to the vehicle (Fig. 7 C ). This cell loss was substantially mitigated in the presence of the NOS‐inhibitor l‐NAME (200 μm). Next, we measured nNOS and PGP9.5 expression by Western blotting to estimate nNOS reduction. nNOS expression was not altered by LPS alone 0.5 ng ml –1 but significantly reduced after incubation with palmitate 0.02 or 0.03 μm ± LPS (Fig. 7 D ) and this decrease was countered by the addition of L‐NAME in the milieu. PGP9.5 expression was slightly reduced after incubation with LPS 0.5 ng ml –1 alone and this reduction was even more pronounced in presence of palmitate 20 μm (Fig. 7 E ), Together, these results suggest that LPS and palmitate act synergistically to damage enteric neurons via nNOS activity.

Because we hypothesized that TLR4 is essential in HFD‐induced neuronal loss, we assessed the effect of WD feeding on NOS neurons and GI motility in mice lacking this receptor. Compared to WT, 12‐week WD‐fed TLR4 −/− mice did not exhibit any increase in BW gain (Fig. 6 A ). Fecal levels of Lcn2 were not affected by the lack of TLR4 (Fig. 6 B ). However, increased levels of fecal LPS and flagellin were observed in WD‐fed TLR4 −/− mice compared to RD‐fed TLR4 −/− and WD‐fed WT mice (Fig. 6 C and D ). Importantly, no change was seen in NADPH‐diaphorase stained myenteric neurons in the proximal colon between RD‐fed or WD‐fed TLR4 −/− mice (Fig. 6 B ), although both exhibited less neurons than control WT mice. The colonic transit was similar between the two groups of TLR4 −/− mice (Fig. 6 F ). By contrast, WD‐fed TLR4 −/− mice exhibited more NADPH‐diaphorase stained cells in the distal ileum than their controls (Fig. 6 G ), without an alteration in GI transit (Fig. 6 H ).

Assessment of GI in mice fed a RD or WD for 3, 6, 9 and 12 weeks, bead expulsion time ( A ) and dye transit ( B ) after oral gavage with Evans blue dye/methyl cellulose solution. Correlations between colonic nitrergic neurons and colonic transit (bead expulsion time) (RD, n = 12; WD, n = 13) ( C ) and between colonic transit and body weight gain (RD, n = 13, WD, n = 12) ( D ). GE ( E ) and dye propagation ( F ) in upper small intestine in WD‐fed mice for 9 weeks. G , GI transit in GF mice fed with a RD or a WD for 6 weeks.

We next investigated how intestinal motor functions were impacted by the WD‐induced nitrergic neurodegeneration. We observed delayed colonic transit in 12‐week WD‐fed mice (+ 144%) but not at other time points (Fig. 5 A ). This was not associated with a delay of the whole GI transit at 12 weeks (Fig. 5 B ). At 6 weeks, there was a delay in whole gut transit (+33%) followed by a faster transit at 9 weeks (–31%). In 12‐week fed mice, a reduced of number of nitrergic neurons was associated with a longer colonic transit time (Fig. 5 C ). In addition, a delayed colonic transit correlated with an increased BW gain (Fig. 5 D ). Next, we assessed the GE in 9‐week fed mice to investigate the faster GI transit; although GE was similar between RD‐fed and WD‐fed mice (Fig. 5 E ), the dye propagation from the stomach tended to be faster, suggesting upper small intestinal acceleration (Fig. 5 F ). Finally, we observed that WD for 6 weeks did not alter GI transit in GF mice (Fig. 5 G ), indicating that gut microbiota may play a role in the delay observed in WT WD‐fed mice.

Proximal colon myenteric neurons stained for peripherin ( A ) and NADPH‐diaphorase ( B ) in RD and WD mice fed for 6, 9 and 12 weeks. C , cleaved‐caspase3/PGP9.5 costaining in colonic cryosections from 6‐week fed RD or WD mice. D , correlation between colonic nitrergic neurons and body weight gain in mice fed for 12 weeks ( n = 13 for each group). Distal ileum myenteric neurons stained for peripherin ( E ) and NADPH‐diaphorase ( F ) in RD and WD mice fed for 6, 9 and 12 weeks.

