br Conclusion Our experiments have confirmed the
Conclusion Our experiments have confirmed the effects of fasting, DIO, and E2 on the expression of Ghsr, Npy, and Agrp in the ARC (Briggs et al., 2013, Briggs et al., 2010, Brown and Clegg, 2010, Coppola et al., 2007, Palou et al., 2009, Roepke et al., 2008, Verhulst et al., 2012). However, gene expression in pools of NPY neurons does not fully reflect findings in the heterogeneous ARC for other genes involved in GHSR signaling. Clearly, neuronal cell type should be considered when studying the expression of ubiquitously expressed genes and proteins in hypothalamic nuclei. The quantitative analysis of pools of GFP-tagged neurons by real-time quantitative PCR from treated males and females will greatly enhance our understanding of the sex-dependent, cell-type-specific effects of fasting, DIO, and E2 on hypothalamic homeostatic functions. A final concern is the relevance of peripheral vs. central ghrelin (from BH ghrelin neurons) (Guan et al., 2003) in the activation of NPY neurons and other ARC neurons (Frazao et al., 2014). Peripheral administration of ghrelin does activate ARC NPY neurons (Wang et al., 2002), but does not eliminate the role of catecholamine hindbrain neurons in mediating the actions of peripheral ghrelin on feeding behavior and NPY activation (Date et al., 2006, Date et al., 2002, Emanuel and Ritter, 2010). Therefore, the alterations in plasma ghrelin may not be a significant contributor to the ARC and NPY changes in gene expression. Results from the current study focusing on the genes involved in the GHSR signaling pathway in NPY neurons provide some insight into the interactions of orexigenic ghrelin signaling and anorexigenic E2 signaling in the control of energy homeostasis in females. Indeed, sex differences in ghrelin and GHSR activity are driven, in part, by the interaction of E2 with fasting and DIO (Priego et al., 2009). In summary, these studies emphasize the importance of considering cell type and sex while delineating the effects of ghrelin and potentially other peripheral hormones on hypothalamic gene expression and homeostatic functions.
Acknowledgments The author must thank Drs. Wendie Cohick and Sara Campbell for their assistance with the 17β-estradiol and ghrelin assays, respectively. This research is supported by funds from NIH R00DK083457, R00DK083457-S1, P30ES005022, and NJ06107 (USDA-NIFA).
Introduction Ghrelin, a 28-amino Autophagy Compound Library sale peptide, was isolated from the stomach and identified as an endogenous ligand for growth hormone secretagogue (GHS) receptor type 1a (GHSR-1a; Kojima et al., 1999). The ghrelin peptides were found to circulate in two distinct forms, acylated ghrelin and unacylated ghrelin. Ghrelin acylation on the third serine residue is essential for GHSR-1a binding and for the main endocrine functions of acylated ghrelin, including stimulation of GH secretion, induction of food intake, and regulation of energy homeostasis (Kojima and Kangawa, 2005). Besides these effects, ghrelin has a broad range of peripheral functions, including roles in milk production, regulation of reproduction and cell proliferation (Nakahara et al., 2003, Garcia et al., 2007, Granata et al., 2010, Isgaard et al., 2008) . Ghrelin is mainly synthesized by X/A-like cells in the stomach (Ariyasu et al., 2001), however, ghrelin expression has also been demonstrated in other tissues, including the placenta, ovary and mammary glands (Harrison et al., 2007, Zhang et al., 2008, David et al., 2005, Malin et al., 2008). Similarly, many peripheral tissues also express GHSR-1a (Papotti et al., 2000). This ubiquitous distribution suggests that locally produced ghrelin might exert autocrine/paracrine effects in different tissues (Korbonits et al., 2004). Studies have shown an important interrelationship between energy homeostasis and mammary function. Lactation is an energetically expensive phase in female mammals because mothers must generate enough energy to maintain their energy homeostasis while meeting the energetic requirements for milk production (Milsom et al., 1992, Barber et al., 1992). Studies have shown that ghrelin plays an important role in energy homeostasis (Wren et al., 2000, Nakazato et al., 2001). These results imply that ghrelin might be a novel member of the regulatory network involved in the regulation of food intake, energy homeostasis and mammary function. It is likely that some of these effects are not due to the direct action of ghrelin but to indirect action through GH secretion. As GH seems to play an important role in mammogenesis, lactogenesis and galactopoiesis in humans and animals (Milsom et al., 1992, Flint et al., 1992, Etherton and Bauman, 1998), ghrelin may affect milk production by increasing GH secretion. In monogastrics and ruminants, ghrelin stimulates the release of GH (Date et al., 2000, Wren et al., 2000, Javed et al., 2006). Much recent evidence suggests numerous other functions for ghrelin, including effects on milk production. Nakahara (2003) reported an increase in both milk yield and β-casein mRNA expression after daily injection of ghrelin in rats. Recent study has reported that long-term infusions of ghrelin increased milk yield and resulted in a significant change in milk composition in early lactation of dairy cows (Roche et al., 2008). In addition, hypothalamic and pituitary expression of GHSR-1a mRNA is increased in lactating animals compared to non-lactating animals (Abizaid et al., 2008). These results suggest that ghrelin may play an important role in regulation of milk production in the hypothalamus and pituitary as target tissues, however, little attention has been directed to the role this hormone may play in the mammary glands. Of particular note, ghrelin positive cells were found in the ducts of the human mammary glands (Malin et al., 2008), and GHSR-1a mRNA was present in the mammary glands of lactating rats (Nakahara et al., 2003). Evidence has been presented that the mammary glands may produce a number of chemical factors that may be involved in local control of secretory activity, it is possible that the gland also elaborates a locally-acting ghrelin and thereby participates in the control of its own secretory activity and development.