Ghrelin-responsive mediobasal hypothalamic neurons mediate exercise-associated food intake and exercise endurance.

Previous studies have implicated the orexigenic hormone ghrelin as a mediator of exercise endurance and the feeding response postexercise. Specifically, plasma ghrelin levels nearly double in mice when they are subjected to an hour-long bout of high-intensity interval exercise (HIIE) using treadmills. Also, growth hormone secretagogue receptor-null (GHSR-null) mice exhibit decreased food intake following HIIE and diminished running distance (time until exhaustion) during a longer, stepwise exercise endurance protocol. To investigate whether ghrelin-responsive mediobasal hypothalamus (MBH) neurons mediate these effects, we stereotaxically delivered the inhibitory designer receptor exclusively activated by designer drugs virus AAV2-hSyn-DIO-hM4(Gi)-mCherry to the MBH of Ghsr-IRES-Cre mice, which express Cre recombinase directed by the Ghsr promoter. We found that chemogenetic inhibition of GHSR-expressing MBH neurons (upon delivery of clozapine-N-oxide) 1) suppressed food intake following HIIE, 2) reduced maximum running distance and raised blood glucose and blood lactate levels during an exercise endurance protocol, 3) reduced food intake following ghrelin administration, and 4) did not affect glucose tolerance. Further, HIIE increased MBH Ghsr expression. These results indicate that activation of ghrelin-responsive MBH neurons is required for the normal feeding response to HIIE and the usual amount of running exhibited during an exercise endurance protocol.

Metabolic insights from a GHSR-A203E mutant mouse model.

OBJECTIVE: Binding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. METHODS: We generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. RESULTS: In COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first ∼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. CONCLUSIONS: Our data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.

Hypothalamic loss of Snord116 and Prader-Willi syndrome hyperphagia: the buck stops here?

Hyperphagia and obesity are the best-known manifestations of Prader-Willi syndrome (PWS) and are responsible for most of the overall morbidity and mortality associated with the disease. Yet these PWS symptoms remain poorly understood and without effective pharmacologic therapies. Mouse models attempting to recapitulate both the genetic alterations and marked hyperphagia plus obesity of PWS have been enigmatic, leading to skepticism about the use of mouse models to investigate PWS. In this issue of the JCI, Polex-Wolf and colleagues challenge the skeptics by successfully inducing hyperphagia following bilateral mediobasal hypothalamic deletion of the Snord116 gene from adult mice. Obesity also resulted, although only in a subset of mice. While this approach represents an exciting advance, highlighting a pathologic effect of loss of mediobasal hypothalamic Snord116 expression on the development of PWS's hallmark symptoms, the variability in the body-weight and body composition responses to this site-selective gene deletion raises several questions.

The role of ghrelin-responsive mediobasal hypothalamic neurons in mediating feeding responses to fasting.

OBJECTIVE: Ghrelin is a stomach-derived hormone that affects food intake and regulates blood glucose. The best-characterized actions of ghrelin are mediated by its binding to and activation of the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Adequate examination of the identity, function, and relevance of specific subsets of GHSR-expressing neurons has been hampered by the absence of a suitable Cre recombinase (Cre)-expressing mouse line with which to manipulate gene expression in a targeted fashion within GHSR-expressing neurons. The present study aims to characterize the functional significance and neurocircuitry of GHSR-expressing neurons in the mediobasal hypothalamus (MBH), as they relate to ghrelin-induced food intake and fasting-associated rebound hyperphagia, using a novel mouse line in which Cre expression is controlled by the Ghsr promoter. METHODS: A Ghsr-IRES-Cre mouse line that expresses Cre directed by the Ghsr promoter was generated. The line was validated by comparing Cre activity in reporter mice to the known brain distribution pattern of GHSR. Next, the requirement of MBH GHSR-expressing neuronal activity in mediating food intake in response to administered ghrelin and in response to fasting was assessed after stereotaxic delivery of inhibitory designer receptor exclusively activated by designer drugs (DREADD) virus to the MBH. In a separate cohort of Ghsr-IRES-Cre mice, stereotaxic delivery of stimulatory DREADD virus to the MBH was performed to assess the sufficiency of MBH GHSR-expressing neuronal activity on food intake. Finally, the distribution of MBH GHSR-expressing neuronal axonal projections was assessed in the DREADD virus-injected animals. RESULTS: The pattern of Cre activity in the Ghsr-IRES-Cre mouse line mostly faithfully reproduced the known GHSR expression pattern. DREADD-assisted inhibition of MBH GHSR neuronal activity robustly suppressed the normal orexigenic response to ghrelin and fasting-associated rebound food intake. DREADD-assisted stimulation of MBH GHSR neuronal activity was sufficient to induce food intake. Axonal projections of GHSR-expressing MBH neurons were observed in a subset of hypothalamic and extra-hypothalamic regions. CONCLUSIONS: These results suggest that 1) activation of GHSR-expressing neurons in the MBH is required for the normal feeding responses following both peripheral administration of ghrelin and fasting, 2) activation of MBH GHSR-expressing neurons is sufficient to induce feeding, and 3) axonal projections to a subset of hypothalamic and/or extra-hypothalamic regions likely mediate these responses. The Ghsr-IRES-Cre line should serve as a valuable tool to further our understanding of the functional significance of ghrelin-responsive/GHSR-expressing neurons and the neuronal circuitry within which they act.

