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1.  TAC3 and TACR3 Mutations in Familial Hypogonadotropic Hypogonadism Reveal a Key Role for Neurokinin B in the Central Control of Reproduction 
Nature genetics  2008;41(3):354-358.
The timely secretion of gonadal sex steroids is essential for the initiation of puberty, the post-pubertal maintenance of secondary sexual characteristics and the normal perinatal development of male external genitalia. Normal gonadal steroid production requires the actions of the pituitary-derived gonatrophins, LH and FSH. We report four human pedigrees with severe congenital gonadotrophin deficiency and pubertal failure in which all affected individuals are homozygous for loss-of-function mutations in TAC3 (encoding Neurokinin B) or its receptor TACR3 (encoding NK3R). Neurokinin B, a member of the substance P-related tachykinin family, is known to be highly expressed in hypothalamic neurons that also express kisspeptin1, a recently identified regulator of gonadotropin-releasing hormone secretion2. These findings implicate Neurokinin B as a critical central regulator of human gonadal function and suggest novel approaches to the pharmacological control of human reproduction and sex hormone-related diseases.
PMCID: PMC4312696  PMID: 19079066
2.  Hypogonadotropic Hypogonadism due to a Novel Missense Mutation in the First Extracellular Loop of the Neurokinin B Receptor 
The neurokinin B (NKB) receptor, encoded by TACR3, is widely expressed within the central nervous system, including hypothalamic nuclei involved in regulating GnRH release. We have recently reported two mutations in transmembrane segments of the receptor and a missense mutation in NKB in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH).
Patients and Methods
We sequenced the TACR3 gene in a family in which three siblings had nIHH. The novel mutant receptor thus identified was studied in a heterologous expression system using calcium flux as the functional readout.
All affected siblings were homozygous for the His148Leu mutation, in the first extracellular loop of the NKB receptor. The His148Leu mutant receptor exhibited profoundly impaired signaling in response to NKB (EC50 = 3 ± 0.1 nm and >5 μm for wild-type and His148Leu, respectively). The location of the mutation in an extracellular part of the receptor led us also to test whether senktide, a synthetic NKB analog, may retain ability to stimulate the mutant receptor. However, the signaling activity of the His148Leu receptor in response to senktide was also severely impaired (EC50 = 1 ± 1 nm for wild-type and no significant response of His148Leu to 10 μm).
Homozygosity for the TACR3 His148Leu mutation leads to failure of sexual maturation in humans, whereas signaling by the mutant receptor in vitro in response to either NKB or senktide is severely impaired. These observations further strengthen the link between NKB, the NKB receptor, and regulation of human reproductive function.
PMCID: PMC4306717  PMID: 19755480
3.  Role of the POZ Zinc Finger Transcription Factor FBI-1 in Human and Murine Adipogenesis 
The Journal of biological chemistry  2003;279(12):11711-11718.
Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2–4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.
PMCID: PMC4303998  PMID: 14701838
4.  The Wnt antagonist Dickkopf-1 and its receptors are coordinately regulated during early human adipogenesis 
Journal of cell science  2006;119(0 12):2613-2620.
Secretion of Wnts by adipose cells has an important role in the control of murine adipogenesis. We present the first evidence that a Wnt antagonist, Dickkopf 1 (Dkk1), is secreted by human preadipocytes and promotes adipogenesis. DKK1 mRNA increases six hours after onset of human adipogenesis and this is followed by an increase in Dkk1 protein. With further differentiation, the mRNA and protein levels progressively decline such that they are undetectable in mature adipocytes. The transient induction in DKK1 correlates with downregulation of cytoplasmic and nuclear β-catenin levels, this being a surrogate marker of canonical Wnt signalling, and Wnt/β-catenin transcriptional activity. In addition, constitutive expression of Dkk1 in 3T3-L1 preadipocytes promotes their differentiation, further supporting the functional significance of increased Dkk1 levels during human adipogenesis. Concomitant downregulation of the Dkk1 receptors LRP5 and LRP6 is likely to potentiate the ability of Dkk1 to inhibit Wnt signalling and promote differentiation. Notably, Dkk1 is not expressed in primary murine preadipocytes or cell lines. The involvement of Dkk1 in human but not murine adipogenesis indicates that inter-species differences exist in the molecular control of this process. Given the public health importance of disorders of adipose mass, further knowledge of the pathways involved specifically in human adipocyte differentiation might ultimately be of clinical relevance.
