Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

To date, genome-wide association studies (GWAS) have identified at least 32 novel loci for obesity and body mass-related traits. However, the causal genetic variant and molecular mechanisms of specific susceptibility genes in relation to obesity are yet to be fully confirmed and characterised. Here, we examined whether the candidate gene NEGR1 encoding the neuronal growth regulator 1, also termed neurotractin or Kilon, accounts for the obesity association. To characterise the function of NEGR1 for body weight control in vivo, we generated two novel mutant mouse lines, including a constitutive NEGR1-deficient mouse line as well as an ENU-mutagenised line carrying a loss-of-function mutation (Negr1-I87N) and performed metabolic phenotypic analyses. Ablation of NEGR1 results in a small but steady reduction of body mass in both mutant lines, accompanied with a small reduction in body length in the Negr1-I87N mutants. Magnetic resonance scanning reveals that the reduction of body mass in Negr1-I87N mice is due to a reduced proportion of lean mass. Negr1-I87N mutants display reduced food intake and physical activity while normalised energy expenditure remains unchanged. Expression analyses confirmed the brain-specific distribution of NEGR1 including strong expression in the hypothalamus. In vitro assays show that NEGR1 promotes cell-cell adhesion and neurite growth of hypothalamic neurons. Our results indicate a role of NEGR1 in the control of body weight and food intake. This study provides evidence that supports the link of the GWAS candidate gene NEGR1 with body weight control.

[1]  A. Lodge,et al.  Identification and Characterization of CEPU-Se—A Secreted Isoform of the IgLON Family Protein, CEPU-1 , 2001, Molecular and Cellular Neuroscience.

[2]  T. Brümmendorf,et al.  Neurotractin/kilon promotes neurite outgrowth and is expressed on reactive astrocytes after entorhinal cortex lesion , 2005, Molecular and Cellular Neuroscience.

[3]  Tanya M. Teslovich,et al.  Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index , 2010 .

[4]  C. Larsson,et al.  The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas. , 2003, Cancer cell.

[5]  C. Wijmenga,et al.  Obesity genes identified in genome-wide association studies are associated with adiposity measures and potentially with nutrient-specific food preference. , 2009, The American journal of clinical nutrition.

[6]  Steve D. M. Brown,et al.  A gene-driven ENU-based approach to generating an allelic series in any gene , 2004, Mammalian Genome.

[7]  J. Salzer,et al.  Neurotrimin Mediates Bifunctional Effects on Neurite Outgrowth via Homophilic and Heterophilic Interactions , 1998, The Journal of Neuroscience.

[8]  U. Ekelund,et al.  Obesity-susceptibility loci have a limited influence on birth weight: a meta-analysis of up to 28,219 individuals. , 2011, The American journal of clinical nutrition.

[9]  J. Chan,et al.  Implication of genetic variants near NEGR1, SEC16B, TMEM18, ETV5/DGKG, GNPDA2, LIN7C/BDNF, MTCH2, BCDIN3D/FAIM2, SH2B1, FTO, MC4R, and KCTD15 with obesity and type 2 diabetes in 7705 Chinese. , 2010, The Journal of clinical endocrinology and metabolism.

[10]  S. Maekawa,et al.  Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family* , 1999, The Journal of Biological Chemistry.

[11]  Christian Gieger,et al.  Six new loci associated with body mass index highlight a neuronal influence on body weight regulation , 2009, Nature Genetics.

[12]  P. Levitt,et al.  Characterization of the genomic structure of the mouse limbic system-associated membrane protein (Lsamp) gene. , 2004, Genomics.

[13]  K. Rajewsky,et al.  Generation and analysis of interleukin-4 deficient mice. , 1991, Science.

[14]  S. Maekawa,et al.  OBCAM, an immunoglobulin superfamily cell adhesion molecule, regulates morphology and proliferation of cerebral astrocytes , 2010, Journal of neurochemistry.

[15]  S. Woods,et al.  Central nervous system control of food intake , 2000, Nature.

[16]  P. Diggle.  Analysis of Longitudinal Data , 1995 .

[17]  T. Tabira,et al.  Neuroblastoma × spinal cord (NSC) hybrid cell lines resemble developing motor neurons , 1992, Developmental dynamics : an official publication of the American Association of Anatomists.

[18]  P. Bates,et al.  OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer , 2003, Nature Genetics.

[19]  Roger D. Cox,et al.  A Mouse Model for the Metabolic Effects of the Human Fat Mass and Obesity Associated FTO Gene , 2009, PLoS genetics.

[20]  Michael W. Schwartz,et al.  Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons , 1998, Nature Neuroscience.

[21]  Nicholette D. Palmer,et al.  Implication of European-derived adiposity loci in African Americans , 2012, International Journal of Obesity.

[22]  P. Levitt,et al.  Genetic deletion of Lsamp causes exaggerated behavioral activation in novel environments , 2007, Behavioural Brain Research.

[23]  Friedrich Buck,et al.  Neurotractin, A Novel Neurite Outgrowth-promoting Ig-like Protein that Interacts with CEPU-1 and LAMP , 1999, The Journal of cell biology.

