Differential gene regulation of GHSR signaling pathway in the arcuate nucleus and NPY neurons by fasting, diet-induced obesity, and 17β-estradiol
Introduction
Obesity is a global health concern due, in part, to changes in diet and lifestyle and is a major risk factor for a variety of clinical conditions including cardiovascular disease, type II diabetes, and metabolic syndrome (Mark, 2006). One hallmark of obesity is the dysregulation of the hypothalamic centers that control feeding behavior, energy expenditure, and the peripheral hormones that mediate the communication between the body and the brain (Cai and Liu, 2011). Normally, neuronal populations in the hypothalamus regulate energy homeostasis by responding to circulating nutrients as well as appetite-regulating hormones such as leptin, insulin, and ghrelin (Gao and Horvath, 2007).
The incidence of metabolic disorders associated with obesity exhibit clear sex differences with premenopausal women having fewer metabolic disorders than men. However, metabolic disorders increase dramatically in postmenopausal women (Ford, 2005, Loucks et al., 2007). The loss of the reproductive steroid 17β-estradiol (E2) is, in part, the major cause of these effects, especially on body weight gain (Rachoń and Teede, 2010, Stefanska et al., 2015). In ovariectomized rodent models, E2 regulates many aspects of energy homeostasis through both peripheral actions and central mechanisms (reviewed in Mauvais-Jarvis et al., 2013, Roepke, 2009, Shi et al., 2009). E2 suppresses feeding and fat accumulation and augments energy expenditure and activity. To control energy homeostasis, E2 primarily uses the nuclear steroid receptor, ERα, which is highly expressed in the hypothalamus (Roepke, 2009). Total body knockout of ERα or deletion in specific hypothalamic neurons produces an obese phenotype with hyperphagia, higher visceral adiposity, and lower activity and energy expenditure in mice (Geary et al., 2001, Heine et al., 2000, Mamounis et al., 2014, Musatov et al., 2007, Xu et al., 2011).
Among the hypothalamic areas involved in energy homeostasis, the arcuate nucleus (ARC) is of special interest as it is an integration center for homeostatic signals from the periphery and the central nervous system (CNS). The “first order” ARC neurons central to the control of energy homeostasis are the proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons (Gao and Horvath, 2007). POMC and NPY neurons have opposing actions in the control of energy homeostasis. POMC neurons are anorexigenic primarily through the actions of α-melanocyte-stimulating hormone (α-MSH) via melanocortin receptors (MC3/4) expressed throughout the hypothalamus (Dietrich and Horvath, 2013). NPY neurons are orexigenic primarily through the actions of its neuropeptides, NPY and agouti-related peptide (AgRP), an antagonist for the MC4 receptors. Thus, the actions of POMC and NPY/AgRP neurons are important for the hypothalamic control of feeding and energy expenditure (Gao and Horvath, 2007).
Ghrelin is a brain-gut peptide hormone secreted from the stomach to stimulate food intake by acting on its receptor, growth hormone secretagogue receptor (GHSR). GHSR is expressed throughout the brain and in NPY/AgRP neurons in the ARC (Cowley et al., 2003, Willesen et al., 1999). Ghrelin-expressing neurons are also found in the periventricular region of the hypothalamus dorsal to the ARC (Guan et al., 2003, Mondal et al., 2005). ARC and peripheral ghrelin administration induces NPY/AgRP gene expression (Chen et al., 2004, Goto et al., 2006, Kamegai et al., 2001, Kamegai et al., 2000), NPY activation (Wang et al., 2002), and potently depolarizes NPY neurons (Andrews et al., 2008, Cowley et al., 2003). Furthermore, ghrelin activation of GHSR potently excites NPY neurons and controls calcium homeostasis (Andrews et al., 2008, Cowley et al., 2003). These rapid effects of ghrelin potentially involve calcium channels (Kohno et al., 2003) and may involve the inhibition of the M-current as recently reported in striatal neurons (Shi et al., 2013).