Estradiol modulates Kiss1 neuronal response to ghrelin.

Ghrelin is a metabolic signal regulating energy homeostasis. Circulating ghrelin levels rise during starvation and fall after a meal, and therefore, ghrelin may function as a signal of negative energy balance. Ghrelin may also act as a modulator of reproductive physiology, as acute ghrelin administration suppresses gonadotropin secretion and inhibits the neuroendocrine reproductive axis. Interestingly, ghrelin's effect in female metabolism varies according to the estrogen milieu predicting an interaction between ghrelin and estrogens, likely at the hypothalamic level. Here, we show that ghrelin receptor (GHSR) and estrogen receptor-α (ERα) are coexpressed in several hypothalamic sites. Higher levels of circulating estradiol increased the expression of GHSR mRNA and the coexpression of GHSR mRNA and ERα selectively in the arcuate nucleus (ARC). Subsets of preoptic and ARC Kiss1 neurons coexpressed GHSR. Increased colocalization was observed in ARC Kiss1 neurons of ovariectomized estradiol-treated (OVX + E2; 80%) compared with ovariectomized oil-treated (OVX; 25%) mice. Acute actions of ghrelin on ARC Kiss1 neurons were also modulated by estradiol; 75 and 22% of Kiss1 neurons of OVX + E2 and OVX mice, respectively, depolarized in response to ghrelin. Our findings indicate that ghrelin and estradiol may interact in several hypothalamic sites. In the ARC, high levels of E2 increase GHSR mRNA expression, modifying the colocalization rate with ERα and Kiss1 and the proportion of Kiss1 neurons acutely responding to ghrelin. Our findings indicate that E2 alters the responsiveness of kisspeptin neurons to metabolic signals, potentially acting as a critical player in the metabolic control of the reproductive physiology.

An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin, tyrosine hydroxylase, and RFamide-related peptide neurons in mice.

Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (<2%). We also observed no coexpression of GHSR-eGFP and RFamide-related peptide-3 (RFRP3) neurons in the dorsomedial hypothalamic nucleus. Importantly, we observed that approximately half of the GHSR-eGFP cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed.

Leptin's effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons.

Studies in humans and rodents indicate that a minimum amount of stored energy is required for normal pubertal development. The adipocyte-derived hormone leptin is a key metabolic signal to the neuroendocrine reproductive axis. Humans and mice lacking leptin or the leptin receptor (LepR) (ob/ob and db/db mice, respectively) are infertile and fail to enter puberty. Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility, but the exact site or sites of leptin action are unclear. Here, we found that genetic deletion of LepR selectively from hypothalamic Kiss1 neurons in mice had no effect on puberty or fertility, indicating that direct leptin signaling in Kiss1 neurons is not required for these processes. However, bilateral lesions of the ventral premammillary nucleus (PMV) of ob/ob mice blunted the ability of exogenous leptin to induce sexual maturation. Moreover, unilateral reexpression of endogenous LepR in PMV neurons was sufficient to induce puberty and improve fertility in female LepR-null mice. This LepR reexpression also normalized the increased hypothalamic GnRH content characteristic of leptin-signaling deficiency. These data suggest that the PMV is a key site for leptin's permissive action at the onset of puberty and support the hypothesis that the multiple actions of leptin to control metabolism and reproduction are anatomically dissociated.

