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Objective: Rodent models raised at environmental temperatures of 21-22 °C are increasingly switched to thermoneutral housing conditions in adulthood to better capture human physiology. We quantified the developmental effects of rearing mice at an ambient temperature of 22 °C vs. 30 °C on metabolic responses to cold and high fat diet (HFD) in adulthood., Methods: Mice were reared from birth to 8 weeks of age at 22 °C or 30 °C, when they were acclimated to single housing at the same temperature for 2-3 weeks in indirect calorimetry cages. Energy expenditure attributable to basal metabolic rate, physical activity, thermic effect of food, and adaptive cold- or diet-induced thermogenesis was calculated. Responses to cooling were evaluated by decreasing the ambient temperature from 22 °C to 14 °C, while responses to HFD feeding were assessed at 30 °C. Influences of rearing temperature on thermogenic responses that emerge over hours, days and weeks were assessed by maintaining mice in the indirect calorimetry cages throughout the study., Results: At an ambient temperature of 22 °C, total energy expenditure (TEE) was 12-16% higher in mice reared at 22 °C as compared to 30 °C. Rearing temperature had no effect on responses in the first hours or week of the 14 °C challenge. Differences emerged in the third week, when TEE increased an additional 10% in mice reared at 22 °C, but mice reared at 30 °C could not sustain this level of cold-induced thermogenesis. Rearing temperature only affected responses to HFD during the first week, due to differences in the timing but not the strength of metabolic adaptations., Conclusion: Rearing at 22 °C does not have a lasting effect on metabolic adaptations to HFD at thermoneutrality, but it programs an enhanced capacity to respond to chronic cold challenges in adulthood. These findings highlight the need to consider rearing temperature when using mice to model cold-induced thermogenesis., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lori M. Zeltser reports financial support was provided by National Institutes of Health. I am a topic editor for Developmental Biology., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Background/purpose: Litter size is a biological variable that strongly influences adult physiology in rodents. Despite evidence from previous decades and recent studies highlighting its major impact on metabolism, information about litter size is currently underreported in the scientific literature. Here, we urge that this important biological variable should be explicitly stated in research articles., Results/conclusion: Below, we briefly describe the scientific evidence supporting the impact of litter size on adult physiology and outline a series of recommendations and guidelines to be implemented by investigators, funding agencies, editors in scientific journals, and animal suppliers to fill this important gap., Competing Interests: Conflicts of interest None declared., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Stress often affects eating behaviors, leading to increased eating in some individuals and decreased eating in others. Identifying physiological and psychological factors that determine the direction of eating responses to stress has been a major goal of epidemiological and clinical studies. However, challenges of standardizing the stress exposure in humans hinder efforts to uncover the underlying mechanisms. The issue of what determines the direction of stress-induced feeding responses has not been directly addressed in animal models, but assays that combine stress with a feeding-related task are commonly used as readouts of other behaviors, such as anxiety. Sex, estrous cyclicity, circadian cyclicity, caloric restriction, palatable diets, elevated body weight, and properties of the stressors similarly influence feeding behavior in humans and rodent models. Yet, most rodent studies do not use conditions that are most relevant for studying feeding behavior in humans. This review proposes a conceptual framework for incorporating these influences to develop reproducible and translationally relevant assays to study effects of stress on food intake. Such paradigms have the potential to uncover links between emotional eating and obesity as well as to the etiology of eating disorders., (Copyright © 2021 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: The goal of this review is to describe how emerging technological developments in pre-clinical animal research can be harnessed to accelerate research in anorexia nervosa (AN)., Recent Findings: The activity-based anorexia (ABA) paradigm, the best characterized animal model of AN, combines restricted feeding, excessive exercise, and weight loss. A growing body of evidence supports the idea that pathophysiological weight loss in this model is due to cognitive inflexibility, a clinical feature of AN. Targeted manipulations that recapitulate brain changes reported in AN - hyperdopaminergia or hyperactivity of cortical inputs to the nucleus accumbens - exacerbate weight loss in the ABA paradigm, providing the first evidence of causality. The power of preclinical research lies in the ability to assess the consequences of targeted manipulations of neuronal circuits that have been implicated in clinical research. Additional paradigms are needed to capture other features of AN that are not seen in ABA., (© 2022. The Author(s).)

