Loss of transient receptor potential channel 5 causes obesity and postpartum depression.

Hypothalamic neural circuits regulate instinctive behaviors such as food seeking, the fight/flight response, socialization, and maternal care. Here, we identified microdeletions on chromosome Xq23 disrupting the brain-expressed transient receptor potential (TRP) channel 5 (TRPC5). This family of channels detects sensory stimuli and converts them into electrical signals interpretable by the brain. Male TRPC5 deletion carriers exhibited food seeking, obesity, anxiety, and autism, which were recapitulated in knockin male mice harboring a human loss-of-function TRPC5 mutation. Women carrying TRPC5 deletions had severe postpartum depression. As mothers, female knockin mice exhibited anhedonia and depression-like behavior with impaired care of offspring. Deletion of Trpc5 from oxytocin neurons in the hypothalamic paraventricular nucleus caused obesity in both sexes and postpartum depressive behavior in females, while Trpc5 overexpression in oxytocin neurons in knock-in mice reversed these phenotypes. We demonstrate that TRPC5 plays a pivotal role in mediating innate human behaviors fundamental to survival, including food seeking and maternal care.

Neural circuits expressing the serotonin 2C receptor regulate memory in mice and humans.

Declined memory is a hallmark of Alzheimer's disease (AD). Experiments in rodents and human postmortem studies suggest that serotonin (5-hydroxytryptamine, 5-HT) plays a role in memory, but the underlying mechanisms are unknown. Here, we investigate the role of 5-HT 2C receptor (5-HT2CR) in regulating memory. Transgenic mice expressing a humanized HTR2C mutation exhibit impaired plasticity of hippocampal ventral CA1 (vCA1) neurons and reduced memory. Further, 5-HT neurons project to and synapse onto vCA1 neurons. Disruption of 5-HT synthesis in vCA1-projecting neurons or deletion of 5-HT2CRs in the vCA1 impairs neural plasticity and memory. We show that a selective 5-HT2CR agonist, lorcaserin, improves synaptic plasticity and memory in an AD mouse model. Cumulatively, we demonstrate that hippocampal 5-HT2CR signaling regulates memory, which may inform the use of 5-HT2CR agonists in the treatment of dementia.

Chromosomal deletions on 16p11.2 encompassing SH2B1 are associated with accelerated metabolic disease.

New approaches are needed to treat people whose obesity and type 2 diabetes (T2D) are driven by specific mechanisms. We investigate a deletion on chromosome 16p11.2 (breakpoint 2-3 [BP2-3]) encompassing SH2B1, a mediator of leptin and insulin signaling. Phenome-wide association scans in the UK (N = 502,399) and Estonian (N = 208,360) biobanks show that deletion carriers have increased body mass index (BMI; p = 1.3 × 10-10) and increased rates of T2D. Compared with BMI-matched controls, deletion carriers have an earlier onset of T2D, with poorer glycemic control despite higher medication usage. Cystatin C, a biomarker of kidney function, is significantly elevated in deletion carriers, suggesting increased risk of renal impairment. In a Mendelian randomization study, decreased SH2B1 expression increases T2D risk (p = 8.1 × 10-6). We conclude that people with 16p11.2 BP2-3 deletions have early, complex obesity and T2D and may benefit from therapies that enhance leptin and insulin signaling.

A rare human variant that disrupts GPR10 signalling causes weight gain in mice.

Disruption of brain-expressed G protein-coupled receptor-10 (GPR10) causes obesity in animals. Here, we identify multiple rare variants in GPR10 in people with severe obesity and in normal weight controls. These variants impair ligand binding and G protein-dependent signalling in cells. Transgenic mice harbouring a loss of function GPR10 variant found in an individual with obesity, gain excessive weight due to decreased energy expenditure rather than increased food intake. This evidence supports a role for GPR10 in human energy homeostasis. Therapeutic targeting of GPR10 may represent an effective weight-loss strategy.

Human loss-of-function variants in the serotonin 2C receptor associated with obesity and maladaptive behavior.

