Maternal High Fat Diet Induces Myenteric Neuronal Loss and Upregulates Myenteric Glia Prior the Development of Obesity or Inflammation

Abstract

Diet‐induced obesity disrupts gastrointestinal (GI) functions possibly due to a loss of myenteric neurons, although few studies have assessed the effects of diet on the development of these neurons. The aim of the present study was to assess the effects of exposure to a high fat diet (HFD) during the development period on myenteric neurons and glial cells in rats and the possible role of systemic and local inflammation.Sprague‐Dawley rats were fed a control or HFD (14% or 60% kcal from fat, respectively) from embryonic day 13; the fundus, corpus and duodenum were removed and fixed at postnatal ages 2, 4, 6 and 12 weeks (n=4 for each timepoint). Tissues were processed for immunohistochemical detection of total myenteric neurons (protein gene product 9.5; PGP9.5) as well as cholinergic (choline acetyltransferase; ChAT) and nitrergic (nitric oxide synthase; NOS) neurons, RNA‐binding protein (HuC/D) and glial cells (glial fibrillary acid protein; GFAP). Confocal microscope images were analyzed using Image J software. Serum and gastrointestinal levels of the cytokines IL‐1β and IL‐6 were quantified via multiplex and qRT‐PCR, respectively.HFD rats were overweight but not regarded as obese until 12 weeks of age (292±12g vs 380±12g). By 4 weeks of age, however, a decrease in number of myenteric neurons per ganglia was observed in the fundus (29 ± 3.6 vs. 38 ± 2.6 neurons/ganglia; P<0.05) and corpus (34.6 ± 0.8 vs. 44.3 ± 1.2 neurons/ganglia; P<0.05), and by 6 weeks of age in the duodenum (26 ± 1.5 vs. 34.6 ± 2.9 neurons/ganglia; P<0.05). A selective loss of nitrergic neurons was observed by 12 weeks of age in the fundus (13.5 ± 0.9% vs 20 ± 0.9% of total neurons; P<0.05), corpus (10.7 ± 0.7% vs 19 ± 3.1%; P<0.05), and duodenum (8.5 ± 2.5% vs 16.5 ± 2.0%; P<0.05). HFD exposure increased myenteric glial cell density by at 4 weeks of age in the all regions studied (fundus 69.9±2.6 vs 62.32±3.1 pixels/cm2; corpus 87.0±7.5 vs 62.9±4.9 pixels/cm2; and duodenum (96.1±5.9 vs 56.9±4.0 pixels/cm2; P<0.05 for each), suggesting a possible diet‐induced inflammation of the gastrointestinal tract. While nuclear aggregation of HuC/D, suggesting compromised neuronal health, was observed in myenteric neurons at 12 weeks of age in all regions studied (fundus 68.6 ± 8.5% vs 41.6 ± 6.6% nuclear localization; corpus 55.0 ± 5.4% vs 23.5 ± 3.4%; and duodenum 52.4 ± 5.5% vs 34.7 ± 1.0%; P<0.05 for each), there were no consistent alterations in serum or gastrointestinal expression of IL‐1β (22.98±2.73 vs 28.57±2.03 pg/mL and 0.67±0.19 vs 0.32±0.09 2DDCT, respectively; P>0.05) and IL‐6 (88±10.17 vs 115±7.34 pg/mL; 0.94±0.39 vs 1.00±0.34 vs ± 2DDCT;P>0.05).The present study suggests that exposure of rats to a HFD from the developmental period induces loss of inhibitory nitrergic neurons accompanied by glial proliferation even prior to the onset of obesity and in the absence of elevated cytokine levels, suggesting that diet may alter gastrointestinal functions independently of increased adiposity or adipose‐related inflammation.Support or Funding InformationFunded by NIH 078364 and NSF IOS1148978