5-HT2CRs expressed by pro-opiomelanocortin neurons regulate insulin sensitivity in liver.

Mice lacking 5-HT 2C receptors (5-HT(2C)Rs) displayed hepatic insulin resistance, a phenotype normalized by re-expression of 5-HT(2C)Rs only in pro-opiomelanocortin (POMC) neurons. 5-HT(2C)R deficiency also abolished the anti-diabetic effects of meta-chlorophenylpiperazine (a 5-HT(2C)R agonist); these effects were restored when 5-HT(2C)Rs were re-expressed in POMC neurons. Our findings indicate that 5-HT(2C)Rs expressed by POMC neurons are physiologically relevant regulators of insulin sensitivity and glucose homeostasis in the liver.

Direct insulin and leptin action on pro-opiomelanocortin neurons is required for normal glucose homeostasis and fertility.

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 leptin and insulin receptors in POMC neurons (Pomc-Cre, Lepr(flox/flox) IR(flox/flox) mice) display systemic insulin resistance, which is distinct from the single deletion of either receptor. In addition, Pomc-Cre, Lepr(flox/flox) IR(flox/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.

A beta3-adrenergic-leptin-melanocortin circuit regulates behavioral and metabolic changes induced by chronic stress.

BACKGROUND: Obesity has been associated with an increased risk of developing several psychiatric illnesses, including major depression and posttraumatic stress disorder. Likewise, these stress-related disturbances are associated with a higher rate of obesity; yet, the neurobiological mechanisms linking obesity and stress remain incompletely understood. METHODS: Following exposure to chronic social defeat stress (CSDS), mice were given free access to either regular chow or a Western-style diet high in triglycerides and cholesterol. Comprehensive metabolic and behavioral testing was then conducted. RESULTS: Mice subjected to CSDS and then fed a high-fat diet for 30 days display severe behavioral deficits accompanied by redistribution of body fat. Stressed mice have decreased adipose tissue as well as decreased serum leptin levels compared with control mice. Pharmacological inhibition of beta(3)-adrenergic signaling during CSDS normalizes these metabolic abnormalities but worsens behavioral symptoms. Furthermore, mice subjected to CSDS display central leptin resistance including reduced expression of pro-opiomelanocortin in hypothalamus. Administration of a central melanocortin agonist worsens stress-induced behavioral deficits, while mice lacking the melanocortin-4 receptor display attenuated symptoms. CONCLUSIONS: These results indicate that chronic signaling through beta(3)-adrenergic receptors during social stress is an adaptive response that improves behavioral function. However, these responses come at the expense of central leptin resistance and melanocortin signaling alterations that contribute to significant and long-lasting metabolic abnormalities.

Monitoring FoxO1 localization in chemically identified neurons.

The PI3K-Akt-FoxO1 pathway contributes to the actions of insulin and leptin in several cell types, including neurons in the CNS. However, identifying these actions in chemically identified neurons has proven difficult. To address this problem, we have developed a reporter mouse for monitoring PI3K-Akt signaling in specific populations of neurons, based on FoxO1 nucleocytoplasmic shuttling. The reporter, FoxO1 fused to green fluorescent protein (FoxO1GFP), is expressed under the control of a ubiquitous promoter that is silenced by a loxP flanked transcriptional blocker. Thus, the expression of the reporter in selected cells is dependent on the action of Cre recombinase. Using this model, we found that insulin treatment resulted in the nuclear exclusion of FoxO1GFP within POMC and AgRP neurons in a dose- and time-dependent manner. FoxO1GFP nuclear exclusion was also observed in POMC neurons following in vivo administration of insulin. In addition, leptin induced transient nuclear export of FoxO1GFP in POMC neurons in a dose dependent manner. Finally, insulin-induced nuclear export was impaired in POMC neurons by pretreatment with free fatty acids, a paradigm known to induce insulin resistance in peripheral insulin target tissues. Thus, our FoxO1GFP mouse provides a tool for monitoring the status of PI3K-Akt signaling in a cell-specific manner under physiological and pathophysiological conditions.

5-HT2CRs expressed by pro-opiomelanocortin neurons regulate energy homeostasis.