PMCID: PMC4304001  PMID: 16763196
Adipocyte; Adipogenesis; Wnt; Dickkopf 1; LRP5; Human
5.  The Peroxisome Proliferator-activated Receptor-γ Regulates Murine Pyruvate Carboxylase Gene Expression in Vivo and in Vitro* 
The Journal of biological chemistry  2005;280(29):27466-27476.
Pyruvate carboxylase (PC) plays a crucial role in various metabolic pathways, including gluconeogenesis, lipogenesis, and glucose-induced insulin secretion. Here we showed for the first time that the PC gene is transcriptionally regulated by peroxisome proliferator-activated receptor-γ (PPARγ) in vitro and in vivo in white and brown adipose tissue. PC mRNA and protein are markedly increased during differentiation of 3T3-L1 cells and HIB-1B, in parallel with the expression of the adipogenic transcription factors, CCAAT-enhancer binding protein α, PPARγ1, and PPARγ2. Tumor necrosis factor-α, a cytokine that blocks differentiation of 3T3-L1 cells, suppressed PC expression. Co-transfection studies in 3T3-L1 preadipocytes or HEK293T cells with a 2.3-kb promoter fragment of mouse PC gene linked to a luciferase reporter construct and with plasmids overexpressing retinoid X receptor α/PPARγ1 or retinoid X receptor α/PPARγ2 showed a 6–8-fold increase above the basal promoter activity. Furthermore, treatment of these transfected cells with the PPARγ agonist doubled the promoter activity. Mutation of the putative PPAR-response element-(−386/−374) of this 2.3-kb PC promoter fragment abolished the PPARγ response. Gel shift and chromatin immunoprecipitation assays demonstrated that endogenous PPARγ binds to this functional PPAR-response element of the PC promoter. Mice with targeted disruption of the PPARγ2 gene displayed ~50–60% reduction of PC mRNA and protein in white adipose tissue. Similarly, in brown adipose tissue of PPARγ2-deficient mice subjected to cold exposure, PC mRNA was 40% lower than that of wild type mice. Impaired in vitro differentiation of white adipocytes of PPARγ2 knock-out mice was also associated with a marked reduction of PC mRNA. Our findings identified PC as a PPARγ-regulated gene and suggested a role for PPARγ regulating intermediary metabolism.
PMCID: PMC4304003  PMID: 15917242
6.  The Link Between Nutritional Status and Insulin Sensitivity Is Dependent on the Adipocyte-Specific Peroxisome Proliferator–Activated Receptor-γ2 Isoform 
Diabetes  2005;54(6):1706-1716.
The nuclear receptor peroxisome proliferator–activated receptor-γ (PPARγ) is critically required for adipogenesis. PPARγ exists as two isoforms, γ1 and γ2. PPARγ2 is the more potent adipogenic isoform in vitro and is normally restricted to adipose tissues, where it is regulated more by nutritional state than PPARγ1. To elucidate the relevance of the PPARγ2 in vivo, we generated a mouse model in which the PPARγ2 isoform was specifically disrupted. Despite similar weight, body composition, food intake, energy expenditure, and adipose tissue morphology, male mice lacking the γ2 isoform were more insulin resistant than wild-type animals when fed a regular diet. These results indicate that insulin resistance associated with ablation of PPARγ2 is not the result of lipodystrophy and suggests a specific role for PPARγ2 in maintaining insulin sensitivity independently of its effects on adipogenesis. Furthermore, PPARγ2 knockout mice fed a high-fat diet did not become more insulin resistant than those on a normal diet, despite a marked increase in their mean adipocyte cell size. These findings suggest that PPARγ2 is required for the maintenance of normal insulin sensitivity in mice but also raises the intriguing notion that PPARγ2 may be necessary for the adverse effects of a high-fat diet on carbohydrate metabolism.
PMCID: PMC4304004  PMID: 15919792
7.  Pharmacological Inhibition of Glucosylceramide Synthase Enhances Insulin Sensitivity 
Diabetes  2007;56(5):1341-1349.