Activation of GHSR in NPY neurons initiates a signaling cascade that involves the mitochondrial enzymes uncoupling protein 2 (UCP2) and carnitine palmitoyltransferase 1 (CPT-1) to control Npy/Agrp gene expression through a forkhead box O1 (FoxO1)-mediated mechanism (Andrews, 2011, Andrews et al., 2008, López et al., 2008a). CPT-1 is involved in malonyl-CoA sensing and fatty acid oxidation, while UCP2 is necessary for reactive oxidation species (ROS) buffering and mitochondrial biogenesis (Andrews, 2011, López et al., 2008b). Of the three types of CPT-1, hypothalamic neurons express CPT-1c, which does not have acyltransferase activity, while hypothalamic astrocytes express CPT-1a, which does have acyltransferase activity (Wolfgang et al., 2006). FoxO1 is a transcription factor that negatively regulates adipogenesis, mediates insulin-induced gluconeogenesis, and is an effector of Npy/Agrp gene expression (Cao et al., 2011, Ren et al., 2012).
Fasting and diet-induced obesity (DIO) regulate expression of Npy, Agrp, Ghsr, and Ucp2 in the ARC (Briggs et al., 2014, Briggs et al., 2013, Briggs et al., 2010, Coppola et al., 2007, Verhulst et al., 2012), which may be a contributing mechanism underlying the ghrelin resistance seen in DIO (Briggs et al., 2014, Briggs et al., 2010, Perreault et al., 2004). Furthermore, a high fat diet (HFD) or DIO inhibits ghrelin's augmentation of hyperphagia (Gardiner et al., 2010, Perez-Tilve et al., 2011). Recently, E2 was shown to increase Ghsr expression in the ARC (Frazao et al., 2014). Interestingly, E2 is an anorexigenic steroid and is known to induce Pomc and suppress Npy gene expression (Roepke, 2009). Because ghrelin is orexigenic, this E2-induced increase in Ghsr expression in the ARC may be independent of its effects on feeding and occur in other ARC neurons.
Because GHSR and the components of its signaling pathway are widely expressed in the heterogeneous cell types of the ARC, determinations of gene expression only in the ARC may lead to incorrect assumptions about their modulation in NPY neurons. Therefore, our hypothesis is that there will be significant differences in gene expression between the ARC and NPY neurons due to fasting and DIO. We also hypothesize that there will be distinct sex differences in the response to these dietary influences due, in part, to E2 in females. To address these hypotheses, we characterized the expression of Ghsr, Ucp2, Cpt1c, Foxo1, and Npy/Agrp in both the ARC and NPY neurons in males after fasting and DIO and in ovariectomized females with or without E2 replacement after fasting and DIO using wild type (WT) C57 mice and GFP-NPY transgenic mice.
Section snippets
Animal care and experimental design
Animal experiments described in this project are in accordance with institutional guidelines based on National Institutes of Health standards and have been approved by The Rutgers University Institutional Animal Care and Use Committee. All animals were maintained under controlled temperature and photoperiod (12 h:12 h). All mice, both WT C57BL/6J (Jackson Laboratory) and GFP-NPY (Dr. Bradford Lowell, Harvard University, (van den Pol et al., 2009)), were given free access to food and water
Linearity of Ghsr expression in NPY neurons
To initially determine the detectability of Ghsr in NPY neurons and its regulation by fasting in males, we harvested individual GFP-NPY neurons from 6 fed males and 6 fasted males and conducted single-cell RT-PCR for Ghsr (Fig. 1A and B). Fasting increased the detection of Ghsr from 63.3 ± 4.2% in fed males to 86.7 ± 4.2% in fasted males (p < 0.01, Fig. 1B). These data suggest that fasting increases the expression of Ghsr specifically in NPY neurons. Furthermore, to determine if the linearity
Discussion
Understanding the impact of caloric restriction and high fat diets on the neuroendocrine control of energy homeostasis is key to addressing the obesity epidemic and other metabolic diseases. Most studies in this field only examine male rodent models due, in part, to the influence of circulating E2 on energy homeostasis in females during the estrous cycle. Another weakness of this field is the prevalence of studies examining genomic effects of these conditions on the heterogeneous nuclei of 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
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).
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