The position statement is issued by The Obesity Society in response to published literature, as well as inquiries made to the Society by patients, providers, Society members, policy makers, and others regarding the efficacy of vaccines in persons with obesity against SARS-CoV-2, the virus that causes COVID-19. The Obesity Society has critically evaluated data from published peer-reviewed literature and briefing documents from Emergency Use Authorization applications submitted by Pfizer-BioNTech, Moderna, and Johnson & Johnson. We conclude that these vaccines are highly efficacious, and their efficacy is not significantly different in people with and without obesity, based on scientific evidence available at the time of publication. The Obesity Society believes there is no definitive way to determine which of these three COVID-19 vaccines is "best" for any weight subpopulation (because of differences in the trial design and outcome measures in the phase 3 trials, elapsed time between doses, and regional differences in the presence of SARS-CoV-2 variants [e.g., South Africa B.1.351 in Johnson & Johnson trial]). All three trials have demonstrated high efficacy against COVID-19-associated hospitalization and death. Therefore, The Obesity Society encourages adults with obesity ≥18 years (≥16 years for Pfizer-BioNTech) to undergo vaccination with any one of the currently available vaccines authorized for emergency use by the US Food and Drug Administration as soon as they are able., (© 2021 The Obesity Society.)

Hypothalamic AgRP and POMC neurons are conventionally viewed as the yin and yang of the body's energy status, since they act in an opposite manner to modulate appetite and systemic energy metabolism. However, although AgRP neurons' functions are comparatively well understood, a unifying theory of how POMC neuronal cells operate has remained elusive, probably due to their high level of heterogeneity, which suggests that their physiological roles might be more complex than initially thought. In this Perspective, we propose a conceptual framework that integrates POMC neuronal heterogeneity with appetite regulation, whole-body metabolic physiology and the development of obesity. We highlight emerging evidence indicating that POMC neurons respond to distinct combinations of interoceptive signals and food-related cues to fine-tune divergent metabolic pathways and behaviours necessary for survival. The new framework we propose reflects the high degree of developmental plasticity of this neuronal population and may enable progress towards understanding of both the aetiology and treatment of metabolic disorders.

Although long-studied in the central nervous system, there is increasing evidence that dopamine (DA) has important roles in the periphery including in metabolic regulation. Insulin-secreting pancreatic β-cells express the machinery for DA synthesis and catabolism, as well as all five DA receptors. In these cells, DA functions as a negative regulator of glucose-stimulated insulin secretion (GSIS), which is mediated by DA D 2 -like receptors including D 2 (D2R) and D 3 (D3R) receptors. However, the fundamental mechanisms of DA synthesis, storage, release, and signaling in pancreatic β-cells and their functional relevance in vivo remain poorly understood. Here, we assessed the roles of the DA precursor L-DOPA in β-cell DA synthesis and release in conjunction with the signaling mechanisms underlying DA's inhibition of GSIS. Our results show that the uptake of L-DOPA is essential for establishing intracellular DA stores in β-cells. Glucose stimulation significantly enhances L-DOPA uptake, leading to increased DA release and GSIS reduction in an autocrine/paracrine manner. Furthermore, D2R and D3R act in combination to mediate dopaminergic inhibition of GSIS. Transgenic knockout mice in which β-cell D2R or D3R expression is eliminated exhibit diminished DA secretion during glucose stimulation, suggesting a new mechanism where D 2 -like receptors modify DA release to modulate GSIS. Lastly, β-cell-selective D2R knockout mice exhibit marked postprandial hyperinsulinemia in vivo. These results reveal that peripheral D2R and D3R receptors play important roles in metabolism through their inhibitory effects on GSIS. This opens the possibility that blockade of peripheral D 2 -like receptors by drugs including antipsychotic medications may significantly contribute to the metabolic disturbances observed clinically.

Maternal nutritional status and the early growth rates of offspring influence the development of the melanocortin system and later susceptibility to metabolic dysregulation, but it is difficult to assess causality. A recent study by van der Klaauw et al. (Cell 2019;176:729-742) provides direct evidence that disrupting systems regulating neuronal circuit formation leads to early-onset obesity in zebrafish, mouse, and humans., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Biosynthesis of insulin - critical to metabolic homeostasis - begins with folding of the proinsulin precursor, including formation of three evolutionarily conserved intramolecular disulfide bonds. Remarkably, normal pancreatic islets contain a subset of proinsulin molecules bearing at least one free cysteine thiol. In human (or rodent) islets with a perturbed endoplasmic reticulum folding environment, non-native proinsulin enters intermolecular disulfide-linked complexes. In genetically obese mice with otherwise wild-type islets, disulfide-linked complexes of proinsulin are more abundant, and leptin receptor-deficient mice, the further increase of such complexes tracks with the onset of islet insulin deficiency and diabetes. Proinsulin-Cys(B19) and Cys(A20) are necessary and sufficient for the formation of proinsulin disulfide-linked complexes; indeed, proinsulin Cys(B19)-Cys(B19) covalent homodimers resist reductive dissociation, highlighting a structural basis for aberrant proinsulin complex formation. We conclude that increased proinsulin misfolding via disulfide-linked complexes is an early event associated with prediabetes that worsens with ß-cell dysfunction in type two diabetes., Competing Interests: AA, LH, AP, FP, SK, LZ, AP, JP, BT, PI, RK, ML, PA No competing interests declared, (© 2019, Arunagiri et al.)

In leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC) 1 , a critical brain area for energy and glucose homeostasis 2,3 . As this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period (CP) for leptin-dependent development of ARC neurocircuits has been proposed 4 . In other brain areas, CP closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh 5 . Here we report that in humans as well as rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed by PNN-like structures. In mice, these neurons are densely-packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both GABAergic and leptin receptor-positive, including a majority of Agrp neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the CP for Agrp neuron maturation and is dependent on input from circulating leptin, as postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the CP. We conclude that neurons crucial to metabolic homeostasis are enmeshed by PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC-ME junction, where metabolically-relevant humoral signals are sensed., Competing Interests: Competing Interests Statement The authors declare no competing financial or non-financial interests in relation to the work described here.

Pro-opiomelanocortin (POMC)-expressing neurons regulate energy balance and mediate the effects of some classes of anti-obesity therapeutics. In this issue of Cell Metabolism, D'Agostino et al. (2018) demonstrate that a small and often overlooked population of POMC neurons in the brainstem contributes to satiation induced by the FDA-approved drug lorcaserin., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A wide range of maternal exposures - undernutrition, obesity, diabetes, stress and infection - are associated with an increased risk of metabolic disease in offspring. Developmental influences can cause persistent structural changes in hypothalamic circuits regulating food intake in the service of energy balance. The physiological relevance of these alterations has been called into question because maternal impacts on daily caloric intake do not persist to adulthood. Recent behavioural and epidemiological studies in humans provide evidence that the relative contribution of appetitive traits related to satiety, reward and the emotional aspects of food intake regulation changes across the lifespan. This Opinion article outlines a neurodevelopmental framework to explore the possibility that crosstalk between developing circuits regulating different modalities of food intake shapes future behavioural responses to environmental challenges.

Obesity is among the most common and costly chronic disorders worldwide. Estimates suggest that in the United States obesity affects one-third of adults, accounts for up to one-third of total mortality, is concentrated among lower income groups, and increasingly affects children as well as adults. A lack of effective options for long-term weight reduction magnifies the enormity of this problem; individuals who successfully complete behavioral and dietary weight-loss programs eventually regain most of the lost weight. We included evidence from basic science, clinical, and epidemiological literature to assess current knowledge regarding mechanisms underlying excess body-fat accumulation, the biological defense of excess fat mass, and the tendency for lost weight to be regained. A major area of emphasis is the science of energy homeostasis, the biological process that maintains weight stability by actively matching energy intake to energy expenditure over time. Growing evidence suggests that obesity is a disorder of the energy homeostasis system, rather than simply arising from the passive accumulation of excess weight. We need to elucidate the mechanisms underlying this "upward setting" or "resetting" of the defended level of body-fat mass, whether inherited or acquired. The ongoing study of how genetic, developmental, and environmental forces affect the energy homeostasis system will help us better understand these mechanisms and are therefore a major focus of this statement. The scientific goal is to elucidate obesity pathogenesis so as to better inform treatment, public policy, advocacy, and awareness of obesity in ways that ultimately diminish its public health and economic consequences., (Copyright © 2017 Endocrine Society.)

This corrects the article DOI: 10.1038/nature21697.

Bone has recently emerged as a pleiotropic endocrine organ that secretes at least two hormones, FGF23 and osteocalcin, which regulate kidney function and glucose homeostasis, respectively. These findings have raised the question of whether other bone-derived hormones exist and what their potential functions are. Here we identify, through molecular and genetic analyses in mice, lipocalin 2 (LCN2) as an osteoblast-enriched, secreted protein. Loss- and gain-of-function experiments in mice demonstrate that osteoblast-derived LCN2 maintains glucose homeostasis by inducing insulin secretion and improves glucose tolerance and insulin sensitivity. In addition, osteoblast-derived LCN2 inhibits food intake. LCN2 crosses the blood-brain barrier, binds to the melanocortin 4 receptor (MC4R) in the paraventricular and ventromedial neurons of the hypothalamus and activates an MC4R-dependent anorexigenic (appetite-suppressing) pathway. These results identify LCN2 as a bone-derived hormone with metabolic regulatory effects, which suppresses appetite in a MC4R-dependent manner, and show that the control of appetite is an endocrine function of bone.

Diet-induced obesity (DIO) resulting from consumption of a high fat diet (HFD) attenuates normal neuronal responses to leptin and may contribute to the metabolic defense of an acquired higher body weight in humans; the molecular bases for the persistence of this defense are unknown. We measured the responses of 23 brain regions to exogenous leptin in 4 different groups of weight- and/or diet-perturbed mice. Responses to leptin were assessed by quantifying pSTAT3 levels in brain nuclei 30 minutes following 3 mg/kg intraperitoneal leptin. HFD attenuated leptin sensing throughout the brain, but weight loss did not restore central leptin signaling to control levels in several brain regions important in energy homeostasis, including the arcuate and dorsomedial hypothalamic nuclei. Effects of diet on leptin signaling varied by brain region, with results dependent on the method of weight loss (restriction of calories of HFD, ad lib intake of standard mouse chow). High fat diet attenuates leptin signaling throughout the brain, but some brain regions maintain their ability to sense leptin. Weight loss restores leptin sensing to some degree in most (but not all) brain regions, while other brain regions display hypersensitivity to leptin following weight loss. Normal leptin sensing was restored in several brain regions, with the pattern of restoration dependent on the method of weight loss., Competing Interests: The role of AstraZeneca in the funding and conduct of this study is described in Role of Funders. This participation of AstraZeneca scientists in no way alters our adherence to PLOS ONE policies on sharing data and materials. There are no other relevant competing interests with regard to any of the authors.