A growing body of evidence implicates ceramide and/or its glycosphingolipid metabolites in the pathogenesis of insulin resistance. We have developed a highly specific small molecule inhibitor of glucosylceramide synthase, an enzyme that catalyzes a necessary step in the conversion of ceramide to glycosphingolipids. In cultured 3T3-L1 adipocytes, the iminosugar derivative N-(5′-adamantane-1′-yl-methoxy)-pentyl-1-deoxynojirimycin (AMP-DNM) counteracted tumor necrosis factor-α-induced abnormalities in glycosphingo-lipid concentrations and concomitantly reversed abnormalities in insulin signal transduction. When administered to mice and rats, AMP-DNM significantly reduced glycosphin-golipid but not ceramide concentrations in various tissues. Treatment of ob/ob mice with AMP-DNM normalized their elevated tissue glucosylceramide levels, markedly lowered circulating glucose levels, improved oral glucose tolerance, reduced A1C, and improved insulin sensitivity in muscle and liver. Similarly beneficial metabolic effects were seen in high fat-fed mice and ZDF rats. These findings provide further evidence that glycosphingolipid metabolites of ceramide may be involved in mediating the link between obesity and insulin resistance and that interference with glycosphingolipid biosynthesis might present a novel approach to the therapy of states of impaired insulin action such as type 2 diabetes.
PMCID: PMC4298701  PMID: 17287460
8.  Leptin Mediates the Increase in Blood Pressure Associated with Obesity 
Cell  2014;159(6):1404-1416.
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.
Graphical Abstract
•Leptin is the link between obesity and increased blood pressure•Leptin acts through the dorsomedial hypothalamus to increase blood pressure•Blockade of leptin signaling reduces blood pressure in obese mice•Humans with defects in leptin signaling are protected from obesity hypertension
Leptin is found to be the link between obesity and increased blood pressure. Blocking leptin action reduces blood pressure in obese mice with clinical studies in humans, suggesting that defects in leptin signaling may protect against hypertension associated with obesity.
PMCID: PMC4259491  PMID: 25480301
9.  Fat mass and obesity-related (FTO) shuttles between the nucleus and cytoplasm 
Bioscience Reports  2014;34(5):e00144.
SNPs (single nucleotide polymorphisms) on a chromosome 16 locus encompassing FTO, as well as IRX3, 5, 6, FTM and FTL are robustly associated with human obesity. FTO catalyses the Fe(II)- and 2OG-dependent demethylation of RNA and is an AA (amino acid) sensor that couples AA levels to mTORC1 (mammalian target of rapamycin complex 1) signalling, thereby playing a key role in regulating growth and translation. However, the cellular compartment in which FTO primarily resides to perform its biochemical role is unclear. Here, we undertake live cell imaging of GFP (green fluorescent protein)-FTO, and demonstrate that FTO resides in both the nucleus and cytoplasm. We show using ‘FLIP’ (fluorescence loss in photobleaching) that a mobile FTO fraction shuttles between both compartments. We performed a proteomic study and identified XPO2 (Exportin 2), one of a family of proteins that mediates the shuttling of proteins between the nucleus and the cytoplasm, as a binding partner of FTO. Finally, using deletion studies, we show that the N-terminus of FTO is required for its ability to shuttle between the nucleus and cytoplasm. In conclusion, FTO is present in both the nucleus and cytoplasm, with a mobile fraction that shuttles between both cellular compartments, possibly by interaction with XPO2.
Exportin interacts with FTO and this interaction might be involved in the nucelocytoplasmic shuttling of FTO in the cell.
PMCID: PMC4206862  PMID: 25242086
Exportin 2; fat mass and obesity-associated protein (FTO); nucelocytoplasmic shuttling; obesity; AA, amino acid; co-IP, co-immunoprecipitation; FLIP, fluorescence loss in photobleaching; GFP, green fluorescent protein; HEK-293 cells, human embryonic kidney cells; IP, immunoprecipitation; MEF, mouse embryonic fibroblast; mTOR, mammalian target of rapamycin; SNP, single nucleotide polymorphism; WT, wild-type; XPO2, Exportin 2
10.  Hypomorphism in human NSMCE2 linked to primordial dwarfism and insulin resistance 
The Journal of Clinical Investigation  2014;124(9):4028-4038.
