Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor

Abstract

Nutritional factors can induce profound neuroplastic changes in the enteric nervous system (ENS), responsible for changes in gastrointestinal (GI) motility. However, long-term effects of a nutritional imbalance leading to obesity, such as Western diet (WD), upon ENS phenotype and control of GI motility remain unknown. Therefore, we investigated the effects of WD-induced obesity (DIO) on ENS phenotype and function as well as factors involved in functional plasticity. Mice were fed with normal diet (ND) or WD for 12 weeks. GI motility was assessed in vivo and ex vivo. Myenteric neurons and glia were analysed with immunohistochemical methods using antibodies against Hu, neuronal nitric oxide synthase (nNOS), Sox-10 and with calcium imaging techniques. Leptin and glial cell line-derived neurotrophic factor (GDNF) were studied using immunohistochemical, biochemical or PCR methods in mice and primary culture of ENS. DIO prevented the age-associated decrease in antral nitrergic neurons observed in ND mice. Nerve stimulation evoked a stronger neuronal Ca(2+) response in WD compared to ND mice. DIO induced an NO-dependent increase in gastric emptying and neuromuscular transmission in the antrum without any change in small intestinal transit. During WD but not ND, a time-dependent increase in leptin and GDNF occurred in the antrum. Finally, we showed that leptin increased GDNF production in the ENS and induced neuroprotective effects mediated in part by GDNF. These results demonstrate that DIO induces neuroplastic changes in the antrum leading to an NO-dependent acceleration of gastric emptying. In addition, DIO induced neuroplasticity in the ENS is likely to involve leptin and GDNF.

Figure 1. Impact of diet-induced obesity upon ENS phenotype in antrum and jejunum

A, immunohistochemical labelling of antral myenteric plexus in mice after 12 weeks of normal diet (ND) and Western diet (WD). Myenteric neurons were stained with anti-Hu C/D and anti-nNOS antibodies. Enteric glial cells were stained with anti-Sox-10 antibody. Scale bar = 50 μm. B–D, quantitative analysis of the number of Hu-IR neurons (B), nNOS-IR neurons (C) and Sox-10-IR cells (D) per ganglion was performed in antrum and jejunum in mice before the beginning of the diet (T0, n= 5) and in mice after 12 weeks of ND or WD (n= 7–11). Values are expressed as means ± SEM. *P < 0.05, Kruskall–Wallis test.

Figure 2. Impact of diet-induced obesity upon µCa²⁺½_i in response to electric stimulation of interganglionic fibre tracts in myenteric neurons

A, typical recordings showing calcium responses in a myenteric neuron from normal diet (ND) mice and from Western diet (WD) mice during and following interganglionic fibre tract train pulse stimulation. Calcium peaks can be observed during each electrical pulse. B, percentage of changes of resting fluorescence (ΔF/F) induced by train pulse stimulations of interganglionic fibre tracts in cells of the myenteric plexus (ND: n= 30 cells, 8 ganglia, 4 mice and WD: n =19 cells, 7 ganglia, 4 mice). Values are expressed as means ± SEM. *P < 0.05, Mann–Whitney test.

Figure 3. Impact of diet-induced obesity upon gastric emptying, small intestinal transit and neuromuscular transmission in antrum

A, gastric emptying of solids in mice after 12 weeks of ND or WD following intraperitoneal injection of saline (n= 16–17) or l-NAME solution (n= 7–9). Values are expressed as means ± SEM. *P < 0.05, Kruskall–Wallis test. B, small intestinal transit in mice after 12 weeks of ND or WD (n= 10). Values are expressed as means ± SEM. *P < 0.05, Mann–Whitney test. C, typical recordings showing EFS-mediated contractile responses in ND and WD mice in basal conditions. D, quantitative analysis of the area under the curve (AUC) measured during EFS in ND and WD mice in basal conditions, in the presence of l-NAME and in the presence of l-NAME and atropine (n= 25–27). Values are expressed as means ± SEM. *P < 0.05, Kruskall–Wallis test. E, quantitative analysis of l-NAME sensitive AUC (ΔAUC; difference between AUC in the presence of l-NAME and AUC without drug) in ND and WD mice (n= 25–27). Values are expressed as means ± SEM. *P < 0.05, Mann–Whitney test. F, quantitative analysis of atropine sensitive AUC (ΔAUC; difference between AUC in the presence of l-NAME and atropine and AUC in the presence of l-NAME) in ND and WD mice (n= 25–27). Values are expressed as means ± SEM. *P < 0.05, Mann–Whitney test.

Figure 4. Impact of diet-induced obesity upon GDNF and leptin content in antrum. Involvement of leptin in the regulation of GDNF production

A, evolution of GDNF concentration in antrum in mice before diet (n= 5), and after 4 weeks (n= 5), 8 weeks (n= 5) and 12 weeks (n= 10) of normal diet (ND) or Western diet (WD). Values are expressed as means ± SEM. *P < 0.05, two-way ANOVA. B, evolution of leptin concentration in antrum in mice before diet (n= 5), and after 4 weeks (n= 5), 8 weeks (n= 5) and 12 weeks (n= 10) of ND or WD. Values are expressed as means ± SEM. *P < 0.05, two-way ANOVA. C, immunohistochemical labelling of antral myenteric plexus with anti-ObR, anti-PGP 9.5 and anti-GFAP antibodies. ObR-IR cells coexpressed PGP 9.5 (arrows) and GFAP (arrowheads). Scale bar = 50 μm. D, GDNF concentration in antrum in 12 week-old WT, db/+ and db/db mice (n= 4–7). Values are expressed as means ± SEM. *P < 0.05, Kruskall–Wallis test. E, GDNF concentration in supernatants of primary cultures of ENS treated with different doses of leptin for 24 h, normalized to control (n= 14–23; control 69.5 ± 5.4 pg ml⁻¹). Values are expressed as means ± SEM. *P < 0.05 vs. control, Kruskall–Wallis test.

Figure 5. Involvement of GDNF in neuroprotective effects of leptin

A, percentage of active caspase-3-IR neurons (of total Hu-IR neurons) in primary cultures of ENS treated with leptin and anti-GDNF neutralizing antibody (GDNF neut. Ab) and normalized to control (n= 10–13; control: 0.48 ± 0.23%). Values are expressed as means ± SEM. *P < 0.05, Kruskall–Wallis test. B, percentage of active caspase-3-IR neurons (of total Hu-IR neurons) in primary cultures of ENS treated with GDNF and normalized to control (n= 5–7; control: 0.25 ± 0.08%). Values are expressed as means ± SEM. *P < 0.05, Mann–Whitney test.