The Inhibitory Effects of Nesfatin-1 in Ventromedial Hypothalamus on Gastric Function and Its Regulation by Nucleus Accumbens

Abstract

Aim: The aim of this study was to investigate the effect of nesfatin-1 signaling in the ventromedial hypothalamus (VMH) on gastric functions, as well as the regulation of these effects by nucleus accumbens (NAc) projections to VMH. Methods: The expression of c-fos in nesfatinergic VMH neurons induced by gastric distension (GD) was measured using the double fluoro-immunohistochemical staining. The firing rates of neurons were monitored with single-unit extracellular electric discharge recording. The projection of nesfatinergic neurons from NAc to VMH was observed by fluorogold retrograde tracer combined with fluoro-immunohistochemical staining. The effect of nesfatin-1 in VMH or electric stimulation in NAc on gastric function was studied by measuring food intake, gastric acid output, gastric motility, and gastric emptying, and the ability of the melanocortin-3/4 receptor antagonist SHU9119 or the anti-nesfatin-1 antibody to block nesfatin-1 in the VMH was assessed. Results: Expression of c-fos was observed in VMH nesfatinergic neurons following GD in rats. Further, nesfatin-1 delivery to single GD-responsive neurons changed the firing rates of these neurons in the VMH. In awake, behaving rats, intra-VMH administration of nesfatin-1 inhibited food intake, gastric acid output, gastric motility, and gastric emptying. These effects were abolished by SHU9119. Fluorogold retrograde tracing showed nesfatinergic neural projection from the NAc to the VMH. Electrical stimulation of NAc modified the firing rates of the VMH neurons and inhibited food intake and gastric functions. The pretreatment with an anti-nesfatin-1 antibody in the VMH reversed the effects of NAc electrical stimulation on the VMH neuronal firing rates and gastric function. Conclusions: Nesfatin-1 in the VMH inhibited food intake, gastric acid output, gastric motility, and gastric emptying. A nesfatinergic pathway between NAc and VMH transmitted metabolism-regulating signals.

Keywords: food intake; gastric function; nesfatin-1; nucleus accumbens; ventromedial hypothalamus.

Figure 1

Effect of gastric distension on c-fos expression in NUCB2/nesfatin-1 positive neurons of VMH. (A,B) c-fos expression in the VMH in control (A) and rats following gastric distension (B). (C,D) Nesfatin-1 expression in the VMH in control (C) and rats following gastric distension (D). (E,F) Double immuno-detection of c-fos and NUCB2/nesfatin-1 in control (E) and rats following gastric distension (F). (G) Quantitative analysis of c-Fos, NUCB2/nesfatin-1, and co-expressing of c-Fos and NUCB2/nesfatin-1 in control and distended rats. ^**P < 0.01 vs. control; ^***P < 0.001 vs. control. White arrow points to neurons co-expressing NUCB2/nesfatin-1 and c-fos.

Figure 2

Effect of nesfatin-1 on the firing rate of GD-responsive neurons in the VMH. (A) GD-E neurons were mostly excited by nesfatin-1, (B) GD-I neurons were mostly inhibited by nesfatin-1, (C) shows the change in firing rate (%) of GD-responsive neurons in the VMH induced by nesfatin-1. Pre-treatment with melanocortin 3/4 receptors antagonist SHU9119 inhibited the effect of nesfatin-1. ^*P < 0.05 vs. NS; ^#P < 0.05 vs. nesfatin-1.

Figure 3

Effect of VMH injection of nesfatin-1 on food intake and gastric function. (A) VMH injection of nesfatin-1 decreased food intake during night period, ^*P < 0.05 vs. NS. (B) Nesfatin-1 attenuated gastric acid secretion induced by 2-deoxy-D-glucose, ^*P < 0.05 vs. NS, ^#P < 0.05 vs. 2-DG + NS,^&P < 0.05 vs. 2-DG + nesfatin-1; (C,D) Gastric motility was inhibited by nesfatin-1, ^*P < 0.05 vs. NS, ^#P < 0.05 vs. Nesfatin-1. (E) Nesfatin-1 delayed gastric emptying of a viscous non-caloric meal in conscious rats.^*P < 0.05 vs. NS, ^#P < 0.05 vs. nesfatin-1.

Figure 4

Co-localization of retrograde labeling from VMH and NUCB2/nesfatin-1 in the neurons of the shell of NAc. (A) The distribution of fluorogold retrotracing from the VMH to the NAc, white arrows indicate FG-labeled neurons, (B) The expression of NUCB2/nesfatin-1 in the NAc, white arrows indicate nesfatin-1positive neurons, (C) Double visualization of fluorogold and nesfatin-1 in NAc shell, white arrows indicate co-labeling of FG and nesfatin-1 in some neurons, (D) Schematic drawing illustrating the position of immunohistochemical figures in the NAc. Scale bars, 100 μm. Ac, core of NAc.

Figure 5

Effect of NAc electrical stimulation on the firing rate of GD-responsive neurons in the VMH (n = 80). (A) GD-E neurons in the VMH were mostly excited by NAc electrical stimulation, (B) GD-I neurons in the VMH were mostly excited by NAc electrical stimulation. (C) Shows the change in firing rate (%) of GD-responsive neurons in the VMH induced by NAc electrical stimulation. Pre-treatment with NUCB2/nesfatin-1 antibody in the VMH reduced the effect of NAc electrical stimulation on VMH GD-responsive neurons. ^*P < 0.05 vs. electrical stimulation.

Figure 6

Effect of electrical stimulation (ES) in NAc shell on food intake and gastric function. (A) high current ES in NAc inhibited food intake, which was reversed by antibody pretreatment in VMH. ^*P < 0.05 vs. Control, ^#P < 0.05 vs. 50 μA. (B) ES in NAc decreased gastric acid secretion induced by 2-DG, which was partially blocked by a nesfatin-1 antibody. ^*P < 0.05 vs. Control, ^#P < 0.05 vs. 2-DG, ^ΔP < 0.05 vs. 2-DG + 50 μA. (C) Gastric motility was decreased by ES in NAc, ^*P < 0.05 vs. Control, ^#P < 0.05 vs. 50 μA. (D) ES in NAc delayed gastric motility, ^*P < 0.05 vs. Control, ^#P < 0.05 vs. 50 μA.

Figure 7

Schematic of the results of this study. Gastric distension activates VMH neurons, including nesfatin-1-positive neurons. VMH injection of nesfatin-1 inhibited food intake, gastric motility, gastric acid output, and emptying. Electrical stimulation of NAc modulates VMH neurons and gastric functions via nesfatinergic projection from the NAc to VMH.