Hypothalamic neurons expressing the anorectic peptide Pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here, we show that Steroid Receptor Coactivator-1 (SRC-1) interacts with a target of leptin receptor activation, phosphorylated STAT3, to potentiate Pomc transcription. Deletion of SRC-1 in Pomc neurons in mice attenuates their depolarization by leptin, decreases Pomc expression and increases food intake leading to high-fat diet-induced obesity. In humans, fifteen rare heterozygous variants in SRC-1 found in severely obese individuals impair leptin-mediated Pomc reporter activity in cells, whilst four variants found in non-obese controls do not. In a knock-in mouse model of a loss of function human variant (SRC-1L1376P), leptin-induced depolarization of Pomc neurons and Pomc expression are significantly reduced, and food intake and body weight are increased. In summary, we demonstrate that SRC-1 modulates the function of hypothalamic Pomc neurons, and suggest that targeting SRC-1 may represent a useful therapeutic strategy for weight loss.

Sexual dimorphism exists in energy balance, but the underlying mechanisms remain unclear. Here we show that the female mice have more pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of hypothalamus than males, and female POMC neurons display higher neural activities, compared to male counterparts. Strikingly, deletion of the transcription factor, TAp63, in POMC neurons confers "male-like" diet-induced obesity (DIO) in female mice associated with decreased POMC neural activities; but the same deletion does not affect male mice. Our results indicate that TAp63 in female POMC neurons contributes to the enhanced POMC neuron functions and resistance to obesity in females. Thus, TAp63 in POMC neurons is one key molecular driver for the sexual dimorphism in energy homeostasis.

BackgroundNeural networks that regulate binge eating remain to be identified, and effective treatments for binge eating are limited.MethodsWe combined neuroanatomic, pharmacologic, electrophysiological, Cre-lox, and chemogenetic approaches to investigate the functions of 5-hydroxytryptamine (5-HT) 2C receptor (5-HT2CR) expressed by dopamine (DA) neurons in the regulation of binge-like eating behavior in mice.ResultsWe showed that 5-HT stimulates DA neural activity through a 5-HT2CR-mediated mechanism, and activation of this midbrain 5-HT→DA neural circuit effectively inhibits binge-like eating behavior in mice. Notably, 5-HT medications, including fluoxetine, d-fenfluramine, and lorcaserin (a selective 5-HT2CR agonist), act on 5-HT2CRs expressed by DA neurons to inhibit binge-like eating in mice.ConclusionsWe identified the 5-HT2CR population in DA neurons as one potential target for antibinge therapies, and provided preclinical evidence that 5-HT2CR agonists could be used to treat binge eating.

Framingham Heart Study suggests that dysfunction of steroid receptor coactivator-1 may be involved in the development of hypertension. However, there is no functional evidence linking steroid receptor coactivator-1 to the regulation of blood pressure. We used immunohistochemistry to map the expression of steroid receptor coactivator-1 protein in mouse brain, especially in regions implicated in the regulation of blood pressure. Steroid receptor coactivator-1 protein was found in central amygdala, medial amygdala, supraoptic nucleus, arcuate nucleus, ventromedial, dorsomedial, paraventricular hypothalamus, and nucleus of the solitary tract. To determine the effects of steroid receptor coactivator-1 protein on cardiovascular system we measured blood pressures, blood flow velocities, echocardiographic parameters, and aortic input impedance in female steroid receptor coactivator-1 knockout mice and their wild type littermates. Steroid receptor coactivator-1 knockout mice had higher blood pressures and increased aortic stiffness when compared to female wild type littermates. Additionally, the hearts of steroid receptor coactivator-1 knockout mice seem to consume higher energy as evidenced by increased impedance and higher heart rate pressure product when compared to female wild type littermates. Our results demonstrate that steroid receptor coactivator-1 may be functionally involved in the regulation of blood pressure and aortic stiffness through the regulation of sympathetic activation in various neuronal populations.

Psychological stress contributes to the development of hypertension in humans. The ovarian hormone, estrogen, has been shown to prevent stress-induced pressor responses in females by unknown mechanisms. Here, we showed that the antihypertensive effects of estrogen during stress were blunted in female mice lacking estrogen receptor-α in the brain medial amygdala. Deletion of estrogen receptor-α in medial amygdala neurons also resulted in increased excitability of these neurons, associated with elevated ionotropic glutamate receptor expression. We further demonstrated that selective activation of medial amygdala neurons mimicked effects of stress to increase blood pressure in mice. Together, our results support a model where estrogen acts on estrogen receptor-α expressed by medial amygdala neurons to prevent stress-induced activation of these neurons, and therefore prevents pressor responses to stress.

