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Abstract The hormone GDF15 is secreted in response to cellular stressors. Metformin elevates circulating levels of GDF15, an action important for the drug’s beneficial effects on body weight. Metformin can also inhibit mammalian respiratory complex I, leading to decreases in ATP:AMP ratio, activation of AMP Kinase (AMPK), and increased GDF15 production. We undertook studies using a range of mice with tissue-specific loss of Gdf15 (namely gut, liver and global deletion) to determine the relative contributions of two classical metformin target tissues, the gut and liver, to the elevation of GDF15 seen with metformin. In addition, we performed comparative studies with another pharmacological agent, the AMP kinase pan-activator, MK-8722. Deletion of Gdf15 from the intestinal epithelium significantly reduced the circulating GDF15 response to oral metformin, whereas deletion of Gdf15 from the liver had no effect. In contrast, deletion of Gdf15 from the liver, but not the gut, markedly reduced circulating GDF15 responses to MK-8722. Further, our data show that, while GDF15 restricts high-fat diet-induced weight gain, the intestinal production of GDF15 is not necessary for this effect. These findings add to the body of evidence implicating the intestinal epithelium in key aspects of the pharmacology of metformin action.

Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

Abstract Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat +/−p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat +/−p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat +/−p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat +/−p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat +/−p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat +/−p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.

Objectives: Obesity in humans and mice is associated with elevated levels of two hormones responsive to cellular stress, namely GDF15 and FGF21. Over-expression of each of these is associated with weight loss and beneficial metabolic changes but where they are secreted from and what they are required for physiologically in the context of overfeeding remains unclear. Methods: Here we used tissue selective knockout mouse models and human transcriptomics to determine the source of circulating GDF15 in obesity. We then generated and characterized the metabolic phenotypes of GDF15/FGF21 double knockout mice. Results: Circulating GDF15 and FGF21 are both largely derived from the liver, rather than adipose tissue or skeletal muscle, in obese states. Combined whole body deletion of FGF21 and GDF15 does not result in any additional weight gain in response to high fat feeding but it does result in significantly greater hepatic steatosis and insulin resistance than that seen in GDF15 single knockout mice. Conclusions: Collectively the data suggest that overfeeding activates a stress response in the liver which is the major source of systemic rises in GDF15 and FGF21. These hormones then activate pathways which reduce this metabolic stress.

Abstract Even though metformin is widely used to treat type2 diabetes, reducing glycaemia and body weight, the mechanisms of action are still elusive. Recent studies have identified the gastrointestinal tract as an important site of action. Here we used intestinal organoids to explore the effects of metformin on intestinal cell physiology. Bulk RNA-sequencing analysis identified changes in hexose metabolism pathways, particularly glycolytic genes. Metformin increased expression of Slc2a1 (GLUT1), decreased expression of Slc2a2 (GLUT2) and Slc5a1 (SGLT1) whilst increasing GLUT-dependent glucose uptake and glycolytic rate as observed by live cell imaging of genetically encoded metabolite sensors and measurement of oxygen consumption and extracellular acidification rates. Metformin caused mitochondrial dysfunction and metformin’s effects on 2D-cultures were phenocopied by treatment with rotenone and antimycin-A, including upregulation of GDF15 expression, previously linked to metformin dependent weight loss. Gene expression changes elicited by metformin were replicated in 3D apical-out organoids and distal small intestines of metformin treated mice. We conclude that metformin affects glucose uptake, glycolysis and GDF-15 secretion, likely downstream of the observed mitochondrial dysfunction. This may explain the effects of metformin on intestinal glucose utilisation and food balance.

Neurons expressing pro-opiomelanocortin (Pomc) regulate food intake and body weight. Here the authors show that Steroid Receptor Coactivator-1 (SRC-1) regulates the function of Pomc expressing neurons, and that rare heterozygous variants found in obese individuals lead to loss of SRC-1 function.

Summary: Adipogenin (Adig) is an adipocyte-enriched transmembrane protein. Its expression is induced during adipogenesis in rodent cells, and a recent genome-wide association study associated body mass index (BMI)-adjusted leptin levels with the ADIG locus. In order to begin to understand the biological function of Adig, we studied adipogenesis in Adig-deficient cultured adipocytes and phenotyped Adig null (Adig−/−) mice. Data from Adig-deficient cells suggest that Adig is required for adipogenesis. In vivo, Adig−/− mice are leaner than wild-type mice when fed a high-fat diet and when crossed with Ob/Ob hyperphagic mice. In addition to the impact on fat mass accrual, Adig deficiency also reduces fat-mass-adjusted plasma leptin levels and impairs leptin secretion from adipose explants, suggesting an additional impact on the regulation of leptin secretion.

