Diabetic patients suffer from gastrointestinal disorders associated with dysmotility, enteric neuropathy and dysbiosis of gut microbiota, however gender differences are not fully known. Previous studies show that high-fat diet (HFD) causes type two diabetes (T2D) in male mice after 4-8 weeks, but only does so in female mice after 16 weeks. This study sought to determine whether sex influences the development of intestinal dysmotility, enteric neuropathy and dysbiosis in mice fed HFD. We fed 8-week old C57BL6 male and female mice a standard chow diet (SCD) or a 72% Kcal HFD for 8 weeks. We analyzed the associations between sex and intestinal dysmotility, neuropathy and dysbiosis using motility assays, immunohistochemistry and next generation sequencing. HFD ingestion caused obesity, glucose intolerance and insulin resistance in male but not female mice. However, HFD ingestion slowed intestinal propulsive motility in both male and female mice. This was associated with decreased inhibitory neuromuscular transmission, loss of myenteric inhibitory motor neurons, and axonal swelling and loss of cytoskeletal filaments. HFD induced dysbiosis and changed the abundance of specific bacteria, especially Allobaculum, Bifidobacterium and Lactobacillus, which correlated with dysmotility and neuropathy. Female mice had higher immunoreactivity and numbers of myenteric inhibitory motor neurons, matching larger amplitudes of inhibitory junction potentials.

Symptoms of diabetic gastrointestinal dysmotility indicate neuropathy of the enteric nervous system. Long-standing diabetic enteric neuropathy has not been fully characterized, however. We used prolonged high fat diet ingestion (20 weeks) in a mouse model to mimic human obese and type 2 diabetic conditions, and analyzed changes seen in neurons of the duodenal myenteric plexus. Ganglionic and neuronal size, number of neurons per ganglionic area, density indices of neuronal phenotypes (immunoreactive nerve cell bodies and varicosities per ganglion or tissue area) and nerve injury were measured. Findings were compared with results previously seen in mice fed the same diet for 8 weeks. Compared to mice fed standard chow, those on a prolonged high fat diet had smaller ganglionic and cell soma areas. Myenteric VIP- and ChAT-immunoreactive density indices were also reduced. Myenteric nerve fibers were markedly swollen and cytoskeletal protein networks were disrupted. The number of nNOS nerve cell bodies per ganglia was increased, contrary to the reduction previously seen after 8 weeks, but the density index of nNOS varicosities was reduced. Mice fed high fat and standard chow diets experienced an age-related reduction in total neurons, biasing towards neurons of sensory phenotype. Meanwhile ageing was associated with an increase in excitatory neuronal markers. Collectively, these results support a notion that nerve damage underlies diabetic symptoms of dysmotility, and reveals adaptive ENS responses to the prolonged ingestion of a high fat diet. This highlights a need to mechanistically study long-term diet-induced nerve damage and age-related impacts on the ENS.

BackgroundHigh‐fat diet, microbial alterations and lipopolysaccharide (LPS) are thought to cause enteric diabetic neuropathy and intestinal dysmotility. However, the role of the gut microbiota, lipoteichoic acid (LTA) from Gram‐positive bacteria and short‐chain fatty acids (SCFAs) in the development of diabetic enteric neuropathy and intestinal dysmotility is not well understood. Our aim was to examine the role of the gut microbiota, LTA and SCFAs in the development of diabetic enteric neuropathy and intestinal dysmotility.MethodsWe fed germ‐free (GF) and conventionally raised (CR) mice either a high‐fat (HFD) or standard chow diet (SCD) for 8 weeks. We analyzed the microbial community composition in CR mice using 16S rRNA sequencing and damage to myenteric neurons using immunohistochemistry. We also studied the effects of LPS, LTA, and SCFAs on duodenal muscularis externa contractions and myenteric neurons using cultured preparations.Key ResultsHigh‐fat diet ingestion reduced the total number and the number of nitrergic myenteric neurons per ganglion in the duodenum of CR but not in GF‐HFD mice. GF mice had fewer neurons per ganglion compared with CR mice. CR mice fed a HFD had increased abundance of Gram‐positive bacteria. LTA and LPS did not affect the frequency of duodenal muscularis contractions after 24 hours of cultured but reduced the density of nitrergic myenteric neurons and increased oxidative stress and TNFα production in myenteric ganglia. SCFAs did not affect muscularis contractions or injure myenteric neurons.Conclusions & InferencesGut microbial alterations induced increase in Gram‐positive bacterial LTA may contribute to enteric neuropathy.