Serotonin reuptake inhibitors and receptor agonists are used to treat obesity, anxiety and depression. Here we studied the role of the serotonin 2C receptor (5-HT2CR) in weight regulation and behavior. Using exome sequencing of 2,548 people with severe obesity and 1,117 control individuals without obesity, we identified 13 rare variants in the gene encoding 5-HT2CR (HTR2C) in 19 unrelated people (3 males and 16 females). Eleven variants caused a loss of function in HEK293 cells. All people who carried variants had hyperphagia and some degree of maladaptive behavior. Knock-in male mice harboring a human loss-of-function HTR2C variant developed obesity and reduced social exploratory behavior; female mice heterozygous for the same variant showed similar deficits with reduced severity. Using the 5-HT2CR agonist lorcaserin, we found that depolarization of appetite-suppressing proopiomelanocortin neurons was impaired in knock-in mice. In conclusion, we demonstrate that 5-HT2CR is involved in the regulation of human appetite, weight and behavior. Our findings suggest that melanocortin receptor agonists might be effective in treating severe obesity in individuals carrying HTR2C variants. We suggest that HTR2C should be included in diagnostic gene panels for severe childhood-onset obesity.

Visualization of sympathetic neural innervation in human white adipose tissue.

Obesity, defined as an excess of adipose tissue that adversely affects health, is a major cause of morbidity and mortality. However, to date, understanding the structure and function of human adipose tissue has been limited by the inability to visualize cellular components due to the innate structure of adipocytes, which are characterized by large lipid droplets. Combining the iDISCO and the CUBIC protocols for whole tissue staining and optical clearing, we developed a protocol to enable immunostaining and clearing of human subcutaneous white adipose tissue (WAT) obtained from individuals with severe obesity. We were able to perform immunolabelling of sympathetic nerve terminals in whole WAT and subsequent optical clearing by eliminating lipids to render the opaque tissue completely transparent. We then used light sheet confocal microscopy to visualize sympathetic innervation of human WAT from obese individuals in a three-dimensional manner. We demonstrate the visualization of sympathetic nerve terminals in human WAT. This protocol can be modified to visualize other structures such as blood vessels involved in the development, maintenance and function of human adipose tissue in health and disease.

Obesity Due to Steroid Receptor Coactivator-1 Deficiency Is Associated With Endocrine and Metabolic Abnormalities.

CONTEXT: Genetic variants affecting the nuclear hormone receptor coactivator steroid receptor coactivator, SRC-1, have been identified in people with severe obesity and impair melanocortin signaling in cells and mice. As a result, obese patients with SRC-1 deficiency are being treated with a melanocortin 4 receptor agonist in clinical trials. OBJECTIVE: Here, our aim was to comprehensively describe and characterize the clinical phenotype of SRC-1 variant carriers to facilitate diagnosis and clinical management. METHODS: In genetic studies of 2462 people with severe obesity, we identified 23 rare heterozygous variants in SRC-1. We studied 29 adults and 18 children who were SRC-1 variant carriers and performed measurements of metabolic and endocrine function, liver imaging, and adipose tissue biopsies. Findings in adult SRC-1 variant carriers were compared to 30 age- and body mass index (BMI)-matched controls. RESULTS: The clinical spectrum of SRC-1 variant carriers included increased food intake in children, normal basal metabolic rate, multiple fractures with minimal trauma (40%), persistent diarrhea, partial thyroid hormone resistance, and menorrhagia. Compared to age-, sex-, and BMI-matched controls, adult SRC-1 variant carriers had more severe adipose tissue fibrosis (46.2% vs 7.1% respectively, P = .03) and a suggestion of increased liver fibrosis (5/13 cases vs 2/13 in controls, odds ratio = 3.4), although this was not statistically significant. CONCLUSION: SRC-1 variant carriers exhibit hyperphagia in childhood, severe obesity, and clinical features of partial hormone resistance. The presence of adipose tissue fibrosis and hepatic fibrosis in young patients suggests that close monitoring for the early development of obesity-associated metabolic complications is warranted.

Obesity due to melanocortin 4 receptor (MC4R) deficiency is associated with delayed gastric emptying.

