Glutamatergic Transmission to Hypothalamic Kisspeptin Neurons Is Differentially Regulated by Estradiol through Estrogen Receptor α in Adult Female Mice.

Estradiol feedback regulates gonadotropin-releasing hormone (GnRH) neurons and subsequent luteinizing hormone (LH) release. Estradiol acts via estrogen receptor α (ERα)-expressing afferents of GnRH neurons, including kisspeptin neurons in the anteroventral periventricular (AVPV) and arcuate nuclei, providing homeostatic feedback on episodic GnRH/LH release as well as positive feedback to control ovulation. Ionotropic glutamate receptors are important for estradiol feedback, but it is not known where they fit in the circuitry. Estradiol-negative feedback decreased glutamatergic transmission to AVPV and increased it to arcuate kisspeptin neurons; positive feedback had the opposite effect. Deletion of ERα in kisspeptin cells decreased glutamate transmission to AVPV neurons and markedly increased it to arcuate kisspeptin neurons, which also exhibited increased spontaneous firing rate. KERKO mice had increased LH pulse frequency, indicating loss of negative feedback. These observations indicate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and neuroendocrine output by estradiol.SIGNIFICANCE STATEMENT The brain regulates fertility through gonadotropin-releasing hormone (GnRH) neurons. Ovarian estradiol regulates the pattern of GnRH (negative feedback) and initiates a surge of release that triggers ovulation (positive feedback). GnRH neurons do not express the estrogen receptor needed for feedback (estrogen receptor α [ERα]); kisspeptin neurons in the arcuate and anteroventral periventricular nuclei are postulated to mediate negative and positive feedback, respectively. Here we extend the network through which feedback is mediated by demonstrating that glutamatergic transmission to these kisspeptin populations is differentially regulated during the reproductive cycle and by estradiol. Electrophysiological and in vivo hormone profile experiments on kisspeptin-specific ERα knock-out mice demonstrate that ERα in kisspeptin cells is required for appropriate differential regulation of these neurons and for neuroendocrine output.

Disruption of leptin receptor-STAT3 signaling enhances leukotriene production and pulmonary host defense against pneumococcal pneumonia.

The adipocyte-derived hormone leptin regulates energy homeostasis and the innate immune response. We previously reported that leptin plays a protective role in bacterial pneumonia, but the mechanisms by which leptin regulates host defense remain poorly understood. Leptin binding to its receptor, LepRb, activates multiple intracellular signaling pathways, including ERK1/2, STAT5, and STAT3. In this study, we compared the responses of wild-type and s/s mice, which possess a mutant LepRb that prevents leptin-induced STAT3 activation, to determine the role of this signaling pathway in pneumococcal pneumonia. Compared with wild-type animals, s/s mice exhibited greater survival and enhanced pulmonary bacterial clearance after an intratracheal challenge with Streptococcus pneumoniae. We also observed enhanced phagocytosis and killing of S. pneumoniae in vitro in alveolar macrophages (AMs) obtained from s/s mice. Notably, the improved host defense and AM antibacterial effector functions in s/s mice were associated with increased cysteinyl-leukotriene production in vivo and in AMs in vitro. Augmentation of phagocytosis in AMs from s/s mice could be blocked using a pharmacologic cysteinyl-leukotriene receptor antagonist. Phosphorylation of ERK1/2 and cytosolic phospholipase A(2) α, known to enhance the release of arachidonic acid for subsequent conversion to leukotrienes, was also increased in AMs from s/s mice stimulated with S. pneumoniae in vitro. These data indicate that ablation of LepRb-mediated STAT3 signaling and the associated augmentation of ERK1/2, cytosolic phospholipase A(2) α, and cysteinyl-leukotriene synthesis confers resistance to s/s mice during pneumococcal pneumonia. These data provide novel insights into the intracellular signaling events by which leptin contributes to host defense against bacterial pneumonia.

Nucleus of the Solitary Tract Serotonin 5-HT2C Receptors Modulate Food Intake.