Objective: Insulin signalling via phosphoinositide 3-kinase (PI3K) requires PIK3R1-encoded regulatory subunits. C-terminal PIK3R1 mutations cause SHORT syndrome, as well as lipodystrophy and insulin resistance (IR), surprisingly without fatty liver or metabolic dyslipidaemia. We sought to investigate this discordance. Methods: The human pathogenic Pik3r1 Y657∗ mutation was knocked into mice by homologous recombination. Growth, body composition, bioenergetic and metabolic profiles were investigated on chow and high-fat diet (HFD). We examined adipose and liver histology, and assessed liver responses to fasting and refeeding transcriptomically. Results: Like humans with SHORT syndrome, Pik3r1WT/Y657∗ mice were small with severe IR, and adipose expansion on HFD was markedly reduced. Also as in humans, plasma lipid concentrations were low, and insulin-stimulated hepatic lipogenesis was not increased despite hyperinsulinemia. At odds with lipodystrophy, however, no adipocyte hypertrophy nor adipose inflammation was found. Liver lipogenic gene expression was not significantly altered, and unbiased transcriptomics showed only minor changes, including evidence of reduced endoplasmic reticulum stress in the fed state and diminished Rictor-dependent transcription on fasting. Increased energy expenditure, which was not explained by hyperglycaemia nor intestinal malabsorption, provided an alternative explanation for the uncoupling of IR from dyslipidaemia. Conclusions: Pik3r1 dysfunction in mice phenocopies the IR and reduced adiposity without lipotoxicity of human SHORT syndrome. Decreased adiposity may not reflect bona fide lipodystrophy, but rather, increased energy expenditure, and we suggest that further study of brown adipose tissue in both humans and mice is warranted.

Some imprinted genes exhibit parental origin specific expression bias rather than being transcribed exclusively from one copy. The physiological relevance of this remains poorly understood. In an analysis of brain-specific allele-biased expression, we identified that Trappc9, a cellular trafficking factor, was expressed predominantly (~70%) from the maternally inherited allele. Loss-of-function mutations in human TRAPPC9 cause a rare neurodevelopmental syndrome characterized by microcephaly and obesity. By studying Trappc9 null mice we discovered that homozygous mutant mice showed a reduction in brain size, exploratory activity and social memory, as well as a marked increase in body weight. A role for Trappc9 in energy balance was further supported by increased ad libitum food intake in a child with TRAPPC9 deficiency. Strikingly, heterozygous mice lacking the maternal allele (70% reduced expression) had pathology similar to homozygous mutants, whereas mice lacking the paternal allele (30% reduction) were phenotypically normal. Taken together, we conclude that Trappc9 deficient mice recapitulate key pathological features of TRAPPC9 mutations in humans and identify a role for Trappc9 and its imprinting in controlling brain development and metabolism.

Objective: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated. Methods: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC. Results: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of β-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P

Recall-by-Genotype (RbG) is an approach to recall participants from genetic studies based on their specific genotype for further, more extensive phenotyping. Here, the authors discuss examples of RbG as well as practical and ethical considerations and provide an online tool to aid in designing RbG studies.

Objective: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. Methods: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. Results: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. Conclusions: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.

