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. 2014 Feb;146(2):473-83.e3.
doi: 10.1053/j.gastro.2013.10.053. Epub 2013 Oct 25.

MicroRNA 375 mediates palmitate-induced enteric neuronal damage and high-fat diet-induced delayed intestinal transit in mice

Behtash Ghazi Nezami  1 , Simon M Mwangi  1 , Jai Eun Lee  1 , Sabrina Jeppsson  1 , Mallappa Anitha  1 , Shadi S Yarandi  1 , Alton B Farris 3rd  2 , Shanthi Srinivasan  3
Affiliations

MicroRNA 375 mediates palmitate-induced enteric neuronal damage and high-fat diet-induced delayed intestinal transit in mice

Behtash Ghazi Nezami et al. Gastroenterology. 2014 Feb.

Abstract

Background & aims: A high-fat diet (HFD) can cause serious health problems, including alteration of gastrointestinal transit, the exact mechanism of which is not clear. Several microRNAs (miRNAs) are involved in energy homeostasis, lipid metabolism, and HFD-induced weight gain. We investigated the role of miRNAs in HFD-induced damage to the enteric nervous system.

Methods: Male mice were fed a HFD (60% calories from fat) or regular diets (18% calories from fat) for 11 weeks. Mice on regular diets and HFDs were given intraperitoneal injections of Mir375 inhibitor or a negative control. Body weights, food intake, stool indices, and gastrointestinal transit (following Evans blue gavage) were measured. An enteric neuronal cell line (immorto-fetal enteric neuronal) and primary enteric neurons were used for in vitro studies.

Results: HFD delayed intestinal transit, which was associated with increased apoptosis and loss of colonic myenteric neurons. Mice fed a low-palmitate HFD did not develop a similar phenotype. Palmitate caused apoptosis of enteric neuronal cells associated with mitochondrial dysfunction and endoplasmic reticulum stress. Palmitate significantly increased the expression of Mir375 in vitro; transfection of cells with a Mir375 inhibitor prevented the palmitate-induced enteric neuronal cell apoptosis. Mir375 expression was increased in myenteric ganglia of mice fed HFD and associated with decreased levels of Mir375 target messenger RNAs, including Pdk1. Systemic injection of a Mir375 inhibitor for 5 weeks prevented HFD-induced delay in intestinal transit and morphologic changes.

Conclusions: HFDs delay colonic transit, partly by inducing apoptosis in enteric neuronal cells. This effect is mediated by Mir375 and is associated with reduced levels of Pdk1. Mir375 might be targeted to increase survival of enteric neurons and gastrointestinal motility.

Keywords: 3-phosphoinositide-dependent protein kinase-1; ChAT; ENS; ER; FFAs; FITC; GI; Gastrointestinal; HFD; Hyperlipidemia; LP-HFD; MicroRNAs; Motility; NADPH diaphorase; Pdk1; RD; SOD; choline acetyltransferase; endoplasmic reticulum; enteric nervous system; fluorescein isothiocyanate; free fatty acids; gastrointestinal; high-fat diet; low-palmitate HFD; mRNA; messenger RNA; miRNAs; microRNAs; nicotinamide adenine dinucleotide phosphate; regular diet; superoxide dismutase.

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Conflict of interest statement