To meet the challenge to human health posed by obesity, a better understanding of the regulation of feeding is essential. Medications targeting 5-hydroxytryptamine (5-HT; serotonin) 2C receptors (htr2c; 5-HT2CR) improve obesity. Here we probed the functional significance of 5-HT2CRs specifically within the brainstem nucleus of the solitary tract (5-HT2CRNTS) in feeding behavior. Selective activation of 5-HT2CRNTS decreased feeding and was sufficient to mediate acute food intake reductions elicited by the 5-HT2CR agonist obesity medication lorcaserin. Similar to pro-opiomelanocortin neurons expressed within the hypothalamic arcuate nucleus (POMCARC), a subset of POMCNTS neurons co-expressed 5-HT2CRs and were activated by 5-HT2CR agonists. Knockdown of POMCNTS prevented the acute appetite-suppressive effect of lorcaserin, whereas POMCARC knockdown prevented the full anorectic effect. These data identify 5-HT2CRNTS as a sufficient subpopulation of 5-HT2CRs in reducing food intake when activated and reveal that 5-HT2CR agonist obesity medications require POMC within the NTS and ARC to reduce food intake.

Activation of Ventral Tegmental Area 5-HT2C Receptors Reduces Incentive Motivation.

Obesity is primarily due to food intake in excess of the body's energetic requirements, intake that is not only associated with hunger but also the incentive value of food. The 5-hydroxytryptamine 2C receptor (5-HT2CR) is a target for the treatment of human obesity. Mechanistically, 5-HT2CRs are positioned to influence both homeostatic feeding circuits within the hypothalamus and reward circuits within the ventral tegmental area (VTA). Here we investigated the role of 5-HT2CRs in incentive motivation using a mathematical model of progressive ratio (PR) responding in mice. We found that the 5-HT2CR agonist lorcaserin significantly reduced both ad libitum chow intake and PR responding for chocolate pellets and increased c-fos expression in VTA 5-HT2CR expressing γ-aminobutyric acid (GABA) neurons, but not 5-HT2CR expressing dopamine (DA) neurons. We next adopted a chemogenetic approach using a 5-HT2CRCRE line to clarify the function of subset of 5-HT2C receptor expressing VTA neurons in the modulation of appetite and food-motivated behavior. Activation of VTA 5-HT2C receptor expressing neurons significantly reduced ad libitum chow intake, operant responding for chocolate pellets, and the incentive value of food. In contrast, chemogenetic inhibition of VTA 5-HT2C receptor expressing neurons had no effect on the feeding behavior. These results indicate that activation of the subpopulation of 5-HT2CR neurons within the VTA is sufficient to significantly reduce homeostatic feeding and effort-based intake of palatable food, and that this subset has an inhibitory role in motivational processes. These findings are relevant to the treatment of obesity.

ERα in Tac2 Neurons Regulates Puberty Onset in Female Mice.

A variety of data suggest that estrogen action on kisspeptin (Kiss1)-containing arcuate nucleus neurons (which coexpress Kiss1, neurokinin B (the product of Tac2) and dynorphin (KNDy) neurons restrains reproductive onset and function, but roles for estrogen action in these Kiss1 neurons relative to a distinct population of rostral hypothalamic Kiss1 neurons (which does not express Tac2 or dynorphin) have not been directly tested. To test the role for estrogen receptor (ER)α in KNDy cells, we thus generated Tac2(Cre) and Kiss1(Cre) knock-in mice and bred them onto the Esr1(flox) background to ablate ERα specifically in Tac2-expressing cells (ERα(Tac2)KO mice) or all Kiss1 cells (ERα(Kiss1)KO mice), respectively. Most ERα-expressing Tac2 neurons represent KNDy cells. Arcuate nucleus Kiss1 expression was elevated in ERα(Tac2)KO and ERα(Kiss1)KO females independent of gonadal hormones, whereas rostral hypothalamic Kiss1 expression was normal in ERα(Tac2)KO but decreased in ERα(Kiss1)KO females; this suggests that ERα in rostral Kiss1 cells is crucial for control of Kiss1 expression in these cells. Both ERα(Kiss1)KO and ERα(Tac2)KO females displayed early vaginal opening, early and persistent vaginal cornification, increased gonadotropins, uterine hypertrophy, and other evidence of estrogen excess. Thus, deletion of ERα in Tac2 neurons suffices to drive precocious gonadal hyperstimulation, demonstrating that ERα in Tac2 neurons typically restrains pubertal onset and hypothalamic reproductive drive.

