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1.  Disruption of hypothalamic leptin signaling in mice leads to early-onset obesity, but physiological adaptations in mature animals stabilize adiposity levels 
The Journal of Clinical Investigation  2010;120(8):2931-2941.
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.
PMCID: PMC2912188  PMID: 20592471
2.  Differentiation of hypothalamic-like neurons from human pluripotent stem cells 
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.
PMCID: PMC4319421  PMID: 25555215
3.  Developmental Switch of Leptin Signaling in Arcuate Nucleus Neurons 
The Journal of Neuroscience  2014;34(30):9982-9994.
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.
PMCID: PMC4107412  PMID: 25057200
arcuate nucleus; development; KATP channels; leptin; mouse; NPY
4.  Central insulin signaling modulates hypothalamus–pituitary–adrenal axis responsiveness 
Molecular Metabolism  2014;4(2):83-92.
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.
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.
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 (IRNkx2.1 KO), we observed elevated arginine vasopressin (AVP) levels at baseline and heightened neuroendocrine responses to restraint stress. IRNkx2.1 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 IRSim1 KO males, in which InsR was disrupted in the PVH. In contrast to observations in the IRNkx2.1 KO males, disrupting InsR signals in ARH neurons expressing Agrp (IRAgrp KO) led to reduced AVP release in the median eminence (ME).
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.
PMCID: PMC4314547  PMID: 25685696
Insulin; Hypothalamus; AgRP; HPA axis; Stress response; ACTH, adrenocorticotropic hormone; AgRP, agouti-related peptide; ARH, arcuate nucleus of the hypothalamus; AVP, arginine vasopressin; CORT, corticosterone; CRH, corticotropin-releasing hormone; FST, forced swim test; Gr, Glucocorticoid receptor; HPA axis, Hypothalamus–Pituitary–Adrenal axis; InsR, insulin receptor; IRAgrp KO, knockout of InsR using Agrp-Cre; IRNkx2.1 KO, knockout of InsR using Nkx2.1-Cre; IRSim1 KO, knockout of InsR using Sim1-Cre; MB, marble burying test; MBH, mediobasal hypothalamus; ME, median eminence; NPY, neuropeptide Y; NSF, novelty suppressed feeding test; OF, open field test; POMC, pro-opiomelanocortin; SIH, stress-induced hyperthermia test
5.  Effects of a novel MC4R agonist on maintenance of reduced body weight in diet induced obese mice 
Obesity (Silver Spring, Md.)  2014;22(5):1287-1295.
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 agonist in such a paradigm.
Design and 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 four weeks, and thereafter released from caloric restriction while continuing to receive the drug (WR).
After release of WR mice to AL feeding, the high-dose group (53.4nmol/day) regained 12.4% less BW than their vehicle-treated controls. In WR mice, 10.8nmol/day of the agonist was sufficient to maintain these animals at 95.1% of initial BW vs 53.4nmol/day required to maintain the BW of AL animals (94.5%).
In the WR state, the MC4R agonist was comparably efficacious to a 5-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.
PMCID: PMC4008720  PMID: 24318934
6.  Functional Organization of Neuronal and Humoral Signals Regulating Feeding Behavior 
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.
PMCID: PMC3991304  PMID: 23642202
food intake; CNS circuits; energy balance; obesity
7.  Respective Contributions of Maternal Insulin Resistance and Diet to Metabolic and Hypothalamic Phenotypes of Progeny 
Obesity (Silver Spring, Md.)  2010;19(3):492-499.
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.
PMCID: PMC3234171  PMID: 20948526
8.  Differential Gene Expression Between Neuropeptide Y Expressing Neurons of the Dorsomedial Nucleus of the Hypothalamus and the Arcuate Nucleus: Microarray Analysis Study 
Brain research  2010;1350:139-150.
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.
PMCID: PMC2917610  PMID: 20380814
10.  Regulation of Fto/Ftm gene expression in mice and humans 
Two recent, large GWAS in European populations have associated a ∼47 Kb region that contains part of the FTO gene with high 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 under/over feeding, and to 4 °C. Fto/Ftm expression was reduced in mesenteric adipose tissue of mice segregating for the Ay, Lepob, Leprdb, Cpefat 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 co-regulated. The putative overlapping regulatory region contains at least 2 canonical CUTL1 binding sites. One of these nominal CUTL1 sites includes rs8050136, a SNP associated with high body mass. The A allele of rs8050136 – associated with lower body mass than the C allele – preferentially bound CUTL1 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.
PMCID: PMC2808712  PMID: 18256137
obesity; hypothalamus; adipose tissue; CUTL1

Results 1-10 (10)