ENU Mutagenesis Identifies Mice with Morbid Obesity and Severe Hyperinsulinemia Caused by a Novel Mutation in Leptin

Abstract

BackgroundObesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals.Methodology/Principal FindingsTo better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin145E/145E mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin145E/145E mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin145E/145E mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region.Conclusions/SignificanceThus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of human obesity syndrome.

Approach to assessing determinants of glucose homeostasis in the conscious mouse

“…Mouse lines can then be established according to a phenotypic criteria, such as hyperglycemia, thereby revealing a mutation as cause for the phenotype. ENU mutations have led to the identification of novel alleles in genes already known to be involved in glucose homeostasis including glucokinase (Inoue et al 2004; van Burck et al 2010) and leptin (Hong et al 2010; Osborn et al 2010) as well as genes previously unrelated to glucose metabolism, such as the gene encoding the histone methyltransferase Mll2 (Goldsworthy et al 2013). ENU-induced mutagenesis is a strong method in that it can generate a wide array of subtle phenotypes and the resulting lines may more closely recapitulate the human phenotype compared to engineered knock-out models.…”

Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel, TRPV5

“…To overcome these difficulties and facilitate the identification of genetic abnormalities causing hypercalciuric nephrolithiasis, we embarked on studies to establish mouse models generated using N -ethyl- N -nitrosourea (ENU), a chemical mutagen that causes point mutations by alkylation of nucleic acids leading to mispairing and subsequent single base substitutions during DNA replication [18]. ENU mouse mutants, which can be associated with loss-of-function, hypomorphic, hypermorphic or dominant-negative effects [18], have been successfully derived for metabolic and renal disorders including a mouse model with obesity and hyperinsulinaemia caused by a V145E substitution in the leptin gene [19], and a mouse model for renal failure due to a C277S substitution in aquaporin-11 [20]. We now report the identification of an ENU-induced mouse mutant model for autosomal dominant hypercalciuria, HCALC1, due to mutation of the transient receptor potential cation channel, subfamily V, member 5 ( Trpv5 ) gene.…”

Genome-Wide Mouse Mutagenesis Reveals CD45-Mediated T Cell Function as Critical in Protective Immunity to HSV-1

“…Furthermore, positional cloning of mutant genes is now facilitated by recent progress in the field of genomics, such as exome sequencing and the availability of the mouse genome sequence. Thus, ENU mutagenesis has been used to successfully identify genes, proteins, and signaling pathways involved in a wide range of biological processes, including susceptibility to infection [40] [41], obesity [42], muscle development and function [43], cardiomyopathy [44], thrombocytopenia [45] and immunodeficiency [46]. This last report, describing a single-nucleotide mutation in the Unc93b1 gene that abrogates signaling via TLR3, 7 and 9, has largely contributed in the discovery of the human UNC93B1 mutations [14].…”