OBJECTIVE: People who are severely obese due to melanocortin-4 receptor (MC4R) deficiency experience hyperphagia and impaired fullness after a meal (satiety). Meal-induced satiety is influenced by hormones, such as peptide-YY (PYY), which are released by enteroendocrine cells upon nutrient delivery to the small intestine. DESIGN: We investigated whether gastric emptying and PYY levels are altered in MC4R deficiency. METHODS: Gastric emptying was measured with a gastric scintigraphy protocol using technetium-99m (99 Tcm )-Tin Colloid for 3.5 h in individuals with loss of function MC4R variants and a control group of similar age and weight. In a separate study, we measured plasma PYY levels before and at multiple time points after three standardised meals given to individuals with MC4R deficiency and controls. Fasting PYY (basal secretion) and postprandial PYY levels were measured and the area under the curve and inter-meal peak were calculated. RESULTS: We found that gastric emptying time was significantly delayed and percentage meal retention increased in individuals with MC4R deficiency compared to obese controls. In addition, fasting and mean PYY secretion throughout the day were decreased in MC4R deficiency, whereas postprandial PYY secretion was unaltered. CONCLUSION: Delayed gastric emptying and reduced basal PYY secretion may contribute to impaired satiety in people with obesity due to MC4R deficiency.

Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila.

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

Obesity-Associated GNAS Mutations and the Melanocortin Pathway.

BACKGROUND: GNAS encodes the Gαs (stimulatory G-protein alpha subunit) protein, which mediates G protein-coupled receptor (GPCR) signaling. GNAS mutations cause developmental delay, short stature, and skeletal abnormalities in a syndrome called Albright's hereditary osteodystrophy. Because of imprinting, mutations on the maternal allele also cause obesity and hormone resistance (pseudohypoparathyroidism). METHODS: We performed exome sequencing and targeted resequencing in 2548 children who presented with severe obesity, and we unexpectedly identified 22 GNAS mutation carriers. We investigated whether the effect of GNAS mutations on melanocortin 4 receptor (MC4R) signaling explains the obesity and whether the variable clinical spectrum in patients might be explained by the results of molecular assays. RESULTS: Almost all GNAS mutations impaired MC4R signaling. A total of 6 of 11 patients who were 12 to 18 years of age had reduced growth. In these patients, mutations disrupted growth hormone-releasing hormone receptor signaling, but growth was unaffected in carriers of mutations that did not affect this signaling pathway (mean standard-deviation score for height, -0.90 vs. 0.75, respectively; P = 0.02). Only 1 of 10 patients who reached final height before or during the study had short stature. GNAS mutations that impaired thyrotropin receptor signaling were associated with developmental delay and with higher thyrotropin levels (mean [±SD], 8.4±4.7 mIU per liter) than those in 340 severely obese children who did not have GNAS mutations (3.9±2.6 mIU per liter; P = 0.004). CONCLUSIONS: Because pathogenic mutations may manifest with obesity alone, screening of children with severe obesity for GNAS deficiency may allow early diagnosis, improving clinical outcomes, and melanocortin agonists may aid in weight loss. GNAS mutations that are identified by means of unbiased genetic testing differentially affect GPCR signaling pathways that contribute to clinical heterogeneity. Monogenic diseases are clinically more variable than their classic descriptions suggest. (Funded by Wellcome and others.).

Human MC4R variants affect endocytosis, trafficking and dimerization revealing multiple cellular mechanisms involved in weight regulation.

The Melanocortin-4 Receptor (MC4R) plays a pivotal role in energy homeostasis. We used human MC4R mutations associated with an increased or decreased risk of obesity to dissect mechanisms that regulate MC4R function. Most obesity-associated mutations impair trafficking to the plasma membrane (PM), whereas obesity-protecting mutations either accelerate recycling to the PM or decrease internalization, resulting in enhanced signaling. MC4R mutations that do not affect canonical Gαs protein-mediated signaling, previously considered to be non-pathogenic, nonetheless disrupt agonist-induced internalization, ß-arrestin recruitment, and/or coupling to Gαs, establishing their causal role in severe obesity. Structural mapping reveals ligand-accessible sites by which MC4R couples to effectors and residues involved in the homodimerization of MC4R, which is disrupted by multiple obesity-associated mutations. Human genetic studies reveal that endocytosis, intracellular trafficking, and homodimerization regulate MC4R function to a level that is physiologically relevant, supporting the development of chaperones, agonists, and allosteric modulators of MC4R for weight loss therapy.

