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

Chen-Jee Hong,Yau-Sheng Tsai,Chuan-Kai Chou,Irene H Cheng,Shiow-Yi Chen,Pei-Jane Tsai,Chih-Ya Cheng,Chia-Ning Yang,Chih-Wei Hsu,Ying-Jay Liou,Shih-Jen Tsai,Ya-Tzu Lin,You-Chung Chuang,Huei-Fen Jheng,Gian Paolo Fadini

doi:10.1371/journal.pone.0015333

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.

Highlights

Leptin, a 16-kDa protein produced mainly in adipose tissue and secreted into the bloodstream, plays an important role in regulating body weight, metabolism and reproductive function [1]
ENU-induced obese mice harbor a mutation in leptin During an ENU mutagenesis screen, we found several obese mice from the same G1 founder inherited in a recessive pattern (Figure 1A)
Using the random ENU-mutagenesis, we created a novel mouse model inherited in a recessive pattern for obesity and insulin resistance due to a missense V145E mutation in the Leptin gene

Summary

Introduction

A 16-kDa protein produced mainly in adipose tissue and secreted into the bloodstream, plays an important role in regulating body weight, metabolism and reproductive function [1]. Circulating leptin levels are highly correlated with white adipose tissue mass. The lack of leptin action causes a disruption in energy balance with hyperphagia and decreased energy expenditure, leading to morbid obesity and development of type 2 diabetes. Leptin administration decreases food intake and body weight while preserving metabolic energy utilization. Mouse and human leptin cDNA encodes a 167 amino acid residue protein with a 21 amino acid residue signal sequence that is cleaved to yield the 146 amino acid residue mature protein. Mouse leptin shares approximately 96% and 84% sequence identity with the rat and human protein, respectively. Leptin is central to the regulation of energy metabolism and control of body weight in mammals

Methods

Results

Conclusion