Distinct Hypothalamic and Brainstem Contributions to Lorcaserin Action.

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.

MC4R-dependent suppression of appetite by bone-derived lipocalin 2.

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.

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.

Synaptic plasticity in neuronal circuits regulating energy balance.

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.

Defining POMC neurons using transgenic reagents: impact of transient Pomc expression in diverse immature neuronal populations.

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.

Respective contributions of maternal insulin resistance and diet to metabolic and hypothalamic phenotypes of progeny.

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.

Disruption of hypothalamic leptin signaling in mice leads to early-onset obesity, but physiological adaptations in mature animals stabilize adiposity levels.

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.

Pomc-expressing progenitors give rise to antagonistic neuronal populations in hypothalamic feeding circuits.

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.

Regulation of Fto/Ftm gene expression in mice and humans.

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.