Objective: Pro-opiomelanocortin (POMC)-derived peptides act on neurons expressing the Melanocortin 4 receptor (MC4R) to reduce body weight. Setmelanotide is a highly potent MC4R agonist that leads to weight loss in diet-induced obese animals and in obese individuals with complete POMC deficiency. While POMC deficiency is very rare, 1–5% of severely obese individuals harbor heterozygous mutations in MC4R. We sought to assess the efficacy of Setmelanotide in human MC4R deficiency. Methods: We studied the effects of Setmelanotide on mutant MC4Rs in cells and the weight loss response to Setmelanotide administration in rodent studies and a human clinical trial. We annotated the functional status of 369 published MC4R variants. Results: In cells, we showed that Setmelanotide is significantly more potent at MC4R than the endogenous ligand alpha-melanocyte stimulating hormone and can disproportionally rescue signaling by a subset of severely impaired MC4R mutants. Wild-type rodents appear more sensitive to Setmelanotide when compared to MC4R heterozygous deficient mice, while MC4R knockout mice fail to respond. In a 28-day Phase 1b clinical trial, Setmelanotide led to weight loss in obese MC4R variant carriers. Patients with POMC defects upstream of MC4R show significantly more weight loss with Setmelanotide than MC4R deficient patients or obese controls. Conclusions: Setmelanotide led to weight loss in obese people with MC4R deficiency; however, further studies are justified to establish whether Setmelanotide can elicit clinically meaningful weight loss in a subset of the MC4R deficient obese population.

Abstract Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GNAS, which were sometimes found with accelerated growth rather than short stature as described previously. Nominally significant associations were found for rare functional variants in BBS1, BBS9, GNAS, MKKS, CLOCK and ANGPTL6. The p.S284X variant in ANGPTL6 drives the association signal (rs201622589, MAF~0.1%, odds ratio = 10.13, p-value = 0.042) and results in complete loss of secretion in cells. Further analysis including additional case-control studies and population controls (N = 260,642) did not support association of this variant with obesity (odds ratio = 2.34, p-value = 2.59 × 10−3), highlighting the challenges of testing rare variant associations and the need for very large sample sizes. Further validation in cohorts with severe obesity and engineering the variants in model organisms will be needed to explore whether human variants in ANGPTL6 and other genes that lead to obesity when deleted in mice, do contribute to obesity. Such studies may yield druggable targets for weight loss therapies.

Objective: Arcuate proopiomelanocortin (POMC) neurons are critical nodes in the control of body weight. Often characterized simply as direct targets for leptin, recent data suggest a more complex architecture. Methods: Using single cell RNA sequencing, we have generated an atlas of gene expression in murine POMC neurons. Results: Of 163 neurons, 118 expressed high levels of Pomc with little/no Agrp expression and were considered “canonical” POMC neurons (P+). The other 45/163 expressed low levels of Pomc and high levels of Agrp (A+P+). Unbiased clustering analysis of P+ neurons revealed four different classes, each with distinct cell surface receptor gene expression profiles. Further, only 12% (14/118) of P+ neurons expressed the leptin receptor (Lepr) compared with 58% (26/45) of A+P+ neurons. In contrast, the insulin receptor (Insr) was expressed at similar frequency on P+ and A+P+ neurons (64% and 55%, respectively). Conclusion: These data reveal arcuate POMC neurons to be a highly heterogeneous population.Accession Numbers: GSE92707. Author Video: Author Video Watch what authors say about their articles Keywords: POMC, Melanocortin, AGRP, Leptin, Insulin, Hypothalamus, Arcuate nucleus, Gene expression, Neuron, Transcriptome

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.

Hypothalamic melanocortin-4-receptors (MC4R) regulate food preference in rodents, but their role in humans is unclear. Here, the authors perform food preference and liking tests in humans with MC4R mutations and find that they prefer fatty food more, but sweet food less, than people without MC4R mutations.

McKittrick–Wheelock syndrome (MWS) is a rare consequence of severe dehydration and electrolyte depletion due to mucinous diarrhoea secondary to a rectosigmoid villous adenoma. Reported cases of MWS commonly describe hypersecretion of mucinous diarrhoea in association with dehydration, hypokalaemia, hyponatraemia, hypochloraemia and pre-renal azotemia. Hyperglycaemia and diabetes are rarely reported manifestations of MWS. Herein we describe the case of a 59-year-old woman who presented with new-onset diabetes and severe electrolyte derangement due to a giant rectal villous adenoma. Subsequent endoscopic resection of the tumour cured her diabetes and normalised electrolytes. This case describes a rare cause of ‘curable diabetes’ and indicates hyperaldosteronism and/or whole-body potassium stores as important regulators of insulin secretion and glucose homeostasis.