Structural maintenance of chromosomes (SMC) complexes are essential for maintaining chromatin structure and regulating gene expression. Two the three known SMC complexes, cohesin and condensin, are important for sister chromatid cohesion and condensation, respectively; however, the function of the third complex, SMC5–6, which includes the E3 SUMO-ligase NSMCE2 (also widely known as MMS21) is less clear. Here, we characterized 2 patients with primordial dwarfism, extreme insulin resistance, and gonadal failure and identified compound heterozygous frameshift mutations in NSMCE2. Both mutations reduced NSMCE2 expression in patient cells. Primary cells from one patient showed increased micronucleus and nucleoplasmic bridge formation, delayed recovery of DNA synthesis, and reduced formation of foci containing Bloom syndrome helicase (BLM) after hydroxyurea-induced replication fork stalling. These nuclear abnormalities in patient dermal fibroblast were restored by expression of WT NSMCE2, but not a mutant form lacking SUMO-ligase activity. Furthermore, in zebrafish, knockdown of the NSMCE2 ortholog produced dwarfism, which was ameliorated by reexpression of WT, but not SUMO-ligase–deficient NSMCE. Collectively, these findings support a role for NSMCE2 in recovery from DNA damage and raise the possibility that loss of its function produces dwarfism through reduced tolerance of replicative stress.
PMCID: PMC4151221  PMID: 25105364
11.  Obesity-Associated Melanocortin-4 Receptor Mutations Are Associated With Changes in the Brain Response to Food Cues 
Mutations in the melanocortin-4 receptor (MC4R) represent the commonest genetic form of obesity and are associated with hyperphagia.
The aim of this study was to investigate whether melanocortin signaling modulates anticipatory food reward by studying the brain activation response to food cues in individuals with MC4R mutations.
Design/Setting/Participants/Main Outcome Measure:
We used functional magnetic resonance imaging to measure blood oxygen level-dependent responses to images of highly palatable, appetizing foods, bland foods, and non-food objects in eight obese individuals with MC4R mutations, 10 equally obese controls, and eight lean controls with normal MC4R genotypes. Based on previous evidence, we performed a region-of-interest analysis centered on the caudate/putamen (dorsal striatum) and ventral striatum.
Compared to non-foods, appetizing foods were associated with activation in the dorsal and ventral striatum in lean controls and in MC4R-deficient individuals. Surprisingly, we observed reduced activation of the dorsal and ventral striatum in obese controls relative to MC4R-deficient patients and lean controls. There were no group differences for the contrast of disgusting foods with bland foods or non-foods, suggesting that the effects observed in response to appetizing foods were not related to arousal.
We identified differences in the striatal response to food cues between two groups of obese individuals, those with and those without MC4R mutations. These findings are consistent with a role for central melanocortinergic circuits in the neural response to visual food cues.
PMCID: PMC4258603  PMID: 25062455
13.  Functional Characterization of Obesity-Associated Variants Involving the α and β Isoforms of Human SH2B1 
Endocrinology  2014;155(9):3219-3226.
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.
PMCID: PMC4138566  PMID: 24971614
14.  Knockdown of Diacylglycerol Kinase Delta Inhibits Adipocyte Differentiation and Alters Lipid Synthesis 
Obesity (Silver Spring, Md.)  2013;21(9):1823-1829.
Decreased expression of diacylglycerol kinase delta (DGKδ) has been linked to insulin resistance in humans and mice and is abundantly expressed in adipose tissue. We therefore examined its role in adipogenesis.
Design and Methods
We generated 3T3-L1 preadipocytes in which DGKδ expression had been knocked down and determined the effect of this on adipogenesis. We also performed lipidomic analysis to determine levels of the DGKδ product phosphatidic acid (PA), its substrate diacylglycerol (DAG) and triglyceride (TG).
Inhibiting DGKδ expression prevents adipogenesis. DGKδ knockdown in differentiating adipocytes blunted the increase in total levels of PA and DAG but did not affect the early rise in TG levels. DAG or PA species acting as TG precursors were only modestly reduced by DGKδ knockdown which significantly impaired the accumulation of DAG or PA species implicated in intracellular signaling. We also observed stimulation of the DAG activated kinase PKCδ in DGKδ knockdown cells, despite no increase in detectable species of DAG.
DGKδ is a novel regulator of adipogenesis and phosphorylates a quantitatively small pool of signaling DAG important for differentiation and indirectly affects overall levels of signaling DAG and PA species distinct from those acting as precursors for TG synthesis.
PMCID: PMC3763967  PMID: 23703849
Diacylglycerol; adipogenesis; lipids; phosphatidic acid; DGKδ
15.  An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy 
Nature genetics  2013;45(8):947-950.
