Psychopharmacology (2013) 229:275–283 DOI 10.1007/s00213-013-3096-7
ORIGINAL INVESTIGATION
Central manipulation of dopamine receptors attenuates the orexigenic action of ghrelin Amparo Romero-Picó & Marta G. Novelle & Cintia Folgueira & Miguel López & Ruben Nogueiras & Carlos Diéguez
Received: 3 September 2012 / Accepted: 1 April 2013 / Published online: 28 April 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Objective Recent evidence suggests that ghrelin, a peptidic hormone stimulating food intake, interacts with the dopamine signaling. This interaction has been demonstrated to modulate several effects of ghrelin, such as locomotor activity, memory, and food intake. Ghrelin increases dopamine levels in the shell of the nucleus accumbens stimulating food intake, while ablation of the ghrelin receptor attenuates the hypophagia caused by the activation of dopamine receptor 2. However, it is not known whether the orexigenic action of ghrelin is due to changes in central dopamine receptors. Materials and methods We used Sprague–Dawley rats injected with different dopamine receptor agonists, antagonists, and ghrelin. Results We demonstrate that the specific central blockade of dopamine receptor 1, 2, and 3 (D1, D2, and D3, respectively) reduces the orexigenic action of ghrelin. Similarly, specific central stimulation, either singly of dopamine receptor 1 or dopamine receptors 2 and 3 simultaneously, causes a significant decrease in ghrelin-induced food intake. Costimulation of all three receptors (D1, D2, and D3) also led to a marked attenuation in ghrelin-induced food intake.
A. Romero-Picó : M. G. Novelle : C. Folgueira : M. López : R. Nogueiras (*) : C. Diéguez (*) Department of Physiology, School of Medicine-CIMUS, Instituto de Investigación Sanitaria (IDIS), University of Santiago de Compostela, Avda. Barcelona 3, 15782 Santiago de Compostela A Coruña, Spain e-mail:
[email protected] e-mail:
[email protected] A. Romero-Picó : M. G. Novelle : C. Folgueira : M. López : R. Nogueiras : C. Diéguez CIBER “Fisiopatología de la Obesidad y Nutrición”, Instituto de Salud Carlos III, San Francisco s/nSantiago de Compostela A Coruña, Spain
Importantly, the reduction in ghrelin-induced feeding was not caused by malaise or any type of behavioral alteration. Conclusion Taken together, these data indicate that dopamine receptors play an important role in acute stimulation of feeding behavior induced by central injection of ghrelin. Keywords Appetite . Dopamine receptor . Food intake
Introduction The stomach-derived peptidic hormone ghrelin increases food intake and adiposity (Tschop et al. 2000; Nakazato et al. 2001). Ghrelin stimulates eating when injected into different hypothalamic and extrahypothalamic areas. Within the hypothalamus, injections of ghrelin into the arcuate nucleus (ARC) and paraventricular nucleus were more effective than injections into the ventromedial nucleus (VMH) (Currie et al. 2012). However, the metabolic pathways modulating ghrelin feeding behavior have been studied mainly on hypothalamic neurons of the ARC and VMH and are mediated by the growth hormone secretagogue receptor 1a (GHS-R1a) (Sun et al. 2004). Signal transduction of ghrelin involves hypothalamic SIRT1 (Velasquez et al. 2011), which deacetylates p53, stimulating phosphorylation of AMPK and inactivation of several enzymatic steps in the novo fatty acid bio-synthesis pathway in the VMH (Lopez et al. 2008). These molecular events induce changes in UCP2 (Andrews et al. 2008) and up-regulation of the transcription factors Bsx (Sakkou et al. 2007; Nogueiras et al. 2008), FoxO1, and pCREB (Lage et al. 2010). Activation of these transcription factors ultimately leads to increased rates of transcription rate of NPY and AgRP (Wren et al. 2000). In addition to hypothalamic nuclei, the GHS-R1a is also expressed in dopaminergic neurons particularly in the ventral tegmental area (VTA) (Abizaid et al. 2006). Dopamine (DA) is the core neurotransmitter implicated in reward-associated