Background: Although nausea and vomiting are very common in pregnancy, their pathogenesis is poorly understood. We tested the hypothesis that circulating growth and differentiation factor 15 (GDF15) concentrations in early pregnancy, whose gene is implicated in hyperemesis gravidarum, are associated with nausea and vomiting. Methods: Blood samples for the measurement of GDF15 and human chorionic gonadotrophin (hCG) concentrations were obtained early in the second trimester (median 15.1 (interquartile range 14.4-15.7) weeks) of pregnancy from 791 women from the Cambridge Baby Growth Study, a prospective pregnancy and birth cohort. During each trimester participants completed a questionnaire which included questions about nausea, vomiting and antiemetic use. Associations with pre-pregnancy body mass indexes (BMI) were validated in 231 pregnant NIPTeR Study participants. Results: Circulating GDF15 concentrations were higher in women reporting vomiting in the second trimester than in women reporting no pregnancy nausea or vomiting: 11,581 (10,977-12,219) (n=175) vs. 10,593 (10,066-11,147) (n=193) pg/mL, p=0.02). In women who took antiemetic drugs during pregnancy (n=11) the GDF15 levels were also raised 13,157 (10,558-16,394) pg/mL (p =0.04). Serum GFD15 concentrations were strongly positively correlated with hCG levels but were inversely correlated with maternal BMIs, a finding replicated in the NIPTeR Study. Conclusions: Week 15 serum GDF15 concentrations are positively associated with second trimester vomiting and maternal antiemetic use in pregnancy. Given GDF15’s site of action in the chemoreceptor trigger zone of the brainstem and its genetic associations with hyperemesis gravidarum, these data support the concept that GDF15 may be playing a pathogenic role in pregnancy-associated vomiting.

Objective: Loss of function FTO mutations significantly impact body composition in humans and mice, with Fto-deficient mice reported to resist the development of obesity in response to a high-fat diet (HFD). We aimed to further explore the interactions between FTO and HFD and determine if FTO can influence the adverse metabolic consequence of HFD. Methods: We studied mice deficient in FTO in two well validated models of leptin resistance (HFD feeding and central palmitate injection) to determine how Fto genotype may influence the action of leptin. Using transcriptomic analysis of hypothalamic tissue to identify relevant pathways affected by the loss of Fto, we combined data from co-immunoprecipitation, yeast 2-hybrid and luciferase reporter assays to identify mechanisms through which FTO can influence the development of leptin resistant states. Results: Mice deficient in Fto significantly increased their fat mass in response to HFD. Fto+/− and Fto−/− mice remained sensitive to the anorexigenic effects of leptin, both after exposure to a HFD or after acute central application of palmitate. Genes encoding components of the NFкB signalling pathway were down-regulated in the hypothalami of Fto-deficient mice following a HFD. When this pathway was reactivated in Fto-deficient mice with a single low central dose of TNFα, the mice became less sensitive to the effect of leptin. We identified a transcriptional coactivator of NFкB, TRIP4, as a binding partner of FTO and a molecule that is required for TRIP4 dependent transactivation of NFкB. Conclusions: Our study demonstrates that, independent of body weight, Fto influences the metabolic outcomes of a HFD through alteration of hypothalamic NFкB signalling. This supports the notion that pharmacological modulation of FTO activity might have the potential for therapeutic benefit in improving leptin sensitivity, in a manner that is influenced by the nutritional environment.

Objective: Disruption of the genes encoding either seipin or 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) causes severe congenital generalized lipodystrophy (CGL) in humans. However, the function of seipin in adipogenesis remains poorly defined. We demonstrated recently that seipin can bind the key adipogenic phosphatidic acid (PA) phosphatase lipin 1 and that seipin forms stable dodecamers. As AGPAT2 generates PA, the substrate for lipin 1, we investigated whether seipin might bind both enzymes of this lipid biosynthetic pathway, which is required for adipogenesis to occur. Methods: We employed co-immunoprecipitation and immunofluorescence methods to determine whether seipin can interact with AGPAT2 and the consequences of this in developing adipocytes. Atomic force microscopy was used to determine whether these interactions involved direct association of the proteins and to define the molecular architecture of these complexes. Results: Our data reveal that seipin can bind AGPAT2 during adipogenesis and that stabilizing this interaction during adipogenesis can increase the nuclear accumulation of PPARγ. Both AGPAT2 and lipin 1 can directly associate with seipin dodecamers, and a single seipin complex can simultaneously bind both AGPAT2 and lipin with a defined orientation. Conclusions: Our study provides the first direct molecular link between seipin and AGPAT2, two proteins whose disruption causes CGL. Moreover, it provides the first example of an interaction between seipin and another protein that causally influences a key aspect of adipogenesis. Together our data suggest that the critical role of seipin in adipogenesis may involve its capacity to juxtapose important regulators of this process in a multi-protein complex.

MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies.

BackgroundHigher circulating levels of the branched-chain amino acids (BCAAs; i.e., isoleucine, leucine, and valine) are strongly associated with higher type 2 diabetes risk, but it is not known whether this association is causal. We undertook large-scale human genetic analyses to address this question.Methods and findingsGenome-wide studies of BCAA levels in 16,596 individuals revealed five genomic regions associated at genome-wide levels of significance (p < 5 × 10-8). The strongest signal was 21 kb upstream of the PPM1K gene (beta in standard deviations [SDs] of leucine per allele = 0.08, p = 3.9 × 10-25), encoding an activator of the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) responsible for the rate-limiting step in BCAA catabolism. In another analysis, in up to 47,877 cases of type 2 diabetes and 267,694 controls, a genetically predicted difference of 1 SD in amino acid level was associated with an odds ratio for type 2 diabetes of 1.44 (95% CI 1.26-1.65, p = 9.5 × 10-8) for isoleucine, 1.85 (95% CI 1.41-2.42, p = 7.3 × 10-6) for leucine, and 1.54 (95% CI 1.28-1.84, p = 4.2 × 10-6) for valine. Estimates were highly consistent with those from prospective observational studies of the association between BCAA levels and incident type 2 diabetes in a meta-analysis of 1,992 cases and 4,319 non-cases. Metabolome-wide association analyses of BCAA-raising alleles revealed high specificity to the BCAA pathway and an accumulation of metabolites upstream of branched-chain alpha-ketoacid oxidation, consistent with reduced BCKD activity. Limitations of this study are that, while the association of genetic variants appeared highly specific, the possibility of pleiotropic associations cannot be entirely excluded. Similar to other complex phenotypes, genetic scores used in the study captured a limited proportion of the heritability in BCAA levels. Therefore, it is possible that only some of the mechanisms that increase BCAA levels or affect BCAA metabolism are implicated in type 2 diabetes.ConclusionsEvidence from this large-scale human genetic and metabolomic study is consistent with a causal role of BCAA metabolism in the aetiology of type 2 diabetes.

Summary Work on obesity is evolving, and obesity is a consequence of our evolutionary history. In the space of 50 years, we have become an obese species. The reasons why can be addressed at a number of different levels. These include separating between whether the primary cause lies on the food intake or energy expenditure side of the energy balance equation, and determining how genetic and environmental effects contribute to weight variation between individuals. Opinion on whether increased food intake or decreased energy expenditure drives the obesity epidemic is still divided, but recent evidence favours the idea that food intake, rather than altered expenditure, is most important. There is more of a consensus that genetics explains most (probably around 65%) of weight variation between individuals. Recent advances in genome-wide association studies have identified many polymorphisms that are linked to obesity, yet much of the genetic variance remains unexplained. Finding the causes of this unexplained variation will be an impetus of genetic and epigenetic research on obesity over the next decade. Many environmental factors – including gut microbiota, stress and endocrine disruptors – have been linked to the risk of developing obesity. A better understanding of gene-by-environment interactions will also be key to understanding obesity in the years to come.

The close correspondence between energy intake and expenditure over prolonged time periods, coupled with an apparent protection of the level of body adiposity in the face of perturbations of energy balance, has led to the idea that body fatness is regulated via mechanisms that control intake and energy expenditure. Two models have dominated the discussion of how this regulation might take place. The set point model is rooted in physiology, genetics and molecular biology, and suggests that there is an active feedback mechanism linking adipose tissue (stored energy) to intake and expenditure via a set point, presumably encoded in the brain. This model is consistent with many of the biological aspects of energy balance, but struggles to explain the many significant environmental and social influences on obesity, food intake and physical activity. More importantly, the set point model does not effectively explain the ‘obesity epidemic’ – the large increase in body weight and adiposity of a large proportion of individuals in many countries since the 1980s. An alternative model, called the settling point model, is based on the idea that there is passive feedback between the size of the body stores and aspects of expenditure. This model accommodates many of the social and environmental characteristics of energy balance, but struggles to explain some of the biological and genetic aspects. The shortcomings of these two models reflect their failure to address the gene-by-environment interactions that dominate the regulation of body weight. We discuss two additional models – the general intake model and the dual intervention point model – that address this issue and might offer better ways to understand how body fatness is controlled.