Proximal colon myenteric neurons stained for peripherin ( A ) and NADPH‐diaphorase ( B ) in RD and WD mice fed for 6, 9 and 12 weeks. C , cleaved‐caspase3/PGP9.5 costaining in colonic cryosections from 6‐week fed RD or WD mice. D , correlation between colonic nitrergic neurons and body weight gain in mice fed for 12 weeks ( n = 13 for each group). Distal ileum myenteric neurons stained for peripherin ( E ) and NADPH‐diaphorase ( F ) in RD and WD mice fed for 6, 9 and 12 weeks.

Next, we investigated the effects of WD consumption on colonic and ileal myenteric neurons after 6, 9 and 12 weeks. The total number of myenteric neurons in the proximal colon, as shown by peripherin staining, was reduced by 30% and 31%, respectively, after 9 and 12 weeks of WD (Fig. 4 A ). This loss was also observed for NADPH‐diaphorase stained neurons that were reduced by 41% and 51%, respectively, after 9 and 12 weeks of WD (Fig. 4 B ), although it was not preceded by increased myenteric cleaved‐caspase3 staining at 6 weeks (Fig. 4 C ). Moreover, we observed that the decreased number of nitrergic neurons correlated with a greater BW gain in 12‐week fed mice (Fig. 4 D ). By contrast, neither total, nor nitrergic neurons in the ileum were affected by WD (Fig. 4 E and F ).

We found a positive correlation between fecal LPS and plasma FFA levels in 6‐week fed WD‐mice (Fig. 3 A ) and used germ‐free mice to investigate this phenomenon. In these animals, we observed a slight increase of BW gain in the 6‐week WD‐fed group (Fig. 3 B ), although no change in FFA was observed (Fig. 3 C ).

Fecal levels of LPS ( A ) and flagellin ( B ) in RD and WD mice fed for 3, 6, 9 and 12 weeks. C , heatmap showing fecal metabolites from RD and WD mice fed for 6 weeks. Statistics after data normalization and averaging ( n = 5 per group) are shown on the right. D , fecal Lcn2 in RD and WD mice fed for 3, 6, 9 and 12 weeks.

Because diet plays a key role in microbial ecology, we assessed the concentration of two bacterial products, LPS and flagellin, which are increasingly being used to reflect microbiota dysbiosis. Fecal LPS, but not flagellin, was significantly increased (+160%) in feces after 6 weeks of WD consumption (Fig. 2 A and B ). We next subjected fecal samples of 6‐week fed RD and WD mice to linear trap quadrupole‐Fourier transform mass spectrometer metabolomics analysis from which 3370 metabolites were quantified. Among the metabolites increased in WD feces compared to RD that we identified, there were compounds related to bacterial wall metabolism (muramic acid and diaminopimelic acid); metabolites common to microbiota and host such as N ‐acetyl glucosamine, tetraethylammonium, glucuronic acid, citrulline, 5‐hydroxytryptamine and hypoxanthine; and those resulting from diet consumption (palmitate, triglycerides) (Fig. 2 C ). Taurocholate levels were decreased in WD‐fed mice. Finally, no alteration in fecal Lcn2 was observed, suggesting that WD‐fed mice did not exhibit intestinal inflammation (Fig. 2 D ).

, body weight gain of RD or WD mice fed for 3, 6, 9 and 12 weeks and representative pictures of visceral fat from 12‐week fed‐mice., mesenteric fat pads from 6, 9 and 12 weeks., blood glucose from 12‐week fed mice. Plasma levels of citrulline (), LPS () and FFA () in RD and WD mice fed for 3, 6, 9 and 12 weeks., FFA levels from proximal colon mucosa/submucosa from RD or WD mice fed for 6, 9 and 12 weeks., mesenteric (MAT) and epididymal adipose tissue (EAT) mRNAs expression of IFNγ, IL‐6, KC and MCP1 from WT mice fed for 6 weeks with a RD or a WD. [Color figure can be viewed at wileyonlinelibrary.com