Drugs activating 5-hydroxytryptamine 2C receptors (5-HT2CRs) potently suppress appetite, but the underlying mechanisms for these effects are not fully understood. To tackle this issue, we generated mice with global 5-HT2CR deficiency (2C null) and mice with 5-HT2CRs re-expression only in pro-opiomelanocortin (POMC) neurons (2C/POMC mice). We show that 2C null mice predictably developed hyperphagia, hyperactivity, and obesity and showed attenuated responses to anorexigenic 5-HT drugs. Remarkably, all these deficiencies were normalized in 2C/POMC mice. These results demonstrate that 5-HT2CR expression solely in POMC neurons is sufficient to mediate effects of serotoninergic compounds on food intake. The findings also highlight the physiological relevance of the 5-HT2CR-melanocortin circuitry in the long-term regulation of energy balance.

Central nervous system regulation of energy metabolism: ghrelin versus leptin.

In this brief review, we introduce some major themes in the regulation of energy, lipid, and glucose metabolism by the central nervous system (CNS). Rather than comprehensively discussing the field, we instead will discuss some of the key findings made regarding the interaction of the hormones ghrelin and leptin with the CNS.

Synaptic glutamate release by ventromedial hypothalamic neurons is part of the neurocircuitry that prevents hypoglycemia.

The importance of neuropeptides in the hypothalamus has been experimentally established. Due to difficulties in assessing function in vivo, the roles of the fast-acting neurotransmitters glutamate and GABA are largely unknown. Synaptic vesicular transporters (VGLUTs for glutamate and VGAT for GABA) are required for vesicular uptake and, consequently, synaptic release of neurotransmitters. Ventromedial hypothalamic (VMH) neurons are predominantly glutamatergic and express VGLUT2. To evaluate the role of glutamate release from VMH neurons, we generated mice lacking VGLUT2 selectively in SF1 neurons (a major subset of VMH neurons). These mice have hypoglycemia during fasting secondary to impaired fasting-induced increases in the glucose-raising pancreatic hormone glucagon and impaired induction in liver of mRNAs encoding PGC-1alpha and the gluconeogenic enzymes PEPCK and G6Pase. Similarly, these mice have defective counterregulatory responses to insulin-induced hypoglycemia and 2-deoxyglucose (an antimetabolite). Thus, glutamate release from VMH neurons is an important component of the neurocircuitry that functions to prevent hypoglycemia.

Minireview: From anorexia to obesity--the yin and yang of body weight control.

Over the past decade, there has been a tremendous increase in the understanding of the molecular and neural mechanisms that control food intake and body weight. Yet eating disorders and cachexia are still common, and obesity cases are rising at alarming rates. Thus, despite recent progress, an increased understanding of the molecular and neural substrates that control body weight homeostasis is a major public health goal. In this review, we discuss the mechanisms by which metabolic signals interact with key behavioral, neuroendocrine, and autonomic regulatory regions of the central nervous system. Additionally, we offer a model in which hormones such as leptin and ghrelin interact with similar central nervous system circuits and engage them in such a way as to maintain an appropriate and tight regulation of body weight and food intake. Our model predicts that overstimulation or understimulation of these central pathways can result in obesity, anorexia, or cachexia.

Central serotonin and melanocortin pathways regulating energy homeostasis.

It is now established that the hypothalamus is essential in coordinating endocrine, autonomic, and behavioral responses to changes in energy availability. However, the interaction of key peptides, neuropeptides, and neurotransmitters systems within the hypothalamus has yet to be delineated. Recently, we investigated the mechanisms through which central serotonergic (5-hydroxytryptamine, 5-HT) systems recruit leptin-responsive hypothalamic pathways, such as the melanocortin systems, to affect energy balance. Through a combination of functional neuroanatomy, feeding, and electrophysiology studies in rodents, we found that 5-HT drugs require functional melanocortin pathways to exert their effects on food intake. Specifically, we observed that anorectic 5-HT drugs activate pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (Arc). We provide evidence that the serotonin 2C receptor (5-HT(2C)R) is expressed on POMC neurons and contributes to this effect. Finally, we found that 5-HT drug-induced hypophagia is attenuated by pharmacological or genetic blockade of downstream melanocortin 3 and 4 receptors. We review candidate brain regions expressing melanocortin 3 and 4 receptors that play a role in energy balance. A model is presented in which activation of the melanocortin system is downstream of 5-HT and is necessary to produce the complete anorectic effect of 5-HT drugs. The data reviewed in this paper incorporate the central 5-HT system to the growing list of metabolic signals that converge on melanocortin neurons in the hypothalamus.