There is an urgent need to identify therapeutic targets for anorexia nervosa (AN) because current medications do not impact eating behaviors that drive AN's high mortality rate. A major obstacle to developing new treatments is the lack of animal models that recapitulate the pattern of disease onset typically observed in human populations. Here we describe a translational mouse model to study interactions between genetic, psychological and biological risk factors that promote anorexic behavior. We combined several factors that are consistently associated with increased risk of AN-adolescent females, genetic predisposition to anxiety imposed by the BDNF-Val66Met gene variant, social isolation stress and caloric restriction (CR). Approximately 40% of the mice with all of these risk factors will exhibit severe self-imposed dietary restriction, sometimes to the point of death. We systematically varied the risk factors outlined above to explore how they interact to influence anorexic behavior. We found that the Val66Met genotype markedly increases the likelihood and severity of abnormal feeding behavior triggered by CR, but only when CR is imposed in the peri-pubertal period. Incidence of anorexic behavior in our model is dependent on juvenile exposure to social stress and can be extinguished by adolescent handling, but is discordant from anxiety-like behavior. Thus, this study characterized gene × environment interactions during adolescence that could be the underlying driver of abnormal eating behavior in certain AN patients, and represents a promising system to identify possible targets for therapeutic intervention.

Although most adults can lose weight by dieting, a well-characterized compensatory decrease in energy expenditure promotes weight regain more than 90% of the time. Using mice with impaired hypothalamic leptin signaling as a model of early-onset hyperphagia and obesity, we explored whether this unfavorable response to weight loss could be circumvented by early intervention. Early-onset obesity was associated with impairments in the structure and function of brown adipose tissue mitochondria, which were ameliorated by weight loss at any age. Although decreased sympathetic tone in weight-reduced adults resulted in net reductions in brown adipose tissue thermogenesis and energy expenditure that promoted rapid weight regain, this was not the case when dietary interventions were initiated at weaning. Enhanced energy expenditure persisted even after mice were allowed to resume overeating, leading to lasting reductions in adiposity. These findings reveal a time window when dietary interventions can produce metabolic improvements that are stably maintained.

Objective: Humans and animals exposed to undernutrition (UN) during development often experience accelerated "catch-up" growth when food supplies are plentiful. Little is known about the mechanisms regulating early growth rates. We previously reported that actions of leptin and presynaptic inputs to orexigenic NPY/AgRP/GABA (NAG) neurons in the arcuate nucleus of the hypothalamus are almost exclusively excitatory during the lactation period, since neuronal and humoral inhibitory systems do not develop until after weaning. Moreover, we identified a critical step that regulates the maturation of electrophysiological responses of NAG neurons at weaning - the onset of genes encoding ATP-dependent potassium (KATP) channel subunits. We explored the possibility that UN promotes subsequent catch-up growth, in part, by delaying the maturation of negative feedback systems to neuronal circuits driving food intake., Methods: We used the large litter (LL) size model to study the impacts of postnatal UN followed by catch-up growth. We evaluated the maturation of presynaptic and postsynaptic inhibitory systems in NAG neurons using a combination of electrophysiological and molecular criteria, in conjunction with leptin's ability to suppress fasting-induced hyperphagia., Results: The onset of KATP channel subunit expression and function, the switch in leptin's effect on NAG neurons, the ingrowth of inhibitory inputs to NAG neurons, and the development of homeostatic feedback to feeding circuits were delayed in LL offspring relative to controls. The development of functional KATP channels and the establishment of leptin-mediated suppression of food intake in the peri-weaning period were tightly linked and were not initiated until growth and adiposity of LL offspring caught up to controls., Conclusions: Our data support the idea that initiation of KATP channel subunit expression in NAG neurons serves as a molecular gatekeeper for the maturation of homeostatic feeding circuits.

