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.

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.

Leptin mediates the increase in blood pressure associated with obesity.

Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin's effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species.

KSR2 mutations are associated with obesity, insulin resistance, and impaired cellular fuel oxidation.

Kinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEKERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.

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.).

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 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.

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.

Neural deletion of Sh2b1 results in brain growth retardation and reactive aggression.

Psychiatric disorders are associated with aberrant brain development and/or aggressive behavior and are influenced by genetic factors; however, genes that affect brain aggression circuits remain elusive. Here, we show that neuronal Src-homology-2 (SH2)B adaptor protein-1 ( Sh2b1) is indispensable for both brain growth and protection against aggression. Global and brain-specific deletion of Sh2b1 decreased brain weight and increased aggressive behavior. Global and brain-specific Sh2b1 knockout (KO) mice exhibited fatal, intermale aggression. In a resident-intruder paradigm, latency to attack was markedly reduced, whereas the number and the duration of attacks was significantly increased in global and brain-specific Sh2b1 KO mice compared with wild-type littermates. Consistently, core aggression circuits were activated to a higher level in global and brain-specific Sh2b1 KO males, based on c-fos immunoreactivity in the amygdala and periaqueductal gray. Brain-specific restoration of Sh2b1 normalized brain size and reversed pathologic aggression and aberrant activation of core aggression circuits in Sh2b1 KO males. SH2B1 mutations in humans were linked to aberrant brain development and behavior. At the molecular level, Sh2b1 enhanced neurotrophin-stimulated neuronal differentiation and protected against oxidative stress-induced neuronal death. Our data suggest that neuronal Sh2b1 promotes brain development and the integrity of core aggression circuits, likely through enhancing neurotrophin signaling.-Jiang, L., Su, H., Keogh, J. M., Chen, Z., Henning, E., Wilkinson, P., Goodyer, I., Farooqi, I. S., Rui, L. Neural deletion of Sh2b1 results in brain growth retardation and reactive aggression.

Role of melanocortin signalling in the preference for dietary macronutrients in human beings.

BACKGROUND: Signalling though the melanocortin 4 receptor (MC4R), which is widely expressed in the hypothalamus, mediates food intake and macronutrient preference in rodents. Studies in patients with MC4R deficiency can provide insights into the role of this pathway in man. We investigated the role of melanocortin signalling in fat and sucrose preference in human beings by studying patients with loss of function mutations in MC4R. METHODS: We studied 24 obese patients with MC4R deficiency, and 80 healthy controls (40 obese, 40 lean). We used an ad-libitum meal protocol consisting of three meals covertly manipulated to provide 20% (low), 40% (medium), and 60% (high) fat content. We used the same procedure for meals manipulated to provide 8% (low), 26% (medium), and 54% (high) sucrose content. We measured food intake and rated liking for the meals with visual analogue scores. Data were analysed by ANOVA and Tukey's post-hoc tests or a linear mixed-effects model with an interaction term for study group and study meal when appropriate. FINDINGS: Although the liking of the three different fat meals did not differ between the three groups, patients with MC4R mutations consumed 95% more of the high fat meal than did lean controls and 65% more of the high fat meal than did obese controls (p=0·0222 for the interaction of group by meal). By contrast, although liking ratings for low and medium sucrose meals were comparable in the individuals with MC4R deficiency, liking ratings for the high sucrose meal were significantly reduced (p=0·0252 in linear mixed-effects model, intercept 57·8, MC4R group factor -26·2, factors in the model for MC4R-low sucrose 27·7, MC4R-medium sucrose 22·6). Similarly, patients with MC4R deficiency consumed less of all three sucrose meals than did healthy controls (p=0·0064). INTERPRETATION: Our study shows that the central melanocortin system has divergent effects on macronutrient preference and intake in human beings. FUNDING: Wellcome Trust, NIHR Cambridge Biomedical Research Centre, Bernard Wolfe Health Neuroscience Fund, NeuroFAST consortium, which is funded by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 245009.

Large, rare chromosomal deletions associated with severe early-onset obesity.

