Ghrelin-induced orexigenic effect in rats depends on the metabolic status and is counteracted by peripheral CB1 receptor antagonism

Abstract

Ghrelin is an endogenous regulator of energy homeostasis synthesized by the stomach to stimulate appetite and positive energy balance. Similarly, the endocannabinoid system is part of our internal machinery controlling food intake and energy expenditure. Both peripheral and central mechanisms regulate CB1-mediated control of food intake and a functional relationship between hypothalamic ghrelin and cannabinoid CB1 receptor has been proposed. First of all, we investigated brain ghrelin actions on food intake in rats with different metabolic status (negative or equilibrate energy balance). Secondly, we tested a sub-anxiogenic ultra-low dose of the CB1 antagonist SR141716A (Rimonabant) and the peripheral-acting CB1 antagonist LH-21 on ghrelin orexigenic actions. We found that: 1) central administration of ghrelin promotes food intake in free feeding animals but not in 24 h food-deprived or chronically food-restricted animals; 2) an ultra-low dose of SR141716A (a subthreshold dose 75 folds lower than the EC50 for induction of anxiety) completely counteracts the orexigenic actions of central ghrelin in free feeding animals; 3) the peripheral-restricted CB1 antagonist LH-21 blocks ghrelin-induced hyperphagia in free feeding animals. Our study highlights the importance of the animaĺs metabolic status for the effectiveness of ghrelin in promoting feeding, and suggests that the peripheral endocannabinoid system may interact with ghrelińs signal in the control of food intake under equilibrate energy balance conditions.

Figure 1. Effects of ghrelin (0.25 µg, 0.5 µg and 1.0 µg, i.c.v.) on accumulated food intake in free feeding, 24 h food-deprived (fasted) or chronically food-restricted animals 60 min and 120 min after administration.

Free-feeding animals (n = 9 in each group) had free access to standard pellets of food; fasted rats (n = 9 in each group) were totally deprived for food during 24 h before the experiment; and chronic food-restricted rats (n = 9 in each group) were limited daily for food intake until body weights reached 80% of free-feeding values (20–25 days). The amount of ingested food in vehicle-injected animals was higher in the food-restricted group, following by the fasted group and free feeding rats, respectively. Ghrelin administration induced hyperphagia at any dose tested exclusively in animals fed ad libitum. No effect of ghrelin administration was observed in fasted or food-restricted animals. Data are means ± SEM of accumulated food intake. Different from free feeding vehicle-injected rats: ^###P<0.001. Different from fasted vehicle-injected rats: ^£££P<0.001. Different from vehicle-injected rats in the same condition (free feeding): *P<0.05. **P<0.01. ***P<0.001.

Figure 2. EC₅₀ of a dose-response curve of SR141716A on food intake and anxiety.

A) effect of SR141716A (0, 0.1, 1.0 and 3.0 mg/kg, i.p.) on responses for food; B) effect of SR141716A (0, 0.1, 1.0 and 3.0 mg/kg, i.p.) on anxiety, expressed as latency to emerge a defensive withdrawal behaviour. The figures represent a re-analysis of the data previously published . The half maximal effective concentration (EC₅₀) is 2.2 mg/kg and 2.3 mg/kg for food inhibition and induction of anxiety behavior, respectively.

Figure 3. Pretreatment with an ultra-low dose of SR141716A or the peripheral CB1 antagonist LH-21 on the orexigenic effect induced by ghrelin in free feeding animals.

Administration of ghrelin (0.5 µg, i.c.v.) increased food intake in free feeding animals. A) The subeffective dose of SR141716A (0.03 mg/kg, i.p) counteracted ghrelin-induced orexigenic effect. As expected, the ultra-low dose of SR141716A showed no effect on food intake in vehicle-treated animals. B) Pretreatment with LH-21 (3 mg/kg, i.p.) counteracted the increase in food intake induced by ghrelin and had no effect in vehicle-treated rats. Data are means ± SEM of 6–10 determinations per group. Different from vehicle-injected rats: *P<0.05. ***P<0.001; different from ghrelin treatment: ^# P<0.05, ^## P<0.01, ^### P<0.001.