Human BDNF/TrkB variants impair hippocampal synaptogenesis and associate with neurobehavioural abnormalities.

Brain-derived neurotrophic factor (BDNF) signals through its high affinity receptor Tropomyosin receptor kinase-B (TrkB) to regulate neuronal development, synapse formation and plasticity. In rodents, genetic disruption of Bdnf and TrkB leads to weight gain and a spectrum of neurobehavioural phenotypes. Here, we functionally characterised a de novo missense variant in BDNF and seven rare variants in TrkB identified in a large cohort of people with severe, childhood-onset obesity. In cells, the E183K BDNF variant resulted in impaired processing and secretion of the mature peptide. Multiple variants in the kinase domain and one variant in the extracellular domain of TrkB led to a loss of function through multiple signalling pathways, impaired neurite outgrowth and dominantly inhibited glutamatergic synaptogenesis in hippocampal neurons. BDNF/TrkB variant carriers exhibited learning difficulties, impaired memory, hyperactivity, stereotyped and sometimes, maladaptive behaviours. In conclusion, human loss of function BDNF/TrkB variants that impair hippocampal synaptogenesis may contribute to a spectrum of neurobehavioural disorders.

Exome Sequencing Identifies Genes and Gene Sets Contributing to Severe Childhood Obesity, Linking PHIP Variants to Repressed POMC Transcription.

Obesity is genetically heterogeneous with monogenic and complex polygenic forms. Using exome and targeted sequencing in 2,737 severely obese cases and 6,704 controls, we identified three genes (PHIP, DGKI, and ZMYM4) with an excess burden of very rare predicted deleterious variants in cases. In cells, we found that nuclear PHIP (pleckstrin homology domain interacting protein) directly enhances transcription of pro-opiomelanocortin (POMC), a neuropeptide that suppresses appetite. Obesity-associated PHIP variants repressed POMC transcription. Our demonstration that PHIP is involved in human energy homeostasis through transcriptional regulation of central melanocortin signaling has potential diagnostic and therapeutic implications for patients with obesity and developmental delay. Additionally, we found an excess burden of predicted deleterious variants involving genes nearest to loci from obesity genome-wide association studies. Genes and gene sets influencing obesity with variable penetrance provide compelling evidence for a continuum of causality in the genetic architecture of obesity, and explain some of its missing heritability.

Leptin-Mediated Changes in the Human Metabolome.

CONTEXT: While severe obesity due to congenital leptin deficiency is rare, studies in patients before and after treatment with leptin can provide unique insights into the role that leptin plays in metabolic and endocrine function. OBJECTIVE: The aim of this study was to characterize changes in peripheral metabolism in people with congenital leptin deficiency undergoing leptin replacement therapy, and to investigate the extent to which these changes are explained by reduced caloric intake. DESIGN: Ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) was used to measure 661 metabolites in 6 severely obese people with congenital leptin deficiency before, and within 1 month after, treatment with recombinant leptin. Data were analyzed using unsupervised and hypothesis-driven computational approaches and compared with data from a study of acute caloric restriction in healthy volunteers. RESULTS: Leptin replacement was associated with class-wide increased levels of fatty acids and acylcarnitines and decreased phospholipids, consistent with enhanced lipolysis and fatty acid oxidation. Primary and secondary bile acids increased after leptin treatment. Comparable changes were observed after acute caloric restriction. Branched-chain amino acids and steroid metabolites decreased after leptin, but not after acute caloric restriction. Individuals with severe obesity due to leptin deficiency and other genetic obesity syndromes shared a metabolomic signature associated with increased BMI. CONCLUSION: Leptin replacement was associated with changes in lipolysis and substrate utilization that were consistent with negative energy balance. However, leptin's effects on branched-chain amino acids and steroid metabolites were independent of reduced caloric intake and require further exploration.

Neural networks associated with body composition in frontotemporal dementia.