Background. Dogs are compelling models in which to study obesity since the condition shares many characteristics between humans and dogs. Differences in eating behaviour are recognised to contribute to obesity susceptibility in other species but this has not been systematically studied in dogs.Aim. To develop and validate an owner-reported measure of canine eating behaviour and owner or dog related factors which can alter the development of obesity. Further, to then test variation in food-motivation in dogs and its association with obesity and owner management.Methods. Owner interviews, a literature review and existing human appetite scales were used to identify relevant topics and generate items for the questionnaire. Following a pilot phase, a 75 item online questionnaire was distributed via social media. Responses from 302 dog/owner dyads were analysed and factor structure and descriptive statistics calculated. Results were compared with descriptions of dog behaviour and management from a subset of respondents during semi-structured interviews. The optimum questions were disseminated as a 34 item final questionnaire completed by 213 owners, with a subset of respondents repeating the questionnaire 3 weeks later to assess test–retest reliability.Results. Analysis of responses to the final questionnaire relating to 213 dog/owner dyads showed a coherent factor structure and good test–retest reliability. There were three dog factors (food responsiveness and satiety, lack of selectivity, Interest in food), four owner factors (owner motivation to control dog weight, owner intervention to control dog weight, restriction of human food, exercise taken) and two dog health factors (signs of gastrointestinal disease, current poor health). Eating behaviour differed between individuals and between breed groups. High scores on dog factors (high food-motivation) and low scores on owner factors (less rigorous control of diet/exercise) were associated with obesity. Owners of more highly food-motivated dogs exerted more control over their dogs’ food intake than those of less food-motivated dogs.Conclusions. The DORA questionnaire is a reliable and informative owner-reported measure of canine eating behaviour and health and management factors which can be associated with obesity development. The tool will be applicable to study of the canine obesity model and to clinical veterinarians. Results revealed eating behaviour to be similarly associated with obesity as exercise and owners giving titbits.

The gene encoding the melatonin-related receptor (GPR50) is highly expressed within hypothalamic nuclei concerned with the control of body weight and metabolism. We screened GPR50 for mutations in an obese cohort and identified an insertion of four amino acid residues (TTGH) at position 501, two common coding polymorphisms (T528A and V602I), and one noncoding polymorphism (C-16X2GPR50T). Single-nucleotide polymorphisms were then typed in 500 English Caucasian subjects, and associations were sought to intermediate obesity phenotypes. Although no association was seen with body mass index, carriers of two copies of the mutant allele at C-16X2GPR50T, Ins501Del, and A1582G had significantly higher fasting circulating triglyceride levels (P < 0.05). In a separate set of 585 subjects, the associations were replicated, with statistically significant effects of similar magnitude and direction. The association of C-16X2GPR50T with fasting triglycerides was highly significant (P < 0.001). In addition, a significant association between C-16X2GPR50T and circulating HDL levels was observed in the combined population, with C-16X2GPR50T carriers having significantly lower circulating HDL-cholesterol levels (1.39 mM) than wild-type subjects (1.47 mM) (P < 0.01). These findings suggest a previously unexpected role for this orphan receptor in the regulation of lipid metabolism that warrants further investigation.

The strongest BMI-associated GWAS locus in humans is the FTO gene. Rodent studies demonstrate a role for FTO in energy homeostasis and body composition. The phenotypes observed in loss of expression studies are complex with perinatal lethality, stunted growth from weaning, and significant alterations in body composition. Thus understanding how and where Fto regulates food intake, energy expenditure, and body composition is a challenge. To address this we generated a series of mice with distinct temporal and spatial loss of Fto expression. Global germline loss of Fto resulted in high perinatal lethality and a reduction in body length, fat mass, and lean mass. When ratio corrected for lean mass, mice had a significant increase in energy expenditure, but more appropriate multiple linear regression normalisation showed no difference in energy expenditure. Global deletion of Fto after the in utero and perinatal period, at 6 weeks of age, removed the high lethality of germline loss. However, there was a reduction in weight by 9 weeks, primarily as loss of lean mass. Over the subsequent 10 weeks, weight converged, driven by an increase in fat mass. There was a switch to a lower RER with no overall change in food intake or energy expenditure. To test if the phenotype can be explained by loss of Fto in the mediobasal hypothalamus, we sterotactically injected adeno-associated viral vectors encoding Cre recombinase to cause regional deletion. We observed a small reduction in food intake and weight gain with no effect on energy expenditure or body composition. Thus, although hypothalamic Fto can impact feeding, the effect of loss of Fto on body composition is brought about by its actions at sites elsewhere. Our data suggest that Fto may have a critical role in the control of lean mass, independent of its effect on food intake.