Neurons that co-express agouti-related peptide (AgRP) and neuropeptide Y (NPY) are indispensable for normal feeding behavior. Firing activities of AgRP/NPY neurons are dynamically regulated by energy status and coordinate appropriate feeding behavior to meet nutritional demands. However, intrinsic mechanisms that regulate AgRP/NPY neural activities during the fed-to-fasted transition are not fully understood. We found that AgRP/NPY neurons in satiated mice express high levels of the small-conductance calcium-activated potassium channel 3 (SK3) and are inhibited by SK3-mediated potassium currents; on the other hand, food deprivation suppresses SK3 expression in AgRP/NPY neurons, and the decreased SK3-mediated currents contribute to fasting-induced activation of these neurons. Genetic mutation of SK3 specifically in AgRP/NPY neurons leads to increased sensitivity to diet-induced obesity, associated with chronic hyperphagia and decreased energy expenditure. Our results identify SK3 as a key intrinsic mediator that coordinates nutritional status with AgRP/NPY neural activities and animals’ feeding behavior and energy metabolism.

BACKGROUND/AIMS:Apolipoprotein A-IV (apoA-IV) in the brain potently suppresses food intake. However, the mechanisms underlying its anorexigenic effects remain to be identified.METHODS:We first examined the effects of apoA-IV on cellular activities in hypothalamic neurons that co-express agouti-related peptide (AgRP) and neuropeptide Y (NPY) and in neurons that express pro-opiomelanocortin (POMC). We then compared anorexigenic effects of apoA-IV in wild-type mice and in mutant mice lacking melanocortin 4 receptors (MC4Rs; the receptors of AgRP and the POMC gene product). Finally, we examined expression of apoA-IV in mouse hypothalamus and quantified its protein levels at fed versus fasted states.RESULTS:We demonstrate that apoA-IV inhibited the firing rate of AgRP/NPY neurons. The decreased firing was associated with hyperpolarized membrane potential and decreased miniature excitatory postsynaptic current. We further used c-fos immunoreactivity to show that intracerebroventricular (i.c.v.) injections of apoA-IV abolished the fasting-induced activation of AgRP/NPY neurons in mice. Further, we found that apoA-IV depolarized POMC neurons and increased their firing rate. In addition, genetic deletion of MC4Rs blocked anorexigenic effects of i.c.v. apoA-IV. Finally, we detected endogenous apoA-IV in multiple neural populations in the mouse hypothalamus, including AgRP/NPY neurons, and food deprivation suppressed hypothalamic apoA-IV protein levels.CONCLUSION:Our findings support a model where central apoA-IV inhibits AgRP/NPY neurons and activates POMC neurons to activate MC4Rs, which in turn suppresses food intake.

Estrogens act in the ventromedial hypothalamic nucleus (VMH) to regulate body weight homeostasis. However, the molecular mechanisms underlying these estrogenic effects are unknown. We show that activation of estrogen receptor-α (ERα) stimulates neural firing of VMH neurons expressing ERα, and these effects are blocked with intracellular application of a pharmacological inhibitor of the phosphatidyl inositol 3-kinase (PI3K). Further, we demonstrated that mice with genetic inhibition of PI3K activity in VMH neurons showed a sexual dimorphic obese phenotype, with only female mutants being affected. In addition, inhibition of VMH PI3K activity blocked effects of 17β-estradiol to stimulate energy expenditure, but did not affect estrogen-induced anorexia. Collectively, our results indicate that PI3K activity in VMH neurons plays a physiologically relevant role in mediating estrogenic actions on energy expenditure in females.

Background. Brain estrogen receptor-alpha (ER alpha) is essential for estrogenic regulation of energy homeostasis and reproduction. We previously showed that ER alpha expressed by pro-opiotnelanocortin (POMC) neurons mediates estrogen's effects on food intake, body weight, negative regulation of hypothalamic-pituitary-gonadal axis (HPG axis) and fertility.Results and conclusions. We report here that global deletion of a key downstream receptor for POMC peptide, the melanocortin 4 receptor (MC4R), did not affect normal negative feedback regulation of estrogen on the HPG axis, estrous cyclicity and female fertility. Furthermore, loss of the MC4R did not influence estrogenic regulation on food intake and body weight. These results indicate that the MC4R is not required for estrogen's effects on metabolic and reproductive functions. (C) 2016 Elsevier Inc. All rights reserved.