BACKGROUND: Frontotemporal dementia (FTD) is associated with complex changes in eating behavior and metabolism, which potentially affect disease pathogenesis and survival. It is currently not known if body composition changes and changes in fat deposition also exist in FTD, the relationship of these changes in eating behavior and appetite, and whether these changes are centrally mediated. METHODS: Body composition was measured in 28 people with behavioral-variant frontotemporal dementia (bvFTD), 16 with Alzheimer's disease (AD), and 19 healthy controls, using dual energy x-ray absorptiometry. Changes in body composition were correlated to brain grey matter atrophy using voxel-based morphometry on high-resolution magnetic resonance imaging. RESULTS: Behavioral-variant FTD was characterized by changes in body composition, with increased total fat mass, visceral adipose tissue area (VAT area), and android: gynoid ratio compared to control and AD participants (all P values < 0.05). Changes in body composition correlated to abnormal eating behavior and behavioral change (P < 0.01) and functional decline (P < 0.01). Changes in body composition also correlated to grey matter atrophy involving a distributed neural network that included the hippocampus, amygdala, nucleus accumbens, insula, cingulate, and cerebellum - structures known to be central to autonomic control - as well as the thalamus, putamen, accumbens, and caudate, which are involved in reward processing. CONCLUSIONS: Changes in body composition and fat deposition extend the clinical phenomenology in bvFTD beyond cognition and behavior, with changes associated with changes in reward and autonomic processing suggesting that these deficits may be central in FTD.

Crucial Role of the SH2B1 PH Domain for the Control of Energy Balance.

Disruption of the adaptor protein SH2B1 (SH2-B, PSM) is associated with severe obesity, insulin resistance, and neurobehavioral abnormalities in mice and humans. Here, we identify 15 SH2B1 variants in severely obese children. Four obesity-associated human SH2B1 variants lie in the Pleckstrin homology (PH) domain, suggesting that the PH domain is essential for SH2B1's function. We generated a mouse model of a human variant in this domain (P322S). P322S/P322S mice exhibited substantial prenatal lethality. Examination of the P322S/+ metabolic phenotype revealed late-onset glucose intolerance. To circumvent P322S/P322S lethality, mice containing a two-amino acid deletion within the SH2B1 PH domain (ΔP317, R318 [ΔPR]) were studied. Mice homozygous for ΔPR were born at the expected Mendelian ratio and exhibited obesity plus insulin resistance and glucose intolerance beyond that attributable to their increased adiposity. These studies demonstrate that the PH domain plays a crucial role in how SH2B1 controls energy balance and glucose homeostasis.

Steroid receptor coactivator-1 modulates the function of Pomc neurons and energy homeostasis.

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

Genetic architecture of human thinness compared to severe obesity.

The variation in weight within a shared environment is largely attributable to genetic factors. Whilst many genes/loci confer susceptibility to obesity, little is known about the genetic architecture of healthy thinness. Here, we characterise the heritability of thinness which we found was comparable to that of severe obesity (h2 = 28.07 vs 32.33% respectively), although with incomplete genetic overlap (r = -0.49, 95% CI [-0.17, -0.82], p = 0.003). In a genome-wide association analysis of thinness (n = 1,471) vs severe obesity (n = 1,456), we identified 10 loci previously associated with obesity, and demonstrate enrichment for established BMI-associated loci (pbinomial = 3.05x10-5). Simulation analyses showed that different association results between the extremes were likely in agreement with additive effects across the BMI distribution, suggesting different effects on thinness and obesity could be due to their different degrees of extremeness. In further analyses, we detected a novel obesity and BMI-associated locus at PKHD1 (rs2784243, obese vs. thin p = 5.99x10-6, obese vs. controls p = 2.13x10-6 pBMI = 2.3x10-13), associations at loci recently discovered with much larger sample sizes (e.g. FAM150B and PRDM6-CEP120), and novel variants driving associations at previously established signals (e.g. rs205262 at the SNRPC/C6orf106 locus and rs112446794 at the PRDM6-CEP120 locus). Our ability to replicate loci found with much larger sample sizes demonstrates the value of clinical extremes and suggest that characterisation of the genetics of thinness may provide a more nuanced understanding of the genetic architecture of body weight regulation and may inform the identification of potential anti-obesity targets.

Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance.

Hypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.