Sex difference in physical activity, energy expenditure and obesity driven by a subpopulation of hypothalamic POMC neurons.

OBJECTIVE: Obesity is one of the primary healthcare challenges of the 21st century. Signals relaying information regarding energy needs are integrated within the brain to influence body weight. Central among these integration nodes are the brain pro-opiomelanocortin (POMC) peptides, perturbations of which disrupt energy balance and promote severe obesity. However, POMC neurons are neurochemically diverse and the crucial source of POMC peptides that regulate energy homeostasis and body weight remains to be fully clarified. METHODS: Given that a 5-hydroxytryptamine 2c receptor (5-HT2CR) agonist is a current obesity medication and 5-HT2CR agonist's effects on appetite are primarily mediated via POMC neurons, we hypothesized that a critical source of POMC regulating food intake and body weight is specifically synthesized in cells containing 5-HT2CRs. To exclusively manipulate Pomc synthesis only within 5-HT2CR containing cells, we generated a novel 5-HT 2C R (CRE) mouse line and intercrossed it with Cre recombinase-dependent and hypothalamic specific reactivatable Pomc (NEO) mice to restrict Pomc synthesis to the subset of hypothalamic cells containing 5-HT2CRs. This provided a means to clarify the specific contribution of a defined subgroup of POMC peptides in energy balance and body weight. RESULTS: Here we transform genetically programed obese and hyperinsulinemic male mice lacking hypothalamic Pomc with increased appetite, reduced physical activity and compromised brown adipose tissue (BAT) into lean, healthy mice via targeted restoration of Pomc function only within 5-HT2CR expressing cells. Remarkably, the same metabolic transformation does not occur in females, who despite corrected feeding behavior and normalized insulin levels remain physically inactive, have lower energy expenditure, compromised BAT and develop obesity. CONCLUSIONS: These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc expression within 5-HT2CR expressing neurons is sufficient to regulate energy intake and insulin sensitivity in male and female mice. However, an unexpected sex difference in the function of this subset of POMC neurons was identified with regard to energy expenditure. We reveal that a large sex difference in physical activity, energy expenditure and the development of obesity is driven by this subpopulation, which constitutes approximately 40% of all POMC neurons in the hypothalamic arcuate nucleus. This may have broad implications for strategies utilized to combat obesity, which at present largely ignore the sex of the obese individual.

Leptin action via LepR-b Tyr1077 contributes to the control of energy balance and female reproduction.

Leptin action in the brain signals the repletion of adipose energy stores, suppressing feeding and permitting energy expenditure on a variety of processes, including reproduction. Leptin binding to its receptor (LepR-b) promotes the tyrosine phosphorylation of three sites on LepR-b, each of which mediates distinct downstream signals. While the signals mediated by LepR-b Tyr1138 and Tyr985 control important aspects of energy homeostasis and LepR-b signal attenuation, respectively, the role of the remaining LepR-b phosphorylation site (Tyr1077) in leptin action has not been studied. To examine the function of Tyr1077, we generated a "knock-in" mouse model expressing LepR-b (F1077), which is mutant for LepR-b Tyr1077. Mice expressing LepR-b (F1077) demonstrate modestly increased body weight and adiposity. Furthermore, females display impairments in estrous cycling. Our results suggest that signaling by LepR-b Tyr1077 plays a modest role in the control of metabolism by leptin, and is an important link between body adiposity and the reproductive axis.