Our first step was to characterize whether WD consumption results in obesity and diabetes analogous to that previously observed in response to HFD (60% kcal from fat). Therefore, we monitored BW gain and blood glucose in RD and WD mice fed for 3, 6, 9 and 12 weeks. Compared to RD, WD mice gained 21% and 42%, respectively, after 6 and 12 weeks (Fig. 1 A ). Increased mesenteric adiposity contributed to the BW increase at 12 weeks (Fig. 1 B ), although this was not associated with a significant elevation in blood glucose or plasma citrulline (Fig. 1 C and D ). Next, we measured levels of LPS and FFA, both of which can potentially activate TLR4. Plasma LPS was not altered in all groups of WD‐fed mice (Fig. 1 E ); on the other hand, plasma FFA were found to be increased only in 6‐week WD‐fed mice (+18%) (Fig. 1 F ). In addition, colonic mucosal/submucosal FFA had a tendency to be increased after 6 weeks (Fig. 1 G ). To investigate whether adipose tissue inflammation is responsible for this FFA increase, we measured proinflammatory gene expression in both mesenteric and epididymal tissues from 6‐week fed RD and WD mice. No significant changes were observed in IFNγ, IL‐6, KC and MCP1 expression (Fig. 1 H ), suggesting together that 6 weeks of WD feeding induced neither metabolic endotoxaemia, nor adipose tissue inflammation, as observed previously in HFD‐fed mice (60% kcal from fat) (Kim et al ., 2012 ; Anitha et al ., 2016 ).

Discussion

In the present study, we report that, in mice, the consumption of a WD induces nitrergic myenteric neurodegeneration in the proximal colon via TLR4 signalling. This alteration is consequent to gut microbiota dysbiosis and plasma FFA increases after 6 weeks of WD feeding and leads to delayed colonic transit. Finally, cell culture experiments highlight that palmitate potentiates the LPS‐neurodegenerative action in enteric neurons via NO production.

One major finding is that, within 9 weeks, WD leads to colonic nitrergic myenteric neuronal loss in the absence of overt hyperglycemia, intestinal inflammation or endotoxaemia, which are three possible causes of the initiation of neuronal loss (Lakhan & Kirchgessner, 2010; Chandrasekharan et al., 2011; Anitha et al., 2016). RD and WD mice fed for 12 weeks exhibited not only similar blood glucose levels, but also no alteration of plasma citrulline, a marker of metabolic syndrome development in HFD‐fed mice (Sailer et al., 2013). Enteric cell loss in the absence of glucose intolerance has been recently reported in mice fed a high‐fat high‐cholesterol diet (21% fat, 2% cholesterol); however, this was after 33 weeks of feeding (Rivera et al., 2014). Intestinal inflammation was investigated through fecal Lcn2 measurement and was similar between RD and WD mice. Finally, we observed no variation of plasma LPS in all the groups of WD mice, although endotoxaemia has been suggested after 3 weeks of HFD containing more fat (45–60% kcal) and reported to be increased in mice exhibiting NOS neurodegeneration after 12 weeks (Hamilton et al., 2015; Anitha et al., 2016).

Fecal LPS, in contrast, was increased in WD‐fed mice, although only after 6 weeks, suggesting gut microbial alteration (Chassaing et al., 2014b).Moreover, this alteration was associated with a FFA increase in the plasma of those mice. We measured adipose proinflammatory genes to determine adipose tissue expansion or inflammation (Bjorndal et al., 2011; Cullberg et al., 2014). However, mesenteric fat pads were not heavier after 6 weeks of WD and no alteration of genes of interest was observed in mesenteric and epididymal fat pads. In addition, a positive trend in FFA content in proximal colonic mucosa/submucosa was observed after 6 weeks. We found a positive correlation between fecal LPS and plasma FFA and tested the hypothesis that the plasma FFA increase may be related to the gut microbiota dysbiosis. GF mice fed a RD or a WD for 6 weeks did not exhibit hyperlipidaemia despite a slight body weight increase. Future studies will examine the underlying mechanisms that may involve modulation of lipoprotein lipase activity because it is known that microbiota controls the intestinal expression of a lipoprotein lipase circulating inhibitor (Bäckhed et al., 2004). We will investigate further to what extent the microbial dysbiosis might be related to dyslipidemia in WD‐fed mice.