Sequence variants in the first intron of FTO are strongly associated with human obesity and human carriers of the risk alleles show evidence for increased appetite and food intake. Mice globally lacking Fto display a complex phenotype characterised by both increased energy expenditure and increased food intake. The site of action of FTO on energy balance is unclear. Fasting reduces levels of Fto mRNA in the arcuate nucleus (ARC) of the hypothalamus, a site where Fto expression is particularly high. In this study, we have extended this nutritional link by demonstrating that consumption of a high fat diet (45%) results in a 2.5 fold increase in Arc Fto expression. We have further explored the role of hypothalamic Fto in the control of food intake by using stereotactic injections coupled with AAV technology to bi-directionally modulate Fto expression. An over expression of Fto protein by 2.5-fold in the ARC results in a 14% decrease in average daily food intake in the first week. In contrast, knocking down Arc Fto expression by 40% increases food intake by 16%. mRNA levels of Agrp, Pomc and Npy, ARC-expressed genes classically associated with the control of food intake, were not affected by the manipulation of Fto expression. However, over expression of Fto resulted in a 4-fold increase in the mRNA levels of Stat3, a signalling molecule critical for leptin receptor signalling, suggesting a possible candidate for the mediation of Fto's actions. These data provide further support for the notion that FTO itself can influence key components of energy balance, and is therefore a strong candidate for the mediation of the robust association between FTO intronic variants and adiposity. Importantly, this provide the first indication that selective alteration of FTO levels in the hypothalamus can influence food intake, a finding consistent with the reported effects of FTO alleles on appetite and food intake in man.

The adipocyte-derived protein adiponectin is highly heritable and inversely associated with risk of type 2 diabetes mellitus (T2D) and coronary heart disease (CHD). We meta-analyzed 3 genome-wide association studies for circulating adiponectin levels (n = 8,531) and sought validation of the lead single nucleotide polymorphisms (SNPs) in 5 additional cohorts (n = 6,202). Five SNPs were genome-wide significant in their relationship with adiponectin (P< or =5x10(-8)). We then tested whether these 5 SNPs were associated with risk of T2D and CHD using a Bonferroni-corrected threshold of P< or =0.011 to declare statistical significance for these disease associations. SNPs at the adiponectin-encoding ADIPOQ locus demonstrated the strongest associations with adiponectin levels (P-combined = 9.2x10(-19) for lead SNP, rs266717, n = 14,733). A novel variant in the ARL15 (ADP-ribosylation factor-like 15) gene was associated with lower circulating levels of adiponectin (rs4311394-G, P-combined = 2.9x10(-8), n = 14,733). This same risk allele at ARL15 was also associated with a higher risk of CHD (odds ratio [OR] = 1.12, P = 8.5x10(-6), n = 22,421) more nominally, an increased risk of T2D (OR = 1.11, P = 3.2x10(-3), n = 10,128), and several metabolic traits. Expression studies in humans indicated that ARL15 is well-expressed in skeletal muscle. These findings identify a novel protein, ARL15, which influences circulating adiponectin levels and may impact upon CHD risk.