Neuropathy of the enteric nervous system (ENS) is one of the major underlying causes of debilitating gastrointestinal (GI) motility disorders in diabetic patients. Recent studies suggest that diet-microbiome-host interactions -in particular, excess dietary calories, microbial metabolites, lipopolysaccharide (LPS) and disrupted mucosal barrier -play a fundamental role in the pathobiology of obesity and type II diabetes (Boulangé et al. 2016). Furthermore, the composition of the GI microbiome influences ENS physiology, neurochemistry and nerve cell health, as well as GI motility patterns, and vice versa (Kashyap et al. 2013). However, links between such interactions and the mechanisms underlying this neuropathy are not fully understood.In this issue of The Journal of Physiology, Reichardt et al. (2017) address the question of whether ingesting a Western diet (WD) rich in saturated fatty acids and the associated alteration to the gut microbiome disrupts motility, and induces loss of nitrergic myenteric neurons (NMNs), the phenotype that is commonly damaged in diabetic neuropathy (Yarandi & Srinivasan, 2014). The rationale is that most studies have used a high fat diet (HFD; 60-72% kcal from fat), leading to little understanding of how a normal WD affects GI motility, the ENS and their role in the pathobiology of the metabolic syndrome and diabetes. The authors used C57BL/6 mice fed WD (35% kcal from fat, enriched in palmitate) or a regular diet (RD, 16.9% kcal from fat, 4× less palmitate) for 3, 6, 9 and 12 weeks, and TLR4 -/and germ free mice fed WD and RD diets for 6 weeks. Gastrointestinal motility was measured, and damage to myenteric neurons and NMNs was studied in the ileum and proximal colon. Palmitateand LPS-induced damage to NMNs and the role of nitric oxide synthase (nNOS) in such injury were determined in vitro using immortalized myenteric neurons. Faecal metabolites, systemic and visceral fat and mucosal inflammation were analysed.After ingesting WD for 6 weeks, mice were 'overweight' , developed gut microbiota dysbiosis, altered faecal metabolites, increased intraluminal LPS and increased plasma free fatty acid (FFA) levels. Interestingly, unlike HFD, WD did not elicit hyperglycaemia, endotoxaemia and inflammation, suggesting the need to define key differences between the effect of HFD and WD on gut microbiome and metabolic profiles. Another important observation was that WD caused GI dysmotility before Circular muscle Longitudinal muscle Lumen Mucosa Submucosa Muscularis Myenteric plexus Submucosal plexus Extrinsic nerve fibre Oral aboral Figure 1. Excess saturated FFAs in WD causes dysbiosis of the gut microbiome, dysmotility and nerve cell injury in the myenteric plexusElevated levels of FFAs (red dots) in the intestinal lumen and circulation (arrowheads) cause dysmotility prior to triggering microscopically visible damage to NMNs (red arrow). This suggests that FFAs alter the physiology of the ENS directly, or indirectly via interaction with enterocytes (grey), enteroendocrine cells (red), intersti...

Background: Damage to enteric neurons and impaired gastrointestinal muscle contractions cause motility disorders in 70% of diabetic patients. It is thought that enteric neuropathy and dysmotility occur before overt diabetes, but triggers of these abnormalities are not fully known. We tested the hypothesis that intestinal contents of mice with and without high-fat diet-(HFD-) induced diabetic conditions contain molecules that impair gastrointestinal movements by damaging neurons and disrupting muscle contractions. Methods: Small and large intestinal segments were collected from healthy, standard chow diet (SCD) fed mice. Filtrates of ileocecal contents (ileocecal supernatants; ICS) from HFD or SCD mice were perfused through them. Cultured intact intestinal muscularis externa preparations were used to determine whether ICS and their fractions obtained by solid-phase extraction (SPE) and SPE subfractions collected by high-performance liquid chromatography (HPLC) disrupt muscle contractions by injuring neurons and smooth muscle cells. Key Results: ICS from HFD mice reduced intestinal motility, but those from SCD mice had no effect. ICS, aqueous SPE fractions and two out of twenty HPLC subfractions of aqueous SPE fractions from HFD mice blocked muscle contractions, caused a loss of nitrergic myenteric neurons through inflammation, and reduced smooth muscle excitability. Lipopolysaccharide and palmitate caused a loss of nitrergic myenteric neurons but did not affect muscle contractions. Conclusions & Inferences: Unknown molecules in intestinal contents of HFD mice trigger enteric neuropathy and dysmotility. Further studies are required to identify the toxic molecules and their mechanisms of action.