Multiple Beneficial Effects of Ghrelin Agonist, HM01 on Homeostasis Alterations in 6-Hydroxydopamine Model of Parkinson's Disease in Male Rats

Abstract

Background and objective: Developing therapy for non-motor symptoms of Parkinson's disease (PD) is important for improving patients' quality of life. Previously, we reported that the ghrelin receptor agonist, HM01 normalized the decreased 4-h fecal output and levodopa-inhibited gastric emptying in 6-OHDA rats, and activated selective areas in brain and spinal cord. In this study, we evaluated whether chronic HM01 treatment influences motor functions and/or has beneficial effects on non-motor symptoms including alterations of body weight and composition, defecation, feeding and water intake in 6-OHDA rats. Methods: Male rats were microinjected unilaterally into the medial forebrain bundle with either vehicle or 6-OHDA. Three weeks later, we assessed basal body weight, and 24-h fecal output (pellets, weight, dry weight and water content), water intake and food intake (ingested and spillage). Then, HM01 (3 mg/kg) or vehicle was given per gavage daily for 10-12 days and the same parameters were re-assessed daily. Motor behavior (stepping and rotations tests), body composition were monitored before and after the HM01 treatment. Results: 6-OHDA rats showed motor deficits in rotation test induced by apomorphine and stepping test. They also displayed a significant reduction in body weight, water consumption, fecal weight and water content and an increase in food spillage compared to vehicle microinjected rats. Daily oral treatment of HM01 did not modify motor alterations compared to vehicle but significantly increased the body weight, fat mass, and 24-h fecal weight, fecal water content, food and water intake in 6-OHDA rats, while HM01 had no significant effect in vehicle microinjected rats. Fecal weight and water content were both correlated with water intake, but not with food intake. Fat mass, but not body weight, was correlated with food intake. HM01 effects were significant after 24 h and remained similar during the treatment. Conclusions: Chronic treatment with ghrelin agonist, HM01 improved several non-motor symptoms in the rat PD model induced by 6-OHDA lesion including the decrease in body weight, water consumption, fecal weight and water content, and increased food intake while not improving the motor deficits. These findings provide pre-clinical evidence of potential benefits of ghrelin agonists to alleviate non-motor symptoms in PD patients.

Keywords: 6-hydroxydopamine; body composition; defecation; ghrelin agonist; rat; water intake.

Figure 1

Schematic illustration of experimental protocols time course and related determinations.

Figure 2

Photomicrographs of brain sections with tyrosine hydroxylase (TH) immunostaining at levels of the striatum (A,B) and substantia nigra (SN; C,D) from rats microinjected with vehicle (A,C) or 6-OHDA (B,D) in the right site of medial forebrain bundle (mfb). The immunostaining was processed about 6 weeks after mfb microinjection. Marked reductions in striatal fiber density (B) and nigral DA neurons (D) were shown on the side of 6-OHDA injection (right). SNc: substantia nigra pars compacta; SNr: SN pars reticulata; VTA: ventral tegmental area.

Figure 3

Repeated treatments with ghrelin agonist, HM01 did not modify motor impairments in 6-OHDA rat Parkinson’s disease (PD) model. Rats were microinjected with 6-OHDA or vehicle unilaterally into the medial forebrain bundle. HM01 (3 mg/kg) or vehicle was administered og daily for 12 days starting from 4 weeks after microinjection. Stepping (A) and apomorphine-induced rotation (B) tests were performed on the last 2 days at 4–6 h after HM01 treatment in vehicle and 6-OHDA rats. Data are mean ± standard error of the mean (SEM) and animal numbers per group indicated in the graphs. *p < 0.05 vs. vehicle/vehicle by one-way analysis of variance (ANOVA).

Figure 4

Basal body weight (A), 24-h water intake (B), fecal pellets (C), fecal weight (D), dry fecal weight (E), fecal water content (F) and food intake (G) in rats microinjected with 6-OHDA or vehicle unilaterally into the medial forebrain bundle. The assessments were performed in 2–3 days between 3–4 weeks after the microinjection. Data are mean ± SEM, calculated per 300 g body weight (bw). Number of rats/group indicated in each bar of graph A. *p < 0.05 vs. vehicle by Student t-test.

Figure 5

Food spillage assessed 3 weeks after brain microinjection when the rats were housed in cages with a grid to separate the food spills. Food spills (A) and food intake (B) were measured for 24 h and calculated per 300 g body weight (bw). Data are mean ± SEM and animal numbers per group indicated in the graphs. *p < 0.05 vs. vehicle/vehicle by Student t-test.

Figure 6

Effect of orogastric daily administration of HM01 (3 mg/kg) for 10 days on average daily fecal weight (A), fecal water content (B), fecal dry weight (C), fecal pellets (D), and intake of water (E) and food (F) in rats microinjected with 6-OHDA or vehicle unilaterally into the medial forebrain bundle (3–4 weeks before). Data are mean ± SEM calculated per 300 g of body weight (bw). Rats/group are indicated in each bar of graph (A). p < 0.05 as *: vs. vehicle/vehicle, #: vs. 6-OHDA/vehicle and §: vs. vehicle/HM01 by one-way and two-way ANOVA. Correlation of the average 24-h water intake with fecal weight (G) and with fecal water content (H) during 10 days treatment of HM01 (og at 3 mg/kg) analyzed by linear regression.

Figure 7

Body weight and composition of rats microinjected with 6-OHDA or vehicle unilaterally into the medial forebrain bundle after HM01 or vehicle treatments. HM01 (3 mg/kg) or vehicle was administered og daily for 10–12 days starting from 3 to 4 weeks after microinjection of 6-OHDA. (A) Body weight; (B) fat mass; (C) lean mass; (D) total body water. Body fat mass (E) and body weight (F) are correlated with food intake. Data are mean ± SEM. Rats/group are indicated in each bar of graph (A). p < 0.05 as *: vs. vehicle/vehicle, and #: vs. 6-OHDA/vehicle by one-way ANOVA. Two-way ANOVA revealed an interaction between 6-OHDA and HM01 on changes of body weight, lean mass and total body water (p > 0.05), and the HM01 increased body fat mass (p < 0.05).