Suboptimal maternal nutrition exerts lasting impacts on obesity risk in offspring, but the direction of the effect is determined by the timing of exposure. While maternal undernutrition in early pregnancy is associated with increased body mass index, in later pregnancy it can be protective. The importance of the timing of maternal undernutrition is also observed in rodents, however, many of the processes that occur in the last trimester of human gestation are delayed to the postnatal period. Neonatal leptin administration exerts lasting impacts on susceptibility to obesity in rodents. Although leptin can influence the formation of hypothalamic circuits involved in homeostatic control of feeding during the postnatal period, these effects are too late to account for its ability to reverse adverse metabolic programming due to early gestational exposure to maternal undernutrition. This review presents an alternative framework for understanding the effects of neonatal leptin through influences on developing thermoregulatory circuits., (Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.)

The hypothalamus is the central regulator of systemic energy homeostasis, and its dysfunction can result in extreme body weight alterations. Insights into the complex cellular physiology of this region are critical to the understanding of obesity pathogenesis; however, human hypothalamic cells are largely inaccessible for direct study. Here, we developed a protocol for efficient generation of hypothalamic neurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenetic forms of obesity. Combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition in human ESCs/iPSCs resulted in efficient conversion into hypothalamic NKX2.1+ precursors. Application of a NOTCH inhibitor and brain-derived neurotrophic factor (BDNF) further directed the cells into arcuate nucleus hypothalamic-like neurons that express hypothalamic neuron markers proopiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AGRP), somatostatin, and dopamine. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles defined by a hypothalamic-specific gene expression signature that lacked pituitary markers. Importantly, these cells displayed hypothalamic neuron characteristics, including production and secretion of neuropeptides and increased p-AKT and p-STAT3 in response to insulin and leptin. Our results suggest that these hypothalamic-like neurons have potential for further investigation of the neurophysiology of body weight regulation and evaluation of therapeutic targets for obesity.

Defense of core body temperature (Tc) can be energetically costly; thus, it is critical that thermoregulatory circuits are modulated by signals of energy availability. Hypothalamic leptin and insulin signals relay information about energy status and are reported to promote thermogenesis, raising the possibility that they interact to direct an appropriate response to nutritional and thermal challenges. To test this idea, we used an Nkx2.1-Cre driver to generate conditional knockouts (KOs) in mice of leptin receptor (L(2.1)KO), insulin receptor (I(2.1)KO), and double KOs of both receptors (D(2.1)KO). L(2.1)KOs are hyperphagic and obese, whereas I(2.1)KOs are similar to controls. D(2.1)KOs exhibit higher body weight and adiposity than L(2.1)KOs, solely due to reduced energy expenditure. At 20-22°C, fed L(2.1)KOs maintain a lower baseline Tc than controls, which is further decreased in D(2.1)KOs. After an overnight fast, some L(2.1)KOs dramatically suppress energy expenditure and enter a torpor-like state; this behavior is markedly enhanced in D(2.1)KOs. When fasted mice are exposed to 4°C, L(2.1)KOs and D(2.1)KOs both mount a robust thermogenic response and rapidly increase Tc. These observations support the idea that neuronal populations that integrate information about energy stores to regulate the defense of Tc set points are distinct from those required to respond to a cold challenge., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Objective: Obesity is often accompanied by hyperactivity of the neuroendocrine stress axis and has been linked to an increased risk of psychiatric disorders. Insulin is reciprocally regulated with the stress hormone corticosterone (CORT), raising the possibility that insulin normally provides inhibitory tone to the hypothalamus-adrenal-pituitary (HPA) axis. Here we examined whether disrupting signaling via the insulin receptor (InsR) in hypothalamic subpopulations impacts the neuroendocrine response to acute psychological stress., Methods: We used Nkx2.1-Cre, Sim1-Cre and Agrp-Cre transgenic driver lines to generate conditional knockouts of InsR signaling throughout the hypothalamus, paraventricular nucleus of the hypothalamus (PVH) and in neurons expressing Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARH), respectively. We used a combination of molecular, behavioral and neuroendocrine criteria to evaluate the consequences on HPA axis responsiveness., Results: Endpoints related to body weight and glucose homeostasis were not altered in any of the conditional mutant lines. Consistent with observations in the neuronal Insr knockout mice (NIRKO), baseline levels of serum CORT were similar to controls in all three lines. In male mice with broad disruptions of InsR signals in Nkx2.1-expressing regions of the hypothalamus (IR(Nkx2.1) KO), we observed elevated arginine vasopressin (AVP) levels at baseline and heightened neuroendocrine responses to restraint stress. IR(Nkx2.1) KO males also exhibited increased anxiety-like behaviors in open field, marble burying, and stress-induced hyperthermia testing paradigms. HPA axis responsivity was not altered in IR(Sim1) KO males, in which InsR was disrupted in the PVH. In contrast to observations in the IR(Nkx2.1) KO males, disrupting InsR signals in ARH neurons expressing Agrp (IR(Agrp) KO) led to reduced AVP release in the median eminence (ME)., Conclusions: We find that central InsR signals modulate HPA responsivity to restraint stress. InsR signaling in AgRP/NPY neurons appears to promote AVP release, while signaling in other hypothalamic neuron(s) likely acts in an opposing fashion. Alterations in InsR signals in neurons that integrate metabolic and psychiatric information could contribute to the high co-morbidity of obesity and mental disorders.