[24]  S. Maekawa,et al.  IgLON cell adhesion molecule Kilon is a crucial modulator for synapse number in hippocampal neurons , 2008, Brain Research.

[25]  P. Warnke,et al.  Expression of cellular adhesion molecule ‘OPCML’ is down‐regulated in gliomas and other brain tumours , 2007, Neuropathology and applied neurobiology.

[26]  Y. Durocher,et al.  High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. , 2002, Nucleic acids research.

[27]  Pengxiang She,et al.  Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle. , 2007, Cell metabolism.

[28]  Graham J. Wills,et al.  Introduction to graphical modelling , 1995 .

[29]  S. Maekawa,et al.  Biochemical and ultrastructural analyses of iglon cell adhesion molecules, kilon and obcam in the rat brain , 2003, Neuroscience.

[30]  Michael I. Jordan.  Graphical Models , 1998 .

[31]  Paul W. Franks,et al.  Replication and extension of genome-wide association study results for obesity in 4923 adults from northern Sweden , 2009, Human molecular genetics.

[32]  A. Lodge,et al.  Co-localisation, heterophilic interactions and regulated expression of IgLON family proteins in the chick nervous system. , 2000, Brain research. Molecular brain research.

[33]  S. Kõks,et al.  Lower anxiety and a decrease in agonistic behaviour in Lsamp-deficient mice , 2011, Behavioural Brain Research.

[34]  H. Wichmann,et al.  Genes and lifestyle factors in obesity: results from 12 462 subjects from MONICA/KORA , 2010, International Journal of Obesity.

[35]  J. Ott,et al.  Neuronal Genes for Subcutaneous Fat Thickness in Human and Pig Are Identified by Local Genomic Sequencing and Combined SNP Association Study , 2011, PloS one.

[36]  H. Gabra,et al.  The IgLON Family in Epithelial Ovarian Cancer: Expression Profiles and Clinicopathologic Correlates , 2005, Clinical Cancer Research.

[37]  Young-Bum Kim,et al.  Increased Energy Expenditure, Decreased Adiposity, and Tissue-Specific Insulin Sensitivity in Protein-Tyrosine Phosphatase 1B-Deficient Mice , 2000, Molecular and Cellular Biology.

[38]  M. Frotscher,et al.  Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling , 2006, Journal of neurochemistry.

[39]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[40]  Yusuke Nakamura,et al.  Association between obesity and polymorphisms in SEC16B, TMEM18, GNPDA2, BDNF, FAIM2 and MC4R in a Japanese population , 2009, Journal of Human Genetics.

[41]  N. Drouot,et al.  Pathomechanistic characterization of two exonic L1CAM variants located in trans in an obligate carrier of X-linked hydrocephalus , 2012, neurogenetics.

[42]  S. Ring,et al.  Genetic Markers of Adult Obesity Risk Are Associated with Greater Early Infancy Weight Gain and Growth , 2010, PLoS medicine.

[43]  M. Frotscher,et al.  L1 syndrome mutations impair neuronal L1 function at different levels by divergent mechanisms , 2010, Neurobiology of Disease.

[44]  O. Hobert,et al.  New insights into the diversity and function of neuronal immunoglobulin superfamily molecules. , 2003, Annual review of neuroscience.

[45]  D. Hillman,et al.  Complementary expression and heterophilic interactions between IgLON family members neurotrimin and LAMP. , 2002, Journal of neurobiology.

[46]  D. Bates,et al.  Mixed-Effects Models in S and S-PLUS , 2001 .

[47]  Joseph T. Glessner,et al.  The Role of Obesity‐associated Loci Identified in Genome‐wide Association Studies in the Determination of Pediatric BMI , 2009, Obesity.

[48]  Steve D. M. Brown,et al.  Novel phenotypes identified by plasma biochemical screening in the mouse , 2002, Mammalian Genome.

[49]  D. Moss,et al.  DIgLONs inhibit initiation of neurite outgrowth from forebrain neurons via an IgLON-containing receptor complex , 2011, Brain Research.

[50]  Ellen Kampman,et al.  Genome-wide association yields new sequence variants at seven loci that associate with measures of obesity , 2009, Nature Genetics.

[51]  Naoki Nitta,et al.  Reelin Deficiency and Displacement of Mature Neurons, But Not Neurogenesis, Underlie the Formation of Granule Cell Dispersion in the Epileptic Hippocampus , 2006, The Journal of Neuroscience.

[52]  C. Lopez,et al.  Progressive Motor Neuronopathy: A Critical Role of the Tubulin Chaperone TBCE in Axonal Tubulin Routing from the Golgi Apparatus , 2007, The Journal of Neuroscience.

[53]  S. Kenwrick,et al.  Disease-associated mutations in L1 CAM interfere with ligand interactions and cell-surface expression. , 2002, Human molecular genetics.

[54]  E. Weeber,et al.  Loss of Limbic System-Associated Membrane Protein Leads to Reduced Hippocampal Mineralocorticoid Receptor Expression, Impaired Synaptic Plasticity, and Spatial Memory Deficit , 2010, Biological Psychiatry.