Obesity is a highly heritable and genetically heterogeneous disorder. Here we investigated the contribution of copy number variation to obesity in 300 Caucasian patients with severe early-onset obesity, 143 of whom also had developmental delay. Large (>500 kilobases), rare (<1%) deletions were significantly enriched in patients compared to 7,366 controls (P < 0.001). We identified several rare copy number variants that were recurrent in patients but absent or at much lower prevalence in controls. We identified five patients with overlapping deletions on chromosome 16p11.2 that were found in 2 out of 7,366 controls (P < 5 x 10(-5)). In three patients the deletion co-segregated with severe obesity. Two patients harboured a larger de novo 16p11.2 deletion, extending through a 593-kilobase region previously associated with autism and mental retardation; both of these patients had mild developmental delay in addition to severe obesity. In an independent sample of 1,062 patients with severe obesity alone, the smaller 16p11.2 deletion was found in an additional two patients. All 16p11.2 deletions encompass several genes but include SH2B1, which is known to be involved in leptin and insulin signalling. Deletion carriers exhibited hyperphagia and severe insulin resistance disproportionate for the degree of obesity. We show that copy number variation contributes significantly to the genetic architecture of human obesity.

Failure of sucrose replacement with the non-nutritive sweetener erythritol to alter GLP-1 or PYY release or test meal size in lean or obese people.

There is considerable interest in the effect of foods containing high intensity sweeteners on satiation. However, less is known about low-calorie bulk sweeteners such as erythritol. In this randomized three-way crossover study, we studied 10 lean and 10 obese volunteers who consumed three test meals on separate occasions: (a) control sucrose meal; (b) isovolumic meal with partial replacement of sucrose by erythritol; (c) isocaloric meal which contained more erythritol but equivalent calories to the control meal. We measured gut hormone levels, hunger and satiety scores, ad libitum food intake, sucrose preference and intake after the manipulations. There was a greater post-prandial excursion in glucose and insulin levels after sucrose than after the erythritol meals. There was no difference in GLP-1/PYY levels or subsequent energy intake and sucrose preference between sucrose control and isovolumic erythritol meals. In lean (but not obese) participants, hunger decreased to a greater extent after the isocaloric erythritol meal compared to the control meal (p = 0.003) reflecting the larger volume of this meal. Replacing sucrose with erythritol leads to comparable hunger and satiety scores, GLP-1 and PYY levels, and subsequent sucrose preference and intake.

A mutation in the thyroid hormone receptor alpha gene.

Thyroid hormones exert their effects through alpha (TRα1) and beta (TRß1 and TRß2) receptors. Here we describe a child with classic features of hypothyroidism (growth retardation, developmental retardation, skeletal dysplasia, and severe constipation) but only borderline-abnormal thyroid hormone levels. Using whole-exome sequencing, we identified a de novo heterozygous nonsense mutation in a gene encoding thyroid hormone receptor alpha (THRA) and generating a mutant protein that inhibits wild-type receptor action in a dominant negative manner. Our observations are consistent with defective human TRα-mediated thyroid hormone resistance and substantiate the concept of hormone action through distinct receptor subtypes in different target tissues.

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.

Modulation of blood pressure by central melanocortinergic pathways.

BACKGROUND: Weight gain and weight loss are associated with changes in blood pressure through unknown mechanisms. Central melanocortinergic signaling is implicated in the control of energy balance and blood pressure in rodents, but there is no information regarding such an association with blood pressure in humans. METHODS: We assessed blood pressure, heart rate, and urinary catecholamines in overweight or obese subjects with a loss-of-function mutation in MC4R, the gene encoding the melanocortin 4 receptor, and in equally overweight control subjects. We also examined the effects of an MC4R agonist administered for 7 days in 28 overweight or obese volunteers. RESULTS: The prevalence of hypertension was markedly lower in the MC4R-deficient subjects than in the control subjects (24% vs. 53%, P=0.009). After the exclusion of subjects taking antihypertensive medications, blood-pressure levels were significantly lower in MC4R-deficient subjects than in control subjects, with mean (+/-SE) systolic blood pressures of 123+/-14 mm Hg and 131+/-12 mm Hg, respectively (P=0.02), and mean diastolic blood pressures of 73+/-10 mm Hg and 79+/-7 mm Hg, respectively (P=0.03). As compared with control subjects, MC4R-deficient subjects had a lower increase in heart rate on waking (P=0.007), a lower heart rate during euglycemic hyperinsulinemia (P<0.001), and lower 24-hour urinary norepinephrine excretion (P=0.04). The maximum tolerated daily dose of 1.0 mg of the MC4R agonist led to significant increases of 9.3+/-1.9 mm Hg in systolic blood pressure and of 6.6+/-1.1 mm Hg in diastolic blood pressure (P<0.001 for both comparisons) at 24 hours, as compared with placebo. Differences in blood pressure were not explained by changes in insulin levels; there were no significant adverse events. CONCLUSIONS: Results of our genetic and pharmacologic studies implicate melanocortinergic signaling in the control of human blood pressure through an insulin-independent mechanism.

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.