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behaviors including food intake (Palmiter 2007). The importance of endogenous dopaminergic tone in energy homeostasis has been highlighted by several reports showing that (a) food deprivation decreases extracellular DA in central nervous system (CNS) regions implicated in food intake (Pothos et al. 1995), (b) rats under fasting conditions exhibit an increased dopamine D2-receptor binding in the CNS (Thanos et al. 2008), and (c) food-deprived animals showed decreased mRNA levels and activity of the dopamine reuptake transporter in the VTA (Patterson et al. 1998). Ghrelin has been shown to increase DA levels in the shell of the nucleus accumbens (Jerlhag et al. 2006, 2007), stimulating food intake (Abizaid et al. 2006) and increasing locomotor activity in rodents (Jerlhag et al. 2006, 2007). Selective blockade of GHS-R1a in the VTA, on the other hand, prevents ghrelin-stimulated food intake (Naleid et al. 2005; Abizaid et al. 2006). Corroborating these data, the ability of ghrelin to elicit food-reinforced behavior is suppressed by the administration of the dopaminergic neurotoxin 6-hydroxydopamine into the VTA (Weinberg et al. 2011). Importantly, human studies have also demonstrated that peripheral ghrelin stimulates appetite, increases self-reporting of hunger and food imagery (Cummings et al. 2004; Schmid et al. 2005), and, moreover, enhances the activity in brain regions where VTA neurons synapse (Malik et al. 2008). Indeed, in obese patients, there is a BMI-correlated decrease in the striatal D2-receptor expression with a corresponding deficit in signaling. Furthermore, the decrease in D2-receptor signaling is more marked in individuals with the TaqIA A1 polymorphism in the D2 receptor, which is associated with reduced D2receptor expression (Davis et al. 2011). These reports clearly indicate that ghrelin interacts with the DA-reward system to regulate feeding behavior in humans. GHS-R1a is colocalized with the dopamine receptor subtype 1 (DRD1) (Jiang et al. 2006) and DRD2 (Kern et al. 2012), suggesting a close interaction between ghrelin and the dopamine signaling. Moreover, GHS-R1a and DRDs form heterodimers, and the pharmacological or genetic inhibition of GHS-R1a reverses DRD2-agonist-induced anorexia (Kern et al. 2012). Thus, it seems clear that the interaction between ghrelin and the dopamine system plays an important role in the regulation of food intake. Although previous reports have demonstrated an interaction on ghrelin and the dopamine system, mainly focusing on its effects on locomotor activity (Jerlhag et al. 2006) and the rewarding properties of food (Abizaid et al. 2006; Weinberg et al. 2011), the precise role of the central dopamine system in mediating the acute orexigenic effect of ghrelin remains largely unknown. Here, we hypothesized that alteration in the function of the brain dopamine signaling may regulate the acute orexigenic action of intracerebroventricular (ICV) injection of ghrelin. Our results indicate that the stimulation of DRD1 or DRD2/DRD3 in the central
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nervous system partially attenuates ghrelin-induced food intake, and the coactivation of the three receptors also caused a marked reduction in the orexigenic action of ghrelin.
Material and methods Animals and reagents Male Sprague–Dawley rats were housed individually and maintained on a 12:12-h light–dark cycle (8:00 am– 8:00 pm) at 22 °C. They were allowed ad libitum access to water and standard chow. Rats were anesthetized by an intraperitoneal injection of ketamine–xylacine. ICV cannulation was targeted to the lateral ventricle. Animals were killed by decapitation. All animal procedures were conducted in accordance with the standards approved by the Faculty Animal Committee at the University of Santiago de Compostela, and the experiments were performed within the Rules of Laboratory Animal Care and International Law on Animal Experimentation. Acetylated human ghrelin was purchased from Bachem (Bubendorf, Switzerland). All dopamine receptors agonists/antagonists were purchased from Tocris (St Louis, MO, USA): (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390) hydrochloride (D1 receptor antagonist), eticlopride hydrochloride (D2/D3 receptor antagonist), (±)-1-phenyl-2,3,4,5tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF 38393) hydrobromide (D1 receptor agonist), and (−)-quinpirole hydrochloride (D2-receptor agonist). Impact of ICV administration of dopamine receptor antagonists on food intake In order to test the effect of dopamine receptor antagonists on food intake, dose–response and time–response experiments were performed in fasted rats (n=8–10 per group). Rats were pre-fasted overnight (16 h), and each dopamine receptor antagonist was administered by a single ICV injection at 9:00 am at the following doses: 5, 15, and 30 μg for SCH 23390 and 30, and 60 μg for eticlopride. Doses of dopamine receptor antagonists were based on previous studies in which these compounds were injected ICV and the measurable outcome was effect on exercise after 180 min (Sutoo and Akiyama 1999). Cumulative food intake was measured at 1, 2, 4 and 6 h. Impact of ICV administration of dopamine receptor antagonists on ICV ghrelin-induced food intake To study the effect of dopamine receptor antagonists on ghrelin-induced food intake, rats were fed ad libitum and tested in four groups (n=10 rats per group): (1) vehicle +