Leptin action through hypothalamic nitric oxide synthase-1-expressing neurons controls energy balance.

Few effective measures exist to combat the worldwide obesity epidemic(1), and the identification of potential therapeutic targets requires a deeper understanding of the mechanisms that control energy balance. Leptin, an adipocyte-derived hormone that signals the long-term status of bodily energy stores, acts through multiple types of leptin receptor long isoform (LepRb)-expressing neurons (called here LepRb neurons) in the brain to control feeding, energy expenditure and endocrine function(2-4). The modest contributions to energy balance that are attributable to leptin action in many LepRb populations(5-9) suggest that other previously unidentified hypothalamic LepRb neurons have key roles in energy balance. Here we examine the role of LepRb in neuronal nitric oxide synthase (NOS1)-expressing LebRb (LepRb(NOS1)) neurons that comprise approximately 20% of the total hypothalamic LepRb neurons. Nos1(cre)-mediated genetic ablation of LepRb (Lepr(Nos1KO)) in mice produces hyperphagic obesity, decreased energy expenditure and hyperglycemia approaching that seen in whole-body LepRb-null mice. In contrast, the endocrine functions in Lepr(Nos1KO) mice are only modestly affected by the genetic ablation of LepRb in these neurons. Thus, hypothalamic LepRb(NOS1) neurons are a key site of action of the leptin-mediated control of systemic energy balance.

Testosterone interacts with the feedback mechanisms engaged by Tyr985 of the leptin receptor and diet-induced obesity.

Inhibitory signaling through Tyr985 of the leptin receptor contributes to the attenuation of anorectic leptin action in obese animals. Leptin receptor (LEPR-B) Tyr985Leu homozygote mutant mice (termed l/l) were previously generated to study Tyr985's contributions to inhibition of LEPR-B signaling; young female l/l mice display a lean, leptin-sensitive phenotype, while young male l/l are not significantly different from wild-type. We report here that testosterone (but not estrogen) determines the sex-specificity of the l/l phenotype. This provides additional insight into the cellular mechanism by which gonadal hormones determine central sensitivity to leptin, and may help elucidate the long-noted sex differences in leptin sensitivity. Additionally, we observed that Tyr985 signaling protects against a diet-dependent switch that exacerbates obesity with high fat feeding, such that the enhanced leptin sensitivity of l/l mice on a normal diet leads to increased adiposity in the face of chronic high-fat diet.

Molecular mapping of the neural pathways linking leptin to the neuroendocrine reproductive axis.

Negative energy balance and insufficient adipose energy stores decrease the production of leptin, thereby diminishing the leptin-supported secretion of GnRH from the hypothalamus and promoting decreased reproductive function. Leptin acts via its receptor (LepRb) to support the neuroendocrine reproductive axis, but the nature and location of the relevant LepRb neurons remain poorly understood. Possibilities include the direct or indirect action of leptin on hypothalamic GnRH neurons, or on kisspeptin (Kiss1) neurons that are major regulators of GnRH neurons. To evaluate these potential mechanisms, we employed immunohistochemical analysis of the female brain from various molecular mouse models and sheep. Our analysis revealed no LepRb in GnRH neurons or in anteroventral periventricular Kiss1 neurons, and very limited (0-6%) colocalization with arcuate nucleus Kiss1 cells, suggesting that leptin does not modulate reproduction by direct action on any of these neural populations. LepRb neurons, primarily in the hypothalamic ventral premammillary nucleus and a subregion of the preoptic area, lie in close contact with GnRH neurons, however. Furthermore, an unidentified population or populations of LepRb neurons lie in close contact with arcuate nucleus and anteroventral periventricular Kiss1 neurons. Taken together, these findings suggest that leptin communicates with the neuroendocrine reproductive axis via multiple populations of LepRb neurons that lie afferent to both Kiss1 and GnRH neurons.