We found that increasing ghrelin levels, through subcutaneous injections or calorie restriction, produced anxiolytic-and antidepressant-like responses in the elevated plus maze and forced swim test. Moreover, chronic social defeat stress, a rodent model of depression, persistently increased ghrelin levels, whereas growth hormone secretagogue receptor (Ghsr) null mice showed increased deleterious effects of chronic defeat. Together, these findings demonstrate a previously unknown function for ghrelin in defending against depressive-like symptoms of chronic stress.Chronic stress induces changes in mood, feeding and metabolism by a poorly understood neurobiological mechanism. Recent studies have suggested that key metabolic signals may interact with CNS circuits to regulate reward and mood 1 . To further explore these links, we investigated the potential role of ghrelin, an important feeding peptide, in the development of depressive symptoms. Ghrelin is a hormone synthesized predominantly by specialized gastrointestinal endocrine cells and is released during periods of negative energy balance 2 . In response to energy insufficiency, ghrelin induces a potent feeding response via activation of the growth hormone secretagogue receptor (GHSR, ghrelin receptor) 2,3 .To determine whether ghrelin can affect mood symptoms, we physiologically increased ghrelin levels by restricting the food intake of mice with a diet containing 60% of normal calories for Fig. 1). This resulted in a fourfold increase in circulating levels of acylated ghrelin (calorie restricted wild-type mice: 7.93 ± 1.59 pg mL −1 , n = 6; wildtype mice fed ad libitum: 1.98 ± 0.37 pg mL −1 , n = 5; P < 0.01). Calorie-restricted wild-type mice showed robust anxiolytic-and antidepressant-like behavior in the elevated plus maze (EPM) and forced swim test (FST), respectively, as compared with wild-type mice fed ad libitum (controls; Fig. 1a,c). In contrast, genetic blockade of ghrelin signaling in Ghsr −/− mice negated these calorie restriction-associated anxiolytic-and antidepressant-like effects. Further analyses demonstrated that the observed differences between the two genotypes cannot be attributed to differences in sensorimotor coordination, general locomotor activity or body weight ( Supplementary Figs. 1-3 online).We used a pharmacologic approach to extend our food-restriction results. We subcutaneously injected C57BL6/J mice with a dose of ghrelin that induces potent feeding (Fig. 1f) and tested them in the EPM and FST 45 min later. Mice receiving ghrelin demonstrated significantly less anxiety-and depression-like symptoms in these tests compared with saline-injected controls (Fig. 1b,d).Next, we determined whether ghrelin signaling regulates depressive symptoms in a mouse model of chronic stress. We used the chronic social defeat stress (CSDS) procedure, which subjects mice to ten daily bouts of social defeat by aggressive CD1 male mice 1,4 (Fig. 2). Mice subjected to CSDS showed lasting behavioral deficits, including social avoidance ( Suppl...

Background-Ghrelin is a potent orexigenic hormone that likely impacts eating via several mechanisms. Here, we hypothesized that ghrelin can regulate extra-homeostatic, hedonic aspects of eating behavior.

The popular media and personal anecdotes are rich with examples of stress-induced eating of calorically dense "comfort foods." Such behavioral reactions likely contribute to the increased prevalence of obesity in humans experiencing chronic stress or atypical depression. However, the molecular substrates and neurocircuits controlling the complex behaviors responsible for stress-based eating remain mostly unknown, and few animal models have been described for probing the mechanisms orchestrating this response. Here, we describe a system in which food-reward behavior, assessed using a conditioned place preference (CPP) task, is monitored in mice after exposure to chronic social defeat stress (CSDS), a model of prolonged psychosocial stress, featuring aspects of major depression and posttraumatic stress disorder. Under this regime, CSDS increased both CPP for and intake of high-fat diet, and stress-induced food-reward behavior was dependent on signaling by the peptide hormone ghrelin. Also, signaling specifically in catecholaminergic neurons mediated not only ghrelin's orexigenic, antidepressant-like, and food-reward behavioral effects, but also was sufficient to mediate stress-induced food-reward behavior. Thus, this mouse model has allowed us to ascribe a role for ghrelin-engaged catecholaminergic neurons in stress-induced eating. IntroductionMost humans experience altered feeding behaviors upon stress, with approximately 40% eating more and 40% eating less than usual (1). Furthermore, upon stress, most people report an increase in the intake of highly palatable foods, independent of hyperphagia or hypophagia (2, 3). Altered eating is also a frequent finding in individuals with major depressive disorder, with the "atypical" subtype even containing hyperphagia as a possible distinguishing characteristic (4). In one study, 46% of study subjects who met DSM-IV criteria for major depressive disorder with atypical features reported increased appetite (5). Of the remaining depressed patients without atypical features, 18% reported increased appetite, while 50% reported decreased appetite (5). The complex eating behaviors that are associated with and/or stimulated by stress and major depression undoubtedly contribute to the increased number of overweight and obese individuals who experience or have experienced stress and depression. For example, a longitudinal study from New Zealand showed that major depression in late-adolescent girls was associated with a 2.3-fold increased risk of obesity in adulthood and, furthermore, that the prevalence of obesity in adulthood was positively correlated with the number of major depressive episodes during adolescence in these girls (6). In another study, 47% of a large cohort of subjects with atypical depression reported increased body weight (5). Also, the combined overweight and obesity prevalence in a sample of US veterans with posttraumatic stress disorder was found in a chart review study to exceed that within the US general population by 20% (7).