Estrogen receptor-α (ERα) activity in the brain prevents obesity in both males and females. However, the ERα-expressing neural populations that regulate body weight remain to be fully elucidated. Here we showed that single-minded-1 (SIM1) neurons in the medial amygdala (MeA) express abundant levels of ERα. Specific deletion of the gene encoding ERα (Esr1) from SIM1 neurons, which are mostly within the MeA, caused hypoactivity and obesity in both male and female mice fed with regular chow, increased susceptibility to diet-induced obesity (DIO) in males but not in females, and blunted the body weight-lowering effects of a glucagon-like peptide-1-estrogen (GLP-1-estrogen) conjugate. Furthermore, selective adeno-associated virus-mediated deletion of Esr1 in the MeA of adult male mice produced a rapid body weight gain that was associated with remarkable reductions in physical activity but did not alter food intake. Conversely, overexpression of ERα in the MeA markedly reduced the severity of DIO in male mice. Finally, an ERα agonist depolarized MeA SIM1 neurons and increased their firing rate, and designer receptors exclusively activated by designer drug-mediated (DREADD-mediated) activation of these neurons increased physical activity in mice. Collectively, our results support a model where ERα signals activate MeA neurons to stimulate physical activity, which in turn prevents body weight gain.

Background A variety of biological functions of estrogens, including regulation of energy metabolism, are mediated by neurons expressing estrogen receptor-α (ERα) in the brain. However, complex intracellular processes in these ERα-expressing neurons are difficult to unravel, due to the lack of strategy to visualize ERα-expressing neurons, especially in unfixed brain tissues. Results and conclusions Here we generated a novel ERα-ZsGreen reporter mouse line in which expression of a green fluorescent reporter protein, ZsGreen, is driven by a 241 kb ERα gene promoter. We validated that ZsGreen is highly colocalized with endogenous ERα in the brain. Native ZsGreen signals were visualized in unfixed brain tissue, and were used to assist single cell collection and electrophysiological recordings. Finally, we demonstrated that this ERα-ZsGreen mouse allele can be used in combination with other genetic reporter alleles to allow experiments in highly selective neural populations.

Background and PurposeMost forms of human obesity are characterized by impaired leptin sensitivity and, therefore, the effectiveness of anti-obesity leptin therapy in these leptin-resistant obese patients is marginal. Hence, the development of strategies to increase leptin sensitivity is of high priority in the field of obesity research.Experimental ApproachWe first examined the effects of co-administration of leptin and meta-chlorophenylpiperazine (mCPP), an agonist of 5-HT2C and 5-HT1B receptors, on energy balance in leptin-resistant diet-induced obese (DIO) mice. We further assessed leptin-induced phosphorylation of the STAT-3 (pSTAT3) in various brain regions of DIO mice pretreated with mCPP or in mice genetically lacking 5-HT2C receptors.ResultsCo-administration of mCPP with leptin had an additive effect on reducing body weight in DIO mice. Furthermore, mCPP pretreatment in DIO mice enhanced leptin-induced pSTAT3 in the arcuate nucleus, the ventromedial hypothalamic nucleus, and the ventral premammillary nucleus. Finally, deletion of 5-HT2C receptors significantly blunted leptin-induced pSTAT3 in these same hypothalamic regions.Conclusions and ImplicationsOur study provides evidence that drugs, which activate 5-HT2C receptors, could function as leptin sensitizers and be used in combination with leptin to provide additional weight loss in DIO.

Estrogens act upon estrogen receptor (ER)α to inhibit feeding and improve glucose homeostasis in female animals. However, the intracellular signals that mediate these estrogenic actions remain unknown. Here, we report that anorexigenic effects of estrogens are blunted in female mice that lack ERα specifically in proopiomelanocortin (POMC) progenitor neurons. These mutant mice also develop insulin resistance and are insensitive to the glucose-regulatory effects of estrogens. Moreover, we showed that propyl pyrazole triol (an ERα agonist) stimulates the phosphatidyl inositol 3-kinase (PI3K) pathway specifically in POMC progenitor neurons, and that blockade of PI3K attenuates propyl pyrazole triol-induced activation of POMC neurons. Finally, we show that effects of estrogens to inhibit food intake and to improve insulin sensitivity are significantly attenuated in female mice with PI3K genetically inhibited in POMC progenitor neurons. Together, our results indicate that an ERα-PI3K cascade in POMC progenitor neurons mediates estrogenic actions to suppress food intake and improve insulin sensitivity.

