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

Funder: NIHR [Cambridge Biomedical Research Centre at the Cambridge University Hospitals NHS Foundation TrustFunder: NHS National Institute for Health Research Clinical Research NetworkFunder: Royal Society Darwin Trust Research ProfessorshipFunder: NIHR Senior Investigator AwardFunder: Health Data Research UKFunder: Higher Education Funding Council for England CatalystFunder: NIHR Cambridge Biomedical Research CentreFunder: Bernard Wolfe Health Neuroscience EndowmentFunder: The Botnar FondationThe 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

Divergent effects of central melanocortin signalling on fat and sucrose preference in humans.

Melanocortin-4-receptor (MC4R)-expressing neurons modulate food intake and preference in rodents but their role in human food preference is unknown. Here we show that compared with lean and weight-matched controls, MC4R deficient individuals exhibited a markedly increased preference for high fat, but a significantly reduced preference for high sucrose food. These effects mirror those in Mc4r null rodents and provide evidence for a central molecular circuit influencing human macronutrient preference.This work was supported by the Wellcome Trust (to A.A.v.d.K., P.C.F., I.S.F.), the National Institute for Health Research Cambridge Biomedical Research Centre (to S.O’R., I.S.F.), the Bernard Wolfe Health Neuroscience Fund (to A.A.v.d.K., I.S.F., P.C.F.) and the European Research Council (I.S.F.). This work was supported by the NeuroFAST consortium which is funded by the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 245009.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1305

Severe Early-Onset Obesity Due to Bioinactive Leptin Caused by a p.N103K Mutation in the Leptin Gene.

CONTEXT: Congenital leptin deficiency is a very rare cause of severe early-onset obesity. We recently characterized a mutation in the leptin gene (p.D100Y), which was associated with detectable leptin levels and bioinactivity of the hormone. CASE DESCRIPTION: We now describe two siblings, a 9-year-old girl and a 6-year-old boy with severe early-onset obesity and hyperphagia, both homozygous for a c.309C>A substitution in the leptin gene leading to a p.N103K amino acid exchange in the protein and detectable circulating levels of leptin. In vitro experiments in a heterologous cell system demonstrated that the mutated protein was biologically inactive. Treatment with sc recombinant human leptin led to rapid improvement of eating behavior and weight loss. CONCLUSIONS: Sequencing of the leptin gene may need to be considered in hyperphagic, severely obese children with detectable levels of circulating leptin.This work was supported by Grant BMBF 01GI1120A from the Federal Ministry of Education and Research. Support was also provided by the Wellcome Trust (082390/Z/07/Z), the Medical Research Council, the National Institute for Health Research Cambridge Biomedical Research Centre, the European Research Council, and the Bernard Wolfe Health Neuroscience Fund (all to I.S.F.). J.-B.F. was supported by the International Graduate School in Molecular Medicine Ulm.This is the final version of the article. It first appeared from the Endocrine Society via http://dx.doi.org/10.1210/jc.2015-226

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.Wellcome Trust, National Institute for Health Research Cambridge Biomedical Research Centre, Bernard Wolfe Health Neuroscience Fund, Swiss National Science Foundation (Grant IDs: PBLAP3-145870, P3SMP3-155318), NeuroFAST consortium, European Union's Seventh Framework Programme (FP7/ 2007e2013) Grant ID: 245009), Cargill, Sas van Gent, The NetherlandsThis is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.appet.2016.09.00

A Transcriptomic Signature of the Hypothalamic Response to Fasting and BDNF Deficiency in Prader-Willi Syndrome.

Transcriptional analysis of brain tissue from people with molecularly defined causes of obesity may highlight disease mechanisms and therapeutic targets. We performed RNA sequencing of hypothalamus from individuals with Prader-Willi syndrome (PWS), a genetic obesity syndrome characterized by severe hyperphagia. We found that upregulated genes overlap with the transcriptome of mouse Agrp neurons that signal hunger, while downregulated genes overlap with the expression profile of Pomc neurons activated by feeding. Downregulated genes are expressed mainly in neuronal cells and contribute to neurogenesis, neurotransmitter release, and synaptic plasticity, while upregulated, predominantly microglial genes are involved in inflammatory responses. This transcriptional signature may be mediated by reduced brain-derived neurotrophic factor expression. Additionally, we implicate disruption of alternative splicing as a potential molecular mechanism underlying neuronal dysfunction in PWS. Transcriptomic analysis of the human hypothalamus may identify neural mechanisms involved in energy homeostasis and potential therapeutic targets for weight loss

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

Functional characterization of obesity-associated variants involving the α and β isoforms of human SH2B1.

We have previously reported rare variants in sarcoma (Src) homology 2 (SH2) B adaptor protein 1 (SH2B1) in individuals with obesity, insulin resistance, and maladaptive behavior. Here, we identify 4 additional SH2B1 variants by sequencing 500 individuals with severe early-onset obesity. SH2B1 has 4 alternatively spliced isoforms. One variant (T546A) lies within the N-terminal region common to all isoforms. As shown for past variants in this region, T546A impairs SH2B1β enhancement of nerve growth factor-induced neurite outgrowth, and the individual with the T546A variant exhibits mild developmental delay. The other 3 variants (A663V, V695M, and A723V) lie in the C-terminal tail of SH2B1α. SH2B1α variant carriers were hyperinsulinemic but did not exhibit the behavioral phenotype observed in individuals with SH2B1 variants that disrupt all isoforms. In in vitro assays, SH2B1α, like SH2B1β, enhances insulin- and leptin-induced insulin receptor substrate 2 (IRS2) phosphorylation and GH-induced cell motility. None of the variants affect SH2B1α enhancement of insulin- and leptin-induced IRS2 phosphorylation. However, T546A, A663V, and A723V all impair the ability of SH2B1α to enhance GH-induced cell motility. In contrast to SH2B1β, SH2B1α does not enhance nerve growth factor-induced neurite outgrowth. These studies suggest that genetic variants that disrupt isoforms other than SH2B1β may be functionally significant. Further studies are needed to understand the mechanism by which the individual isoforms regulate energy homeostasis and behavior.This work was supported by the Wellcome Trust (098497/Z/ 12/Z; 077016/Z/05/Z; 096106/Z/11/Z) (to I.S. Farooqi and L.R. Pearce), by the Medical Research Council Metabolic Diseases Unit and NIHR Cambridge Biomedical Research Centre (to I.S. Farooqi, I. Barroso, and S. O’Rahilly) and the Bernard Wolfe Health Neuroscience Fund (I.S. Farooqi); and by NIH grants RO1-DK54222 (to C. Carter-Su), RO1-DK065122 and RO1- DK073601 (to L. Rui), a predoctoral fellowship from the Systems and Integrative Biology Training Grant NIH–T32-GM008322 (to M.E. Doche) and a Rackham Merit Fellowship from the University of Michigan (to R. Joe). Confocal microscopy was performed using the Morphology and Image Analysis Core of the Michigan Diabetes Research Center (NIH grant P60-DK20572).This is the final published version distributed under a Creative Commons Attribution License, which can also be found on the publisher's website at: http://press.endocrine.org/doi/abs/10.1210/en.2014-1264?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubme

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, β-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

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