The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

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Enteroendocrine cells such as duodenal cholecystokinin (CCK cells) are generally thought to be confined to certain segments of the gastrointestinal (GI) tract and to store and release peptides derived from only a single peptide precursor. In the current study, however, transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the CCK promoter demonstrated a distribution pattern of CCK-eGFP positive cells that extended throughout the intestine. Quantitative PCR and liquid chromatography-mass spectrometry proteomic analyses of isolated, FACS-purified CCK-eGFP-positive cells demonstrated expression of not only CCK but also glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP), peptide YY (PYY), neurotensin, and secretin, but not somatostatin. Immunohistochemistry confirmed this expression pattern. The broad coexpression phenomenon was observed both in crypts and villi as demonstrated by immunohistochemistry and FACS analysis of separated cell populations. Single-cell quantitative PCR indicated that approximately half of the duodenal CCK-eGFP cells express one peptide precursor in addition to CCK, whereas an additional smaller fraction expresses two peptide precursors in addition to CCK. The coexpression pattern was further confirmed through a cell ablation study based on expression of the human diphtheria toxin receptor under the control of the proglucagon promoter, in which activation of the receptor resulted in a marked reduction not only in GLP-1 cells, but also PYY, neurotensin, GIP, CCK, and secretin cells, whereas somatostatin cells were spared. Key elements of the coexpression pattern were confirmed by immunohistochemical double staining in human small intestine. It is concluded that a lineage of mature enteroendocrine cells have the ability to coexpress members of a group of functionally related peptides: CCK, secretin, GIP, GLP-1, PYY, and neurotensin, suggesting a potential therapeutic target for the treatment and prevention of diabetes and obesity.

Ghrelin, an octanoylated peptide hormone produced in the stomach, rises dramatically in mouse plasma during chronic severe calorie deprivation, an event that is essential to maintain life. The mechanism for this increase is not understood. Here, we study the control of ghrelin secretion in tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene. We found that the ghrelin-secreting cells express high levels of mRNA encoding β 1 -adrenergic receptors. Addition of norepinephrine or epinephrine to the culture medium stimulated ghrelin secretion, and this effect was blocked by atenolol, a selective β 1 -adrenergic antagonist. When WT mice were treated with reserpine to deplete adrenergic neurotransmitters from sympathetic neurons, the fasting-induced increase in plasma ghrelin was blocked. Inhibition was also seen following atenolol administration. We conclude that ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons and acting directly on β 1 receptors on the ghrelinsecreting cells of the stomach.

Leptin monotherapy reverses the deadly consequences and improves several of the metabolic imbalances caused by insulindeficient type 1 diabetes (T1D) in rodents. However, the mechanism(s) underlying these effects is totally unknown. Here, we report that intracerebroventricular (icv) infusion of leptin reverses lethality and greatly improves hyperglycemia, hyperglucagonemia, hyperketonemia, and polyuria caused by insulin deficiency in mice. Notably, icv leptin administration leads to increased body weight while suppressing food intake, thus correcting the catabolic consequences of T1D. Also, icv leptin delivery improves expression of the metabolically relevant hypothalamic neuropeptides proopiomelanocortin, neuropeptide Y, and agouti-related peptide in T1D mice. Furthermore, this treatment normalizes phosphoenolpyruvate carboxykinase 1 contents without affecting glycogen levels in the liver. Pancreatic β-cell regeneration does not underlie these beneficial effects of leptin, because circulating insulin levels were undetectable at basal levels and following a glucose overload. Also, pancreatic preproinsulin mRNA was completely absent in these icv leptin-treated T1D mice. Furthermore, the antidiabetic effects of icv leptin administration rapidly vanished (i.e., within 48 h) after leptin treatment was interrupted. Collectively, these results unveil a key role for the brain in mediating the antidiabetic actions of leptin in the context of T1D.brain | leptin monotherapy | glucose homeostasis | glucagon suppression A ccording to the Juvenile Diabetes Research Foundation, type 1 diabetes (T1D) afflicts 1-3 million people in the United States alone. Regrettably, for reasons yet to be understood, the incidence of T1D has been increasing at an alarming annual rate of ∼3%, thus indicating that the number of patients with T1D is predicted to rise significantly in the future (1). T1D occurs as a consequence of pancreatic β-cell destruction leading to insulin deficiency, a defect that causes hyperglycemia, hyperglucagonemia, cachexia, ketoacidosis, and other abnormalities (2, 3). T1D is a deadly condition if not treated. Current life-saving interventions include daily insulin administration; insulin therapy reduces hyperglycemia, glycosylated hemoglobin, and cachexia and prevents or delays some T1D-associated morbidities (3, 4). However, even with insulin therapy, T1D secondary complications include debilitating and long-lasting conditions, such as heart disease, neuropathy, and hypertension (5-7). Moreover, probably because of insulin's lipogenic and cholesterologenic actions, longterm insulin treatment is suspected to underlie the increased ectopic lipid deposition (i.e., in nonadipose tissues) (8) and incidence of coronary artery disease (>90% after the age of 55 y) (9, 10) seen in patients with T1D. Furthermore, in part attributable to insulin's potent, fast-acting, glycemia-lowering effects, intensive insulin therapy significantly increases the risk for hypoglycemia, an event that is disabling and can even be fatal (3,(11...