Evidence that resistin acts on the mechanical responses of the mouse gastric fundus

Abstract

Resistin, among its several actions, has been reported to exert central anorexigenic effects in rodents. Some adipokines which centrally modulate food intake have also been reported to affect the activity of gastric smooth muscle, whose motor responses represent a source of peripheral signals implicated in the control of the hunger-satiety cycle through the gut-brain axis. On this basis, in the present experiments, we investigated whether resistin too could affect the mechanical responses in the mouse longitudinal gastric fundal strips. Electrical field stimulation (EFS) elicited tetrodotoxin- and atropine-sensitive contractile responses. Resistin reduced the amplitude of the EFS-induced contractile responses. This effect was no longer detected in the presence of L-NNA, a nitric oxide (NO) synthesis inhibitor. Resistin did not influence the direct muscular response to methacholine. In the presence of carbachol and guanethidine, EFS elicited inhibitory responses whose amplitude was increased by resistin. L-NNA abolished the inhibitory responses evoked by EFS, indicating their nitrergic nature. In the presence of L-NNA, resistin did not have any effect on the EFS-evoked inhibitory responses. Western blot and immunofluorescence analysis revealed a significant increase in neuronal nitric oxide synthase (nNOS) expression in neurons of the myenteric plexus following resistin exposure. In conclusion, the present results offer the first evidence that resistin acts on the gastric fundus, likely through a modulatory action on the nitrergic neurotransmission.

Keywords: gastric motility; neuromodulation; nitric oxide; resistin; satiety signals.

FIGURE 1

Influence of resistin on the neurally-evoked contractions and its lack of effects on the response to methacholine. (A,B): representative traces showing the EFS-evoked contractile responses at 4 and 8 Hz stimulation frequency (left hand traces) and their abolition in the presence of TTX [(A), right hand trace] or atropine [(B), right hand trace]. (C,D): representative trace (C) showing that the amplitude of the neurally-induced excitatory responses at both stimulation frequencies employed (left hand trace) is decreased in the presence of 60 ng/ml resistin (right hand trace). Bar chart (D) of the effects of resistin on the mean amplitude of the EFS-evoked contractions at 4 and 8 Hz stimulation frequency. Amplitude of contractile responses is expressed as percentage of the muscular contraction induced by 2 × 10⁻⁶ M methacholine, taken as 100%. All values are means ± SEM of 8 strips (from 5 animals). *p < 0.05 vs Ctrl (Student’s t-test). (E,F): Representative traces showing the response to methacholine (left hand traces), whose amplitude is not affected by TTX [(E), right hand trace] or resistin [(F), right hand trace].

FIGURE 2

Lack of effects of resistin on the neurally-evoked contractile responses in the presence of L-NNA. (A): representative trace showing the EFS-evoked contractile responses at 4 and 8 Hz stimulation frequency (left hand panel). Addition of 2 × 10⁻⁴ M L-NNA to the bath medium enhances the amplitude of the neurally-evoked contractile responses (middle panel). In the presence of L-NNA, resistin (60 ng/ml) no longer depresses the amplitude of the EFS-evoked contractile responses (right hand panel). (B): bar chart showing the lack of effects of resistin on the mean amplitude of the neurally-evoked contractile responses in the presence of L-NNA. Amplitude of contractile responses is expressed as percentage of the muscular contraction induced by 2 × 10⁻⁶ M methacholine, taken as 100%. All values are means ± SEM of 8 strips (from 4 animals). *p < 0.05 L-NNA vs its own Ctrl; **p < 0.05 L-NNA + resistin vs its own Ctrl and p > 0.05 L-NNA + resistin vs L-NNA (ANOVA with Bonferroni’s post hoc test).

FIGURE 3

Influence of resistin on the neurally-evoked relaxant responses and its lack of effects in the presence of L-NNA. (A,B): representative trace showing that the EFS-evoked relaxant responses at 4 and 8 Hz stimulation frequency [(A), left hand trace] are increased in amplitude in the presence of 60 ng/ml resistin (right hand trace). Bar chart (B) of the effects of resistin on the mean amplitude of the EFS-induced relaxant responses. Amplitude values refer to the maximal peak obtained during the stimulation period and represent percentage decreases relative to the muscular tension induced by CCh (1 × 10⁻⁶ M), taken as 100%. All values are means ± SEM of 8 strips (from 4 mice). *p < 0.05 vs Ctrl (Student’s t-test). (C): Representative tracing showing that the EFS-evoked inhibitory responses at 4 and 8 Hz stimulation frequency (left hand trace) are abolished by 2 × 10⁻⁴ M L-NNA (middle trace) and the subsequent addition of resistin (60 ng/ml) to the bath medium has no longer effects (right hand trace) on the neurally-evoked relaxant responses. Note the absence of responses to EFS at 4 and 8 Hz stimulation frequency in the presence of either L-NNA or L-NNA + resistin.

FIGURE 4

Resistin up-regulates nNOS expression. (A) Effect of resistin exposure (30 min) on nNOS expression in mouse gastric fundus assayed by Western blotting: representative bands from a typical experiment. The two representative lanes for control and resistin samples are biological replicates from separate animals. (B) Quantitative analysis. Columns are means ± SEM. Significance of differences (Student’s t-test for two independent samples from 3 mice): *p < 0.05 vs controls (Ctrl). (C) Representative photomicrographs of gastric tissue from control and resistin-exposed (30 min) preparations showing double immunofluorescence labeling. (A1–A4) Co-localization of nNOS and UCH-L1 in control mice: (A1) nNOS signal (green channel); (A2) UCH-L1 signal (red channel); (A3) DAPI; (A4) merged images. (B1–B4) Co-localization of nNOS and UCH-L1 in resistin-exposed preparations: B1 nNOS signal (green channel); (B2) UCH-L1 signal (red channel); (B3) DAPI; (B4) merged images. Scale bar: 20 μm.