Leptin is well known for its role in the regulation of energy homeostasis in adults, a mechanism that at least partially results from the inhibition of the activity of NPY/AgRP/GABA neurons (NAG) in the arcuate nucleus of the hypothalamus (ARH). During early postnatal development in the rodent, leptin promotes axonal outgrowth from ARH neurons, and preautonomic NAG neurons are particularly responsive to leptin's trophic effects. To begin to understand how leptin could simultaneously promote axonal outgrowth from and inhibit the activity of NAG neurons, we characterized the electrochemical effects of leptin on NAG neurons in mice during early development. Here, we show that NAG neurons do indeed express a functional leptin receptor throughout the early postnatal period in the mouse; however, at postnatal days 13-15, leptin causes membrane depolarization in NAG neurons, rather than the expected hyperpolarization. Leptin action on NAG neurons transitions from stimulatory to inhibitory in the periweaning period, in parallel with the acquisition of functional ATP-sensitive potassium channels. These findings are consistent with the idea that leptin provides an orexigenic drive through the NAG system to help rapidly growing pups meet their energy requirements., (Copyright © 2014 the authors 0270-6474/14/349982-13$15.00/0.)

Objective: The physiology of the weight-reduced (WR) state suggests that pharmacologic agents affecting energy homeostasis may have greater efficacy in WR individuals. Our aim was to establish a protocol that allows for evaluation of efficacy of weight maintenance agents and to assess the effectiveness of AZD2820, a novel melanocortin 4 receptor (MC4R) agonist in such a paradigm., Methods: MC4R agonist was administered in stratified doses to mice who were either fed high-fat diet ad libitum (AL) throughout the study; or stabilized at a 20% reduced body weight (BW), administered the drug for 4 weeks, and thereafter released from caloric restriction while continuing to receive the drug (WR)., Results: After release of WR mice to AL feeding, the high-dose group (53.4 nmol/day) regained 12.4% less BW than their vehicle-treated controls since the beginning of drug treatment. In WR mice, 10.8 nmol/day of the agonist was sufficient to maintain these animals at 95.1% of initial BW versus 53.4 nmol/day required to maintain the BW of AL animals (94.5%)., Conclusions: In the WR state, the MC4R agonist was comparably efficacious to a five-fold higher dose in the AL state. This protocol provides a model for evaluating the mechanisms and quantitative efficacy of weight-maintenance strategies and agents., (Copyright © 2013 The Obesity Society.)

Energy homeostasis--ensuring that energy availability matches energy requirements--is essential for survival. One way that energy balance is achieved is through coordinated action of neural and neuroendocrine feeding circuits, which promote energy intake when energy supply is limited. Feeding behavior engages multiple somatic and visceral tissues distributed throughout the body--contraction of skeletal and smooth muscles in the head and along the upper digestive tract required to consume and digest food, as well as stimulation of endocrine and exocrine secretions from a wide range of organs. Accordingly, neurons that contribute to feeding behaviors are localized to central, peripheral, and enteric nervous systems. To promote energy balance, feeding circuits must be able to identify and respond to energy requirements, as well as the amount of energy available from internal and external sources, and then direct appropriate coordinated responses throughout the body.

Maintaining energy balance is of paramount importance for metabolic health and survival. It is achieved through the coordinated regulation of neuronal circuits that control a wide range of physiological processes affecting energy intake and expenditure, such as feeding, metabolic rate, locomotor activity, arousal, growth and reproduction. Neuronal populations distributed throughout the CNS but highly enriched in the mediobasal hypothalamus, sense hormonal, nutrient and neuronal signals of systemic energy status and relay this information to secondary neurons that integrate the information and regulate distinct physiological parameters in a manner that promotes energy homeostasis. To achieve this, it is critical that neuronal circuits provide information about short-term changes in nutrient availability in the larger context of long-term energy status. For example, the same signals lead to different cellular and physiological responses if delivered under fasted versus fed conditions. Thus, there is a clear need to have mechanisms that rapidly and reversibly adjust responsiveness of hypothalamic circuits to acute changes in nutrient availability.