We next analysed fecal metabolites to investigate the gut microbiota alteration suggested by the increased fecal LPS levels in WD‐mice fed for 6 weeks. Among those with a bacterial origin, we found increased levels of muramic acid reflecting increased bacterial fermentation and excretion (Sepehr et al., 2003), diaminopimelic acid produced by Gram negative and some Gram‐positive bacteria during peptidoglycan synthesis (Gillner et al., 2009) and triamethylammonium associated with gut bacterial ecology disruption (Hong et al., 2010). Moreover, a decreased concentration of the primary bile acid taurocholate in WD‐mice feces, compared to control, highlights changes in gut bacterial metabolism. We also found metabolites in WD feces related to the host metabolism: N‐acetylglucosamine, hypoxanthine produced by intestinal cells during endotoxemia (Schmidt et al., 1997), 5‐HTP suggesting higher serotoninergic metabolism as already reported in diet‐induce obese mice (Reichardt et al., 2013), citrulline and, finally, glucuronic acid, reflecting an enhanced elimination of excessive metabolites from plasma to feces via the bile (Karanam et al., 2007). Finally, we characterized metabolites related to WD consumption such as palmitate and triglycerides, and similar increases in feces have been already reported not only in HFD‐fed WT mice, but also GF mice (Rabot et al., 2010). Certain fecal metabolites, in particular bacterial compounds such as diaminopimelic acid with N‐acetylglucosamine or muramic acid with triglycerides, were simultaneously increased in a majority of WD‐fed mice compared to RD‐fed mice.

Focusing next on myenteric neurons, we observed that their total numbers were reduced in WD‐fed mice by 30% and 31% in the proximal colon after 9 and 12 weeks, respectively. These losses included NOS neurons that were concomitantly reduced by 41% and 51%. Representing 40% of the total colonic myenteric cells (Murphy et al., 2007), nitrergic neurons are more important in the proximal colon where they are essential to induce greater relaxation for fecal storage and excess fluid absorption (Takahashi & Owyang, 1998). We previously reported that they represent ∼50% in the murine proximal colon (Nezami et al., 2014; Anitha et al., 2016). By contrast, no changes were observed in the distal ileum of WD‐fed mice. Neuronal counts have all been reported as counts of nerve cells per microscope field. This method could be affected by changes in intestinal length (e.g. during inflammation); however, we did not observe any evidence of macroscopic inflammation in the intestine.

Colonic nitrergic neurodegeneration is associated with changes in motor function, as illustrated by the correlation between colonic myenteric NOS neuronal loss after 12 weeks of WD and delayed colonic transit. Moreover, a delayed colonic transit correlates with an increased body weight gain, suggesting that colonic dysmotility could be associated with obesity development. We recently highlighted a relationship between constipation and a high saturated fat diet in humans (Taba Taba Vakili et al., 2015).

By contrast to the colonic transit, the whole GI transit was delayed after 6 weeks and accelerated after 9 weeks, but not altered after 12 weeks. The transit delay after 6 weeks was not observed in WD‐fed GF mice, highlighting the interactions among microbiota and GI transit (Kashyap et al., 2013). This suggests that microbiota are critical in the delayed GI transit noted at 6 weeks. This is not neurodegenerative because cleaved‐caspase3 staining in colonic myenteric neurons was not increased. Enhanced NOS activity, however, may lead to NO overproduction within cells and oxidative stress, explaining the neuronal loss occurring in WD‐fed mice between 6 and 9 weeks of feeding (Rivera et al., 2011). The faster GI transit observed in 9‐week WD‐fed mice was first considered to be the consequence of an acceleration of the GE because such dysmotility is reported in pre‐diabetic HFD‐fed mice (Baudry et al., 2012). RD and WD mice fed for 9 weeks exhibited similar GE; however, the proximal intestine transit had a tendency to be accelerated. Enteric neuronal plasticity leading to changes in excitatory neurotransmission may contribute to this accelerated motility phenotype and this needs to be investigated further. Future studies will aim to clarify the impact of HFD on upper intestine motility, in particular the GE that is accelerated in non‐diabetic HFD‐fed models but delayed during diabetes.