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vehicle, (2) vehicle + ghrelin, (3) antagonist + vehicle, and (4) antagonist + ghrelin. Dopamine receptor antagonists were administered ICV, 20 min before ICV ghrelin injection (5 μg=1,500 pmol), and food intake was measured 2 h later, when the response to ICV ghrelin injection was highest. The antagonists were administered at a dose of either 15 and 30 μg (SCH 23390) or 30 and 50 μg (eticlopride), delivered as 5 μl ICV over 1 min and using saline as the vehicle ICV. Impact of ICV administration of dopamine receptor agonists on food intake In order to test the effect of dopamine receptor agonists on food intake, dose–response and time–response experiments were performed in fasted rats (n=8–10 per group). Rats were pre-fasted overnight (16 h), and each dopamine receptor agonist was administered by a single ICV injection at the following doses: 5, 15, 45, and 60 μg for SKF 38393 and 5, 15, and 45 μg for quinpirole. Doses were selected in accordance with previous studies (Carr et al. 2003). Cumulative food intake was measured at 1, 2, 4, and 6 h. Impact of ICV administration of dopamine receptor agonists on ICV ghrelin-induced food intake To study the effect of dopamine receptor agonists on ghrelin-induced food intake, rats were fed ad libitum and four different groups were tested (n=10 rats per group): (1) vehicle + vehicle, (2) vehicle + ghrelin, (3) agonist + vehicle, and (4) agonist + ghrelin. Dopamine receptor agonists were administered ICV 20 min before ICV ghrelin injection, and food intake was measured 2 h after injection of ghrelin. Agonists were administered at a dose of 5 μg (SKF 38393) or 45 μg (quinpirole). Saline was used as the vehicle for delivery and for control injections, and all injections were delivered as 5 μl ICV over 1 min. Conditioned taste aversion Rats with an ICV cannula implant were individually housed with free access to food and water. Three days before being tested, rats had access to water between 10:00 and 12:00 a.m. At fourth day, they had access to 0.15 % sodium saccharin rather than water for 30 min. Immediately afterwards, the experimental groups were injected intraperitoneally (IP) and ICVas follows (n=10–12 rats per group): (1) control (IP saline + ICV saline), (2) LiCl (IP 0.15M LiCl + ICV saline), and (3) agonists (IP saline + ICV quinpirole/SKF 38393), followed by free access to water during the next 90 min. At 10:00 am the following day, rats received free access to both saccharin and water for a period of 2 h. Volumes of saccharin and water were measured, and preference to saccharine was calculated as (100×(saccharine intake/(saccharine intake + water intake)).
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Locomotor activity Locomotor activity was monitored for a 2-h period immediately following ICV administration of ghrelin and dopamine receptor agonists using a custom-made 12-cage indirect calorimetry system (TSE LabMaster, TSE Systems, Germany) (Nogueiras et al. 2009). Rats (seven to eight per group) had been housed individually in TSE cages for 48 h between implantation of the ICV cannula and drug administration. Data collected were used to calculate total movement (XT), ambulatory movement (XA), and fine movement (XF). Results are expressed as total number of beam breaks. Statistical analysis and data presentation Results are expressed as mean ± SEM. To analyze the effect of SCH 23390, eticlopride, SKF 38393, and quinpirole on food intake and locomotor activity, we performed one-way ANOVA followed by Bonferroni’s post hoc statistical comparisons between groups. To ascertain any interaction between dopamine receptor antagonists or agonists and ghrelin, we used two-way ANOVA. Any interactions found to be significant using two-way ANOVA were also subjected to one-way ANOVA followed by Bonferroni’s post hoc test comparing selected pair of columns. Significance is indicated as *P