BackgroundHypertension is the leading cause of cardiovascular disease worldwide. The etiology of essential hypertension is unknown. Psychological stress contributes to development of hypertension in humans. Estrogen has been shown to prevent stress‐induced hypertension in rodents with unknown mechanisms. Amygdala neurons have been implicated in controlling blood pressure. Abundant estrogen receptors, including estrogen receptor‐α (ERα), are expressed in the medial amygdala (MeA). Thus, we hypothesize that ERα expressed by MeA neurons mediates the anti‐hypertensive effects of estrogen during stress.MethodsWe use the Cre‐LoxP system to remove ERα from specific MeA neurons in mice (ERαlox/lox/SIM1‐Cre). Female ERαlox/lox/SIM1‐Cre mice and controls (ERαlox/lox) were subjected to bilateral ovariectomy (OVX), given a vehicle (V) or 17‐β‐Estradiol pellet (E2), and inserted with telemetry probes on day one. After a seven‐day recovery, blood pressure was recorded for 3 hours to establish baseline and then mice were subjected to 1 hour stress restraint.ResultsERαlox/lox OVX + V mice showed increased mean arterial blood pressure (MAP) and systolic arterial blood pressure (SAP) in response to stress, whereas this stress‐induced hypertension was blunted in ERαlox/lox OVX + E mice. Interestingly, ERαlox/lox/SIM1‐Cre OVX + E and OVX + V group both showed increased MAP and SAP in response to stress.ConclusionsOur results suggest that ERα in the MeA is required to mediate estrogenic actions to prevent stress‐induced hypertension.

Circadian disruption has become a significant factor contributing to the epidemics of obesity and insulin resistance. However, interventions to treat metabolic dysfunctions induced by circadian disruptions are limited. The ovarian hormone, estrogen, produces important antiobesity and antidiabetic effects in female animals and has profound effects on daily behavioral rhythms. Here, we show that in female mice depleted with endogenous estrogens, a jet-lag paradigm induced visceral fat accumulation and systemic insulin resistance, which were associated with altered expression of multiple circadian genes in the visceral fat depot. Interestingly, all these jet-lag-induced deficits were completely rescued in female mice supplemented with exogenous estrogens. We further examined 24-hour oscillations of circadian genes in adipose tissues in female mice with estrogen depletion or replacement and showed that expression levels of the circadian gene, period circadian protein homolog 2, oscillate in visceral adipose tissue in an estrogen-dependent manner. Together, our results indicate that estrogens interact with the intrinsic circadian clock in adipose tissue and prevent abnormal lipid accumulation caused by circadian disruptions.

Binge eating afflicts approximately 5% of US adults, though effective treatments are limited. Here, we showed that estrogen replacement substantially suppresses binge-like eating behavior in ovariectomized female mice. Estrogen-dependent inhibition of binge-like eating was blocked in female mice specifically lacking estrogen receptor-α (ERα) in serotonin (5-HT) neurons in the dorsal raphe nuclei (DRN). Administration of a recently developed glucagon-like peptide-1-estrogen (GLP-1-estrogen) conjugate designed to deliver estrogen to GLP1 receptor-enhanced regions effectively targeted bioactive estrogens to the DRN and substantially suppressed binge-like eating in ovariectomized female mice. Administration of GLP-1 alone reduced binge-like eating, but not to the same extent as the GLP-1-estrogen conjugate. Administration of ERα-selective agonist propylpyrazole triol (PPT) to murine DRN 5-HT neurons activated these neurons in an ERα-dependent manner. PPT also inhibited a small conductance Ca2+-activated K+ (SK) current; blockade of the SK current prevented PPT-induced activation of DRN 5-HT neurons. Furthermore, local inhibition of the SK current in the DRN markedly suppressed binge-like eating in female mice. Together, our data indicate that estrogens act upon ERα to inhibit the SK current in DRN 5-HT neurons, thereby activating these neurons to suppress binge-like eating behavior and suggest ERα and/or SK current in DRN 5-HT neurons as potential targets for anti-binge therapies.

Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.