The 14th Ile residue is essential for Leptin function in regulating energy homeostasis in rat.

Shuyang Xu,Guoping Fan,Fang Wu,Tiancheng Liu,Zhigang Xue,Jinping Zhou,Zhen Wang,Yun Feng