Hypothalamic orexin, OX1, αMSH, NPY and MCRs expression in dopaminergic D2R knockout mice
Introduction
Dopamine receptor D2 (D2R) knockout mice (KO) generate anterior pituitary lactotrope hyperplasia and have chronic hyperprolactinemia (Diaz-Torga et al., 2002, Cristina et al., 2005). These mice also present altered body growth, with reduction of pituitary GH, and serum IGF-I and IGF BP3 (Garcia-Tornadu et al., 2006). KO males have no differences in 24 h food intake, however when food intake is normalized to body weight there is actually a significant increase in food intake/gram of body weight, which indicates that food intake efficiency is greater in wildtype males (Diaz-Torga et al., 2002). In KO females there is a catch up in body weight after three months of age, and food intake has not been evaluated.
Many components of the neuroendocrine system act as metabolic regulators of energy balance and food intake (Cone, 1999, Cone et al., 2001). Neuropeptides interact with monoamines in the hypothalamus to control physiologic states such as hunger and satiety (Ramos et al., 2005). In particular, dopamine reduces food intake by acting in specific hypothalamic areas, through the dopamine receptors type 1 and 2 (Leibowitz, 1986, Terry et al., 1995) and different energy states (fasting, obesity, anorexia) modulate dopamine receptor expression (Fetissov et al., 2002, Sato et al., 2001). The anorectic dopamine action may be caused by transinaptic activation of orexin neurons (Fadel and Deutch, 2002).
The hypothalamic peptides orexins A and B are so named for their influence on food intake (de Lecea et al., 1998, Sakurai et al., 1998). An intracerebroventricular injection of orexin A does not merely affect eating behavior, but it also induces an increase in heart rate (Monda et al., 2005), blood pressure (Samson et al., 1999) and metabolic rate (Lubkin and Stricker-Krongrad, 1998). A role for the orexins in sleep regulation has also been demonstrated (Beuckmann and Yanagisawa, 2002), and deficiency in orexin neurotransmission results in the sleep disorder narcolepsy in mice, dogs, and humans (Taheri et al., 2002). Orexins act mainly at the orexin 1 (OX1) G protein-coupled receptor in the brain (Trivedi et al., 1998) (Silveyra et al., 2007).
On the other hand, the D2R negatively regulates α-melanocyte-stimulating hormone (αMSH), an anorexigenic peptide (Cote et al., 1986). αMSH is a 13 amino acid peptide produced by post translational processing of proopiomelanocortin (POMC) in the intermediate lobe of the pituitary. In addition to the skin-darkening effect in amphibians and other vertebrates, to which it owes its name, this peptide has anti-inflammatory and antimicrobial effects and probably contributes to innate immunity (Catania et al., 2000). It participates in the control of feeding behavior, reduces food consumption and stimulates catabolism acting at the melanocortin 3 (MC3R) and melanocortin 4 receptors (MC4R). αMSH is expressed primarily in the arcuate nucleus of the hypothalamus, and expressed and secreted to peripheral circulation by the intermediate lobe of the pituitary. Its synthesis and secretion in the pituitary is mainly controlled by the D2R (Chen et al., 1983). αMSH may modify the expression of the orexigenic peptides, orexin A and B in the hypothalamus (Lopez et al., 2007), and there is anatomical evidence that POMC neurons send projections to neurons within the region expressing orexins (Elias et al., 1998).
Other peptides related to food intake are leptin and neuropeptide Y (NPY). Leptin is an anorexigenic satiety factor, which governs energy balance through a negative feedback loop acting on hypothalamic centers in the brain through its receptor OBR (Ahima and Flier, 2000). There is extensive evidence demonstrating that the central melanocortin system is important in mediating the effects of leptin. First, leptin receptors are expressed on the majority of POMC neurons in the arcuate nucleus (Cheung et al., 1997) and the anorectic effects of exogenously administered leptin in rodents are partially reversed by treatment with a melanocortin receptor antagonist (Seeley et al., 1997, da Silva et al., 2004), indicating that the central melanocortin system is downstream of leptin receptor signaling and plays a key role in mediating the effects of this important anorexigenic hormone (Cowley et al., 2001).
NPY is one of the most abundant peptides of the hypothalamus (Kamiji and Inui, 2007). The major sites of neuronal expression of NPY in the hypothalamus are the arcuate nucleus and the dorso medial hypothalamus (DMH). It is a potent orexigenic neuropeptide, and centrally applied stimulates food intake. Prolactin may activate NPY gene expression in the DMH (Chen and Smith, 2004), and GH also increases NPY hypothalamic expression (Hurley et al., 2003). Finally, a relation of the dopaminergic system and NPY has been proposed, as dopaminergic agonists can decrease feeding behavior by an antagonistic action on hypothalamic NPY-containing neurons (Bina and Cincotta, 2000).
In view of the interdependence of the dopaminergic system with prolactin, αMSH, orexins, NPY and leptin, we sought to evaluate the effect of D2R disruption on food intake in both sexes in relation to hypothalamic orexin precursor (PPO) and OX1 expression, serum leptin levels, and expression of its hypothalamic receptor, OBR. Furthermore, we studied circulating, pituitary and hypothalamic α MSH levels, as well as central MC3 and MC4 receptors, and hypothalamic NPY mRNA levels.
Section snippets
Animals
D2 dopamine receptor knockout mice, official strain designation B6.129S2-Drd2tm1low by the Induced Mutant Resource at The Jackson Laboratory (Bar Harbor, ME), generated by targeted mutagenesis of the D2R gene in embryonic stem cells (Asa et al., 1999, Kelly et al., 1997) were used. The original F2 hybrid strain (129S2/Sv × C57BL/6J) containing the mutated D2 receptor allele was backcrossed for ten generations to wildtype C57BL/6J mice. Mutant and wildtype mice were generally the product of
Food intake in wildtype and knockout mice
Food intake per mouse per day was not modified by the genotype in male or female mice at two or five months of age (Fig. 1A). When food intake was normalized to body weight there was a significant increase in food intake/gram of body weight in KO mice of both sexes at 5 months (Fig. 1B, P = 0.045) and not in 2-month-old females.
Serum PRL and in wildtype and knockout mice
As expected, prolactin levels were higher in KO mice than in wildtype littermates of both sexes at 5 months of age. Furthermore, prolactin was 3-fold higher in female KOs
Discussion
Dopamine regulates hunger and satiety by acting in specific hypothalamic areas. The effects of dopamine on food intake have yielded conflicting results in the literature due to the different actions of dopamine on various hypothalamic nuclei, the involvement of multiple receptors, and different responses in food intake when administered systemically or locally into the hypothalamus (Ramos et al., 2005). Systemic treatment with D1/D2 agonists decrease food intake (Leibowitz, 1986, Terry et al.,
Conflict of interest statement
There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Acknowledgements
We thank NIDDK’s National Hormone and Pituitary Program and Dr. A.F. Parlow for mouse prolactin RIA kits, and leptin standard and antiserum.
This work was supported by grants from CONICET (PIP 5350), Fundación Alberto J. Roemmers, Fundación Fiorini and Agencia Nacional de Promoción Científica y Técnica, PICT 2006-207, Buenos Aires, Argentina (DBV).
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