DNA polymerase delta, whose catalytic subunit is encoded by POLD1, is responsible for lagging strand DNA synthesis during DNA replication1. It achieves this with high fidelity due to its intrinsic 3′ to 5′ exonuclease activity, which confers proofreading ability. Missense mutations in the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancer2. Here we report a recurring heterozygous single amino acid deletion at the polymerase active site of POLD1 that abolishes DNA polymerase activity but only mildly impairs 3′ to 5′ exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This suggests that perturbation of function of the ubiquitously expressed POLD1 polymerase has surprisingly tissue-specific effects in man, and argues for an important role for POLD1 function in adipose tissue homeostasis.
PMCID: PMC3785143  PMID: 23770608
16.  Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia 
The Journal of Experimental Medicine  2013;210(6):1137-1151.
Ablation of stromal cells expressing fibroblast activation protein-α (FAP) results in cachexia and anemia, and loss of these cells is seen in transplantable tumor models.
Fibroblast activation protein-α (FAP) identifies stromal cells of mesenchymal origin in human cancers and chronic inflammatory lesions. In mouse models of cancer, they have been shown to be immune suppressive, but studies of their occurrence and function in normal tissues have been limited. With a transgenic mouse line permitting the bioluminescent imaging of FAP+ cells, we find that they reside in most tissues of the adult mouse. FAP+ cells from three sites, skeletal muscle, adipose tissue, and pancreas, have highly similar transcriptomes, suggesting a shared lineage. FAP+ cells of skeletal muscle are the major local source of follistatin, and in bone marrow they express Cxcl12 and KitL. Experimental ablation of these cells causes loss of muscle mass and a reduction of B-lymphopoiesis and erythropoiesis, revealing their essential functions in maintaining normal muscle mass and hematopoiesis, respectively. Remarkably, these cells are altered at these sites in transplantable and spontaneous mouse models of cancer-induced cachexia and anemia. Thus, the FAP+ stromal cell may have roles in two adverse consequences of cancer: their acquisition by tumors may cause failure of immunosurveillance, and their alteration in normal tissues contributes to the paraneoplastic syndromes of cachexia and anemia.
PMCID: PMC3674708  PMID: 23712428
17.  Leptin Regulates Striatal Regions and Human Eating Behavior 
Science (New York, N.Y.)  2007;317(5843):10.1126/science.1144599.
PMCID: PMC3838941  PMID: 17690262
18.  KSR2 Mutations Are Associated with Obesity, Insulin Resistance, and Impaired Cellular Fuel Oxidation 
Cell  2013;155(4):765-777.
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-MEK-ERK 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.
Graphical Abstract
•Mutations in KSR2 are associated with obesity in humans•Mutations affect ERK signaling and impair the oxidation of glucose and fatty acids•Patients display hyperphagia, insulin resistance, and a reduced basal metabolic rate•KSR2 is an important regulator of energy intake and expenditure in humans
Mutations in KSR2, a scaffolding protein involved in multiple signaling pathways, lead to severe metabolic alterations that cause early onset obesity in humans.
PMCID: PMC3898740  PMID: 24209692
19.  Loss of Function of the Melanocortin 2 Receptor Accessory Protein 2 Is Associated with Mammalian Obesity 
Science (New York, N.Y.)  2013;341(6143):275-278.
Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro. To investigate the physiological role of brain-expressed Melanocortin 2 Receptor Accessory Protein 2 (MRAP2), we characterized mice with whole body and brain-specific targeted deletion of Mrap2, both of which develop severe obesity at a young age. Mrap2 interacts directly with Melanocortin 4 Receptor (Mc4r), a protein previously implicated in mammalian obesity, and it enhances Mc4r-mediated generation of the second messenger cyclic AMP, suggesting that alterations in Mc4r signaling may be one mechanism underlying the association between Mrap2 disruption and obesity. In a study of humans with severe, early-onset obesity, we found four rare, potentially pathogenic genetic variants in MRAP2, suggesting that the gene may also contribute to body weight regulation in humans.
PMCID: PMC3788688  PMID: 23869016
20.  Mapping cis- and trans-regulatory effects across multiple tissues in twins 
Nature genetics  2012;44(10):1084-1089.