Melanocortin signaling plays a central role in the regulation of phenotypes related to body weight and energy homeostasis. To specifically target and study the function of proopiomelanocortin (POMC) neurons, Pomc promoter elements have been utilized to generate reporter and Cre recombinase transgenic reagents. Across gestation, we find that Pomc is dynamically expressed in many sites in the developing mouse forebrain, midbrain, hindbrain, spinal cord, and retina. Although Pomc expression in most embryonic brain regions is transient, it is sufficient to direct Cre-mediated recombination of floxed alleles. We visualize the populations affected by this transgene by crossing Pomc-Cre mice to ROSA reporter strains and identify 62 sites of recombination throughout the adult brain, including several nuclei implicated in energy homeostasis regulation. To compare the relationship between acute Pomc promoter activity and Pomc-Cre-mediated recombination at the single cell level, we crossed Pomc-enhanced green fluorescent protein (eGFP) and Pomc-Cre;ROSA-tdTomato lines. We detect the highest concentration of Pomc-eGFP+ cells in the arcuate nucleus of the hypothalamus and dentate gyrus but also observe smaller populations of labeled cells in the nucleus of the solitary tract, periventricular zone of the third ventricle, and cerebellum. Consistent with the dynamic nature of Pomc expression in the embryo, the vast majority of neurons marked with the tdTomato reporter do not express eGFP in the adult. Thus, recombination in off-target sites could contribute to physiological phenotypes using Pomc-Cre transgenics. For example, we find that approximately 83% of the cells in the arcuate nucleus of the hypothalamus immunoreactive for leptin-induced phosphorylated signal transducer and activator of transcription 3 are marked with Pomc-Cre;ROSA-tdTomato; only 13% of these are eGFP+ POMC neurons.

Maternal obesity can influence susceptibility to obesity and type 2 diabetes in progeny. We examined the relationship of maternal insulin resistance (IR), a metabolically important consequence of increased adiposity, to adverse consequences of obesity for fetal development. We used mice heterozygous for a null allele of the insulin receptor (Insr) to study the contributions of maternal IR to offspring phenotype without the potential confound of obesity per se, and how maternal consumption of high-fat diet (HFD) may, independently and interactively, affect progeny. In progeny fed a 60% HFD, body weight and adiposity were transiently (5-7 weeks) increased in wild-type (+/+) offspring of Insr(+/-) HFD-fed dams compared to offspring of wild-type HFD-fed dams. Offspring of HFD-fed wild-type dams had increased body weight, blood glucose, and plasma insulin concentrations compared to offspring of chow-fed wild-type dams. Quantification of proopiomelanocortin (POMC) and neuropeptide-Y (NPY) populations in the arcuate nucleus of the hypothalamus (ARH) of offspring of wild-type vs. Insr(+/-) dams was performed to determine whether maternal IR affects the formation of central feeding circuits. We found a 20% increase in the number of Pomc-expressing cells at postnatal day 9 in offspring of Insr(+/-) dams. In conclusion, maternal HFD consumption-distinct from overt obesity per se-was a major contributor to increased body weight, adiposity, IR, and liver triglyceride (TG) phenotypes in progeny. Maternal IR played a minor role in predisposing progeny to obesity and IR, though it acted synergistically with maternal HFD to exacerbate early obesity in progeny.

The Dorsomedial Nucleus of the Hypothalamus (DMH) is known to play important roles in ingestive behavior and body weight homeostasis. The DMH contains neurons expressing Neuropeptide Y (NPY) during specific physiological conditions of hyperphagia and obesity, however, the role of DMH-NPY neurons has yet to be characterized. In contrast to the DMH-NPY neurons, NPY expressing neurons have been best characterized in the Arcuate Nucleus of the Hypothalamus (ARH). The purpose of this study is to characterize the chemical phenotype of DMH-NPY neurons by comparing the gene expression profiles of NPY neurons in the DMH and ARH isolated from postnatal NPY-hrGFP mice by microarray analysis. Twenty genes were differentially expressed in the DMH-NPY neurons compared to the ARH. Among them, there were several transcriptional factors that play important roles in the regulation of energy balance. DMH-NPY neurons expressed Glutamic Acid Decarboxylase (GAD) 65 and 67, suggesting that they may be GABAergic, similar to ARH-NPY neurons. While ARH-NPY neurons expressed leptin receptor (ObRb) and displayed the activation of STAT3 in response to leptin administration, DMH-NPY neurons showed neither. These findings strongly suggest that DMH-NPY neurons could play a distinct role in the control of energy homeostasis and are differentially regulated from ARH-NPY neurons through afferent inputs and transcriptional regulators., (2010 Elsevier B.V. All rights reserved.)

Distinct populations of leptin-sensing neurons in the hypothalamus, midbrain, and brainstem contribute to the regulation of energy homeostasis. To assess the requirement for leptin signaling in the hypothalamus, we crossed mice with a floxed leptin receptor allele (Leprfl) to mice transgenic for Nkx2.1-Cre, which drives Cre expression in the hypothalamus and not in more caudal brain regions, generating LeprNkx2.1KO mice. From weaning, LeprNkx2.1KO mice exhibited phenotypes similar to those observed in mice with global loss of leptin signaling (Leprdb/db mice), including increased weight gain and adiposity, hyperphagia, cold intolerance, and insulin resistance. However, after 8 weeks of age, LeprNkx2.1KO mice maintained stable adiposity levels, whereas the body fat percentage of Leprdb/db animals continued to escalate. The divergence in the adiposity phenotypes of Leprdb/db and LeprNkx2.1KO mice with age was concomitant with increased rates of linear growth and energy expenditure in LeprNkx2.1KO mice. These data suggest that remaining leptin signals in LeprNkx2.1KO mice mediate physiological adaptations that prevent the escalation of the adiposity phenotype in adult mice. The persistence of severe adiposity in LeprNkx2.1KO mice, however, suggests that compensatory actions of circuits regulating growth and energy expenditure are not sufficient to reverse obesity established at an early age.