BACKGROUND:Expansion of a CAG repeat in the coding region of exon 1 in the ATXN2 gene located in human chromosome 12q24.1 causes the neurodegenerative disease spinocerebellar ataxia type 2 (SCA2). In contrast to other polyglutamine (polyQ) disorders, the SCA2 repeat is not highly polymorphic in central European (CEU) controls with Q22 representing 90% of alleles, and Q23 contributing between 5-7% of alleles. Recently, the ATXN2 CAG repeat has been identified as a target of adaptive selection in the CEU population. Mouse lines deficient for atxn2 develop marked hyperphagia and obesity raising the possibility that loss-of-function mutations in the ATXN2 gene may be related to energy balance in humans. Some linkage studies of obesity related phenotypes such as antipsychotic induced weight gain have reported significant lod scores on chromosome 12q24. We tested the hypothesis that rare loss-of-function ATXN2 variants cause obesity analogous to rare mutations in the leptin, leptin receptor and MC4R genes. METHODOLOGY/PRINCIPAL FINDINGS:We sequenced the coding region of ATXN2 including intron-exon boundaries in 92 severely obese children with a body mass index (BMI) >3.2 standard deviations above age- and gender-adjusted means. We confirmed five previously identified single nucleotide polymorphisms (SNPs) and three new SNPs resulting in two synonymous substitutions and one intronic polymorphism. Alleles encoding >Q22 were overrepresented in our sample of obese children and contributed 15% of alleles in children identified by their parents as white. SNP rs695872 closely flanking the CAG repeat showed a greatly increased frequency of C/C homozygotes and G/C heterozygotes compared with reported frequencies in the CEU population. CONCLUSIONS/SIGNIFICANCE:Although we did not identify variants leading to novel amino acid substitutions, nonsense or frameshift mutations, this study warrants further examination of variation in the ATXN2 gene in obesity and related phenotypes in a larger case-control study with emphasis on rs695872 and CAG repeat structure.

Stored glycogen is an important source of energy for skeletal muscle. Human genetic disorders primarily affecting skeletal muscle glycogen turnover are well-recognised, but rare. We previously reported that a frameshift/premature stop mutation in PPP1R3A, the gene encoding RGL, a key regulator of muscle glycogen metabolism, was present in 1.36% of participants from a population of white individuals in the UK. However, the functional implications of the mutation were not known. The objective of this study was to characterise the molecular and physiological consequences of this genetic variant.In this study we found a similar prevalence of the variant in an independent UK white population of 744 participants (1.46%) and, using in vivo (13)C magnetic resonance spectroscopy studies, demonstrate that human carriers (n = 6) of the variant have low basal (65% lower, p = 0.002) and postprandial muscle glycogen levels. Mice engineered to express the equivalent mutation had similarly decreased muscle glycogen levels (40% lower in heterozygous knock-in mice, p < 0.05). In muscle tissue from these mice, failure of the truncated mutant to bind glycogen and colocalize with glycogen synthase (GS) decreased GS and increased glycogen phosphorylase activity states, which account for the decreased glycogen content.Thus, PPP1R3A C1984DeltaAG (stop codon 668) is, to our knowledge, the first prevalent mutation described that directly impairs glycogen synthesis and decreases glycogen levels in human skeletal muscle. The fact that it is present in approximately 1 in 70 UK whites increases the potential biomedical relevance of these observations.

Type 2 diabetes is an increasingly common, serious metabolic disorder with a substantial inherited component. It is characterised by defects in both insulin secretion and action. Progress in identification of specific genetic variants predisposing to the disease has been limited. To complement ongoing positional cloning efforts, we have undertaken a large-scale candidate gene association study. We examined 152 SNPs in 71 candidate genes for association with diabetes status and related phenotypes in 2,134 Caucasians in a case-control study and an independent quantitative trait (QT) cohort in the United Kingdom. Polymorphisms in five of 15 genes (33%) encoding molecules known to primarily influence pancreatic beta-cell function-ABCC8 (sulphonylurea receptor), KCNJ11 (KIR6.2), SLC2A2 (GLUT2), HNF4A (HNF4alpha), and INS (insulin)-significantly altered disease risk, and in three genes, the risk allele, haplotype, or both had a biologically consistent effect on a relevant physiological trait in the QT study. We examined 35 genes predicted to have their major influence on insulin action, and three (9%)-INSR, PIK3R1, and SOS1-showed significant associations with diabetes. These results confirm the genetic complexity of Type 2 diabetes and provide evidence that common variants in genes influencing pancreatic beta-cell function may make a significant contribution to the inherited component of this disease. This study additionally demonstrates that the systematic examination of panels of biological candidate genes in large, well-characterised populations can be an effective complement to positional cloning approaches. The absence of large single-gene effects and the detection of multiple small effects accentuate the need for the study of larger populations in order to reliably identify the size of effect we now expect for complex diseases.