Sequence-based variation in gene expression is a key driver of disease risk. Common variants regulating expression in cis have been mapped in many eQTL studies typically in single tissues from unrelated individuals. Here, we present a comprehensive analysis of gene expression across multiple tissues conducted in a large set of mono- and dizygotic twins that allows systematic dissection of genetic (cis and trans) and non-genetic effects on gene expression. Using identity-by-descent estimates, we show that at least 40% of the total heritable cis-effect on expression cannot be accounted for by common cis-variants, a finding which exposes the contribution of low frequency and rare regulatory variants with respect to both transcriptional regulation and complex trait susceptibility. We show that a substantial proportion of gene expression heritability is trans to the structural gene and identify several replicating trans-variants which act predominantly in a tissue-restricted manner and may regulate the transcription of many genes.
PMCID: PMC3784328  PMID: 22941192
21.  Perilipin Deficiency and Autosomal Dominant Partial Lipodystrophy 
The New England journal of medicine  2011;364(8):740-748.
Perilipin is the most abundant adipocyte-specific protein that coats lipid droplets, and it is required for optimal lipid incorporation and release from the droplet. We identified two heterozygous frameshift mutations in the perilipin gene (PLIN1) in three families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes. Subcutaneous fat from the patients was characterized by smaller-than-normal adipocytes, macrophage infiltration, and fibrosis. In contrast to wild-type perilipin, mutant forms of the protein failed to increase triglyceride accumulation when expressed heterologously in preadipocytes. These findings define a novel dominant form of inherited lipodystrophy and highlight the serious metabolic consequences of a primary defect in the formation of lipid droplets in adipose tissue.
PMCID: PMC3773916  PMID: 21345103
22.  Severe obesity and diabetes insipidus in a patient with PCSK1 deficiency☆ 
Molecular Genetics and Metabolism  2013;110(1-2):191-194.
Non-synonymous mutations affecting both alleles of PCSK1 (proprotein convertase 1/3) are associated with obesity and impaired prohormone processing. We report a proband who was compound heterozygous for a maternally inherited frameshift mutation and a paternally inherited 474kb deletion that encompasses PCSK1, representing a novel genetic mechanism underlying this phenotype. Although pro-vasopressin is not a known physiological substrate of PCSK1, the development of central diabetes insipidus in this proband suggests that PCSK1 deficiency can be associated with impaired osmoregulation.
•We identify a fourth patient with PCSK1 deficiency.•The null phenotype of PCSK1 deficiency includes obesity and neuroendocrine abnormalities.•We extend the clinical phenotype to include central diabetes insipidus, as well as severe obesity and impaired prohormone processing.
PMCID: PMC3759845  PMID: 23800642
Obesity; Prohormones
23.  Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms 
Diabetologia  2013;56(11):2498-2506.
In humans, disruption of the gene BSCL2, encoding the protein seipin, causes congenital generalised lipodystrophy (CGL) with severe insulin resistance and dyslipidaemia. While the causative gene has been known for over a decade, the molecular functions of seipin are only now being uncovered. Most pathogenic mutations in BSCL2 represent substantial disruptions including significant deletions and frameshifts. However, several more subtle mutations have been reported that cause premature stop codons or single amino acid substitutions. Here we have examined these mutant forms of seipin to gain insight into how they may cause CGL.
We generated constructs expressing mutant seipin proteins and determined their expression and localisation. We also assessed their capacity to recruit the key adipogenic phosphatidic acid phosphatase lipin 1, a recently identified molecular role of seipin in developing adipocytes. Finally, we used atomic force microscopy to define the oligomeric structure of seipin and to determine whether this is affected by the mutations.
We show that the R275X mutant of seipin is not expressed in pre-adipocytes. While the other premature stop mutant forms fail to bind lipin 1 appropriately, the point mutants T78A, L91P and A212P all retain this capacity. We demonstrate that wild-type human seipin forms oligomers of 12 subunits in a circular configuration but that the L91P and A212P mutants of seipin do not.
Our study represents the most comprehensive analysis so far of mutants of seipin causing lipodystrophy and reveals several different molecular mechanisms by which these mutations may cause disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-013-3029-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
PMCID: PMC3824349  PMID: 23989774
Adipogenesis; Adipose tissue; BSCL2; Lipin; Lipodystrophy; Seipin
25.  Rare variants in single-minded 1 (SIM1) are associated with severe obesity  
The Journal of Clinical Investigation  2013;123(7):3042-3050.
Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.
PMCID: PMC3696558  PMID: 23778139

Results 1-25 (101)