Hypothalamic neuron circuits regulating energy balance are highly plastic and develop in response to nutrient and hormonal cues. To identify processes that might be susceptible to gestational influences in mice, we characterized the ontogeny of proopiomelanocortin (POMC) and neuropeptide Y (NPY) cell populations, which exert opposing influences on food intake and body weight. These analyses revealed that Pomc is broadly expressed in immature hypothalamic neurons and that half of embryonic Pomc-expressing precursors subsequently adopt a non-POMC fate in adult mice. Moreover, nearly one quarter of the mature NPY+ cell population shares a common progenitor with POMC+ cells.

Two recent, large whole-genome association studies (GWAS) in European populations have associated a approximately 47-kb region that contains part of the FTO gene with high body mass index (BMI). The functions of FTO and adjacent FTM in human biology are not clear. We examined expression of these genes in organs of mice segregating for monogenic obesity mutations, exposed to underfeeding/overfeeding, and to 4 degrees C. Fto/Ftm expression was reduced in mesenteric adipose tissue of mice segregating for the Ay, Lep ob, Lepr db, Cpe fat, or tub mutations, and there was a similar trend in other tissues. These effects were not due to adiposity per se. Hypothalamic Fto and Ftm expression were decreased by fasting in lean and obese animals and by cold exposure in lean mice. The fact that responses of Fto and Ftm expression to these manipulations were almost indistinguishable suggested that the genes might be coregulated. The putative overlapping regulatory region contains at least two canonical CUTL1 binding sites. One of these nominal CUTL1 sites includes rs8050136, a SNP associated with high body mass. The A allele of rs8050136 preferentially bound CUTL1[corrected] in human fibroblast DNA. 70% knockdown of CUTL1 expression in human fibroblasts decreased FTO and FTM expression by 90 and 65%, respectively. Animals and humans with various genetic interruptions of FTO or FTM have phenotypes reminiscent of aspects of the Bardet-Biedl obesity syndrome, a confirmed "ciliopathy." FTM has recently been shown to be a ciliary basal body protein.

The zona limitans intrathalamica (ZLI) is located at the border between the prospective ventral thalamus and dorsal thalamus, and functions as a diencephalic signaling center. Little is known about the mechanism controlling ZLI formation. Using a combination of fate-mapping studies and in vitro assays, I show that the differentiation of the ZLI from progenitor cells in the alar plate is initiated by a Shh-dependent signal from the basal plate. The subsequent dorsal progression of ZLI differentiation requires ongoing Shh signaling, and is constrained by inhibitory factors derived from the dorsal diencephalon. These studies demonstrate that self-organizing signals from the basal plate regulate the formation of a potential patterning center in the ZLI in an orthogonal orientation in the alar plate, and thus create the potential for coordinated thalamic patterning in two dimensions.

The diencephalon comprises three functionally distinct regions: synencephalon, dorsal thalamus, and ventral thalamus. Patterning of the diencephalon has been proposed to involve subdivision of its anteroposterior axis into segments, neuromeres or prosomeres (Bergquist and Kallen, 1954; Vaage, 1969; Figdor and Stern, 1993; Rubenstein et al., 1994; Redies et al., 2000; Yoon et al., 2000). However, the number and sequence of diencephalic neuromeres, or even their existence, are uncertain. We have examined the proposed subdivisions by morphology, gene expression, acquisition of boundary-specific phenotypes, and cell lineage restriction. We find that at stage 16 in chick the diencephalon is divided into synencephalon and parencephalon. The synencephalon exhibits neuromeric morphology, expresses Prox, and acquires neuromere boundary properties at its interface with both the midbrain and the parencephalon. Although the mesencephalic/synencephalic boundary restricts cell mixing, the synencephalic/parencephalic boundary does not. Similarly, there is no lineage restriction between the parencephalon and the more rostral forebrain (secondary prosencephalon). Subdivision of the parencephalon into ventral and dorsal thalamus involves the formation of a narrow intraparencephalic territory, the zona limitans intrathalamica (zli). This is correlated with the acquisition of cell lineage restriction at both anterior and posterior borders of the zli, the appearance of boundary-specific properties, and Gbx2 and Dlx2 expression in dorsal thalamic and ventral thalamic territories, respectively. At stage 22, the synencephalon is divided into two domains, distinguished by differential gene expression and tissue morphology, but associated with neither a boundary phenotype nor cell lineage restriction. Our results suggest that the diencephalon does not have an overt segmental pattern.