Another major point is that mice lacking TLR4 do not exhibit WD colonic NOS neuron loss and the resulting colonic delay. In the ENS, both neurons and glial cells express TLR4 (Di Liddo et al., 2015). This expression is mainly found on inhibitory nerves and is stronger in the distal large bowel than in the proximal (Arciszewski et al., 2005; Barajon et al., 2009), suggesting why ileal neurons could be less sensitive to WD‐induced neurodegeneration. In addition, this receptor is important for NOS neurons development because we showed that TLR4−/− mice possessed reduced numbers of colonic nitrergic neurons (Anitha et al., 2014). This was confirmed in the present study, where NADPH‐diaphorase neurons were reduced in TLR4−/−. This may indicate that there are two populations of myenteric NOS neurons, expressing or not expressing TLR4, and further experiments will help to characterize this point.

Finally, our in vitro experiments highlight that low palmitate, similar to circulating values reported in healthy patients (Normand‐Lauziere et al., 2010), potentiates LPS‐induced enteric neurodegeneration. We observed first that increasing doses of palmitate induce neuronal loss but not LPS. Next, we saw that a low dose of LPS (0.5 ng·ml–1) amplifies the neurodegeneration cell loss initiated by palmitate (20 or 30 μm). This synergic action was confirmed by the increased reduction of PGP9.5 expression after co‐treatment with palmitate (20 μm) and LPS. This suggested that ingested saturated fat, once in the plasma, may enhance nitrergic myenteric neuron TLR4 activation by basal LPS. We next confirmed that an enhancement of NOS activity is responsible for cultured neuronal loss because this phenomenon was prevented by the addition of l‐NAME (200 μm). nNOS expression was reduced after treatment with palmitate, without further reduction when LPS was also added.

A similar synergic action between LPS and palmitate has been already reported in macrophages where they trigger inflammation (Schilling et al., 2013). Future studies will focus on mechanisms by which palmitate enhances enteric TLR4 signalling. Saturated fatty acids, in addition to being activators, also facilitate TLR4 dimerization (Wong et al., 2009). Variation at a genetic level may be also involved because we showed that TLR4 expression is increased within myenteric ganglia from HFD‐fed mice (Anitha et al., 2016). In addition, these new data demonstrate that enteric TLR4 signalling controls nNOS activity. LPS‐induced nNOS activation has been already reported in a cultured primary oligodendrocyte precursor (Yao et al., 2010). The question remains as to whether enteric TLR4 induction not only leads to nNOS over activity, but also inducible nitric oxide synthase activation as shown in macrophages, resulting in NO production and oxidative stress (Lee et al., 2005).

Taken together, our data support the idea that WD‐induced neuronal loss is initiated by a plasma FFA increase and is dependent on TLR4 signalling. We propose that the transient plasma FFA increase observed after 6 weeks of WD feeding, along with gut microbiota dysbiosis, enhances LPS action and TLR4 signalling within nitrergic myenteric neurons. This initiates nNOS overactivity, leading to neurodegeneration and colonic transit delay, respectively, as observed after 9 and 12 weeks (Fig. 8). Therefore, hyperlipidaemia is essential in HFD‐induced neuropathies. The key findings of the present study are the development of a new model representing a typical WD not associated with diabetes or inflammation, but by dysbiosis as illustrated by fecal metabolomics. There was evidence of colonic neuronal loss in this model that is prevented by lack of TLR4 and requires the synergic action of LPS and saturated fatty acids. Further studies from our laboratory will evaluate the proportion of vasoactive intestinal peptide neurons affected by WD feeding and also other neuronal phenotypes such as intrinsic primary afferent neurons, excitatory motor neurons, interneurons or interstitial cells of Cajal. Moreover, we plan to investigate liver physiology in WD‐fed mice because it was recently suggested by Rivera et al. (2014) that enteric neuron damage may contribute to the GI complications of fatty liver disease. In conclusion, the results of the present study suggest that a WD induces colonic dysmotility through myenteric neurodegeneration. Understanding these mechanisms can lead to new strategies to prevent colonic dysmotility in countries where HFDs are widespread.