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).

Summary Circulating leptin and insulin convey information regarding energy stores to the central nervous system, particularly the hypothalamus. Hypothalamic pro-opiomelanocortin (POMC) neurons regulate energy balance and glucose homeostasis and express leptin and insulin receptors. However, the physiological significance of concomitant leptin and insulin action on POMC neurons remains to be established. Here we show that mice lacking both insulin and LepRs in POMC neurons (Pomc-Cre, Leprflox/flox IRflox/flox mice) display systemic insulin resistance, which is distinct from the single deletion of either receptor. In addition, Pomc-Cre, Leprflox/flox IRflox/flox female mice display elevated serum testosterone levels and ovarian abnormalities resulting in reduced fertility. We conclude that direct action of insulin and leptin on POMC neurons is required to maintain normal glucose homeostasis and reproductive function.

Leptin action on its receptor (LEPR) stimulates energy expenditure and reduces food intake, thereby lowering body weight. One leptin-sensitive target cell mediating these effects on energy balance is the proopiomelanocortin (POMC) neuron. Recent evidence suggests that the action of leptin on POMC neurons regulates glucose homeostasis independently of its effects on energy balance. Here, we have dissected the physiological impact of direct leptin action on POMC neurons using a mouse model in which endogenous LEPR expression was prevented by a LoxP-flanked transcription blocker (loxTB), but could be reactivated by Cre recombinase. Mice homozygous for the Lepr loxTB allele were obese and exhibited defects characteristic of LEPR deficiency. Reexpression of LEPR only in POMC neurons in the arcuate nucleus of the hypothalamus did not reduce food intake, but partially normalized energy expenditure and modestly reduced body weight. Despite the moderate effects on energy balance and independent of changes in body weight, restoring LEPR in POMC neurons normalized blood glucose and ameliorated hepatic insulin resistance, hyperglucagonemia, and dyslipidemia.Collectively, these results demonstrate that direct leptin action on POMC neurons does not reduce food intake, but is sufficient to normalize glucose and glucagon levels in mice otherwise lacking LEPR. IntroductionLeptin is an adipose-derived hormone that acts on its cognate receptors (LEPR) expressed by multiple neuronal groups in distinct areas of the brain (1). The canonical effect of leptin action in the brain is to regulate food intake and energy expenditure and thus body weight (2-4). In addition, leptin regulates several other physiological processes, including hepatic glucose production, insulin action, and glucagon levels (5-10). It is still unclear, however, which neurons mediate the varied physiological effects of leptin.One population of neurons targeted by leptin is proopiomelanocortin (POMC) cells in the arcuate nucleus of the hypothalamus (ARH) and nucleus of the solitary tract (NTS) (2, 3). Leptin action on POMC neurons in the ARH is considered a prototypical site of action in the control of energy balance. This view is partly based on results showing that loss of LEPR in POMC neurons increases body weight (8,11,12). Conversely, LEPR reexpression in the ARH (13), overexpression in the ARH (14-17), and transgenic expression in POMC neurons (18) lower body weight. Interestingly, these latter studies also show lowered blood glucose, suggesting that leptin-sensitive POMC neurons in the ARH directly modulate metabolism (13-18). In the current study, we developed what we believe to be a novel LEPR-null mouse model in which endogenous LEPR expression can be reexpressed in cells that normally express leptin receptors. Here, we reexpress LEPR only in POMC neurons to delineate the physiological effects on energy and metabolic homeostasis.

The hormone ghrelin stimulates eating and helps maintain blood glucose upon caloric restriction. While previous studies have demonstrated that hypothalamic arcuate AgRP neurons are targets of ghrelin, the overall relevance of ghrelin signaling within intact AgRP neurons is unclear. Here, we tested the functional significance of ghrelin action on AgRP neurons using a new, tamoxifen-inducible AgRP-CreERT2 transgenic mouse model that allows spatiotemporally-controlled re-expression of physiological levels of ghrelin receptors (GHSRs) specifically in AgRP neurons of adult GHSR-null mice that otherwise lack GHSR expression. AgRP neuron-selective GHSR re-expression partially restored the orexigenic response to administered ghrelin and fully restored the lowered blood glucose levels observed upon caloric restriction. The normalizing glucoregulatory effect of AgRP neuron-selective GHSR expression was linked to glucagon rises and hepatic gluconeogenesis induction. Thus, our data indicate that GHSR-containing AgRP neurons are not solely responsible for ghrelin's orexigenic effects but are sufficient to mediate ghrelin's effects on glycemia.