Conflict of interest: The authors have no conflict of interests to declare

Figures

Figure 1
Figure 1. Mice fed a HFD develop higher body weight and lipid profile and slower GI motility
(A) Body weight and percentage weight gain in mice fed a HFD or RD for 11 weeks. (B) Serum lipid concentrations were determined in the same groups of mice as in figure. 1A after overnight fasting. (C) The stool characteristics in the HFD and RD mice, and in ob/ob mice. (D) Comparison of the mean transit times of gastrointestinal transit marker between mice fed a HFD or RD showing significantly slower whole-intestinal transit in HFD (greater transit time). (E) Colonic bead propulsion test revealing slower colonic transit (higher time to expel) in HFD compared with RD. HFD indicates high-fat diet; RD, regular diet. Results presented as mean ± SEM. *P<0.05, ** P<0.01, ***P<0.001, n=5.
Figure 2
Figure 2. HFD decreases number of myenteric neurons in the proximal colon by inducing apoptosis
(A) Whole mount preparations of proximal colon from mice on HFD and RD for 11 weeks stained for peripherin, nNOS and ChAT. (B) Representative photographs of peripherin, nNOS and ChAT stained neurons. Scale bar: 100 μm. (C) Proximal colon of mice fed a HFD or RD for 11 weeks assessed for nNOS protein. (D) Proximal colon sections stained for Cleaved caspase-3 and peripherin. (E) Representative immunofluorescence staining photos. Original magnifications: 20x. Scale bar: 50 μm. Results presented as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001, n=5.
Figure 3
Figure 3. Palmitate is taken up by enteric neuronal cells and induces cell death
(A) Representative microphotographs of neuronal cell line treated with palmita te and stained with oil red O. (B) Primary cell cultures of enteric neurons treated with palmitate with arrows indicate lipid deposits in the neuronal clusters. (C) MTS survival assay in enteric neurons after 24 hours of palmitate exposure. (D) Western blot analysis of Cleaved caspase-3 protein level in enteric neuronal cell lines. Graph depicts the average of five separate experiments. (E) IF staining of primary enteric neuronal cells. Arrows indicate cells positive for Cleaved caspase-3. Results presented as mean ± SEM, *P<0.05, ** P<0.01, ***P<0.001.
Figure 4
Figure 4. Palmitate induces ER stress and mitochondrial damage and causes up-regulation of autophagy as a survival mechanism
(A) Palmitate significantly increases CHOP levels in enteric neuronal cell line, and Ultra-structure of ER in primary en teric neurons treated with palmitate shows a marked ER swelling, associated with a dose-dependent decrease in the number of ER. Representative Electron micrographs in which ER area is traced in green. (B) Representative photographs of enteric neurons treated with palmitate for 24 hours and stained for mitochondrial SOD marker (MitoSOX) to assess oxidative stress. Western blot analysis also shows a decrease in the COX IV as a marker of mitochondrial mass. (C) Ultra-structural study of the number and area occupied by mitochondria (traced in red) in primary enteric neuronal culture. Scale bar: 0.5 μm. (D) Western blot assessing LC3B protein level after enteric neurons are exposed to palmitate. Graph depicts the average of five separate experiments. (E) Autophagy flux measured by IF analysis of IM-PEN enteric neuronal cell line stained for LC3B after palmitate treatment in the presence of chloroquine. Bottom panel is high magnification representative photos of primary enteric neurons showing the cells positive for LC3B as red. (F) The level of mitochondrial SOD in the enteric neurons pre-treated with rapamycin followed by exposure to palmitate. Results presented as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5. In vitro palmitate increases microRNA expressions and Mir375 target mRNAs decrease in myenteric ganglia of the HFD fed mice
(A) Quantitative RT-PCR analysis of Mir375, Mir146a and Mir34a in the enteric neuronal cell line after treating with palmitate. (B) The expression of Mir375 and its target mRNAs in the myenteric ganglia isolated by LCM from the proximal colon of the mice treated with RD or HFD. Right panel shows how myenteric ganglion was isolated by LCM for the PCR. n= at least 3 (C) The decrease in Pdk1 mRNA in myenteric neurons was associated with a significant decrease in the protein level of Pdk1 protein in the proximal colon. (D) Similar decrease was observed in the Pdk1 protein level after palmitate treatment in enteric neuronal cells. (E) In vitro Mir375 inhibitor prevents the detrimental effect of palmitate as evident by preventing the increase of Cleaved caspase-3 in neuronal cells. HFD indicates high-fat diet; RD, regular diet. Results presented as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001.
Figure 6
Figure 6. Systemic Mir375 injection causes GI dysmotility and delayed transit, which is associated by myenteric neuronal loss and apoptosis
(A) Stool indices of Mir375 and control miRNA injected mice. (B) Comparison of the mean transit times of Evans blue. (C) Geometric center determination for small intestine. Distribution of FITC-dextran marker 1 hour after gavage displaying slower transit in Mir375 injected mice. (D) Whole mount preparations of proximal colon from Mir375 and control miRNA injected mice stained for NADPH-diaphorase and AChE. (E) Analysis of proximal colon sections stained for Cleaved caspase-3 and peripherin. Original magnifications: 20x. Scale bar: 50 μm. Results presented as mean ± SEM, *P<0.05, **P<0.01, n=6.
Figure 7
Figure 7. Systemic Mir375 inhibitor prevents the development of delayed intestinal transit and protects against the myenteric neuronal loss
(A) Stool indices of the mice fed HFD or RD, treated with Mir375 inhibitor or negative control or vehicle. (B) Comparison of the mean transit times of Evans blue. (C) Geometric center determination for small intestine and distribution of FITC-dextran marker 1 hour after gavage suggesting slower intestinal transit. (D) Whole mount preparation of proximal colon for NADPH-diaphorase. HFD indicates high-fat diet; RD, regular diet. Original magnifications: 10x. Scale bar: 100 μm. Results presented as mean ± SEM, *P<0.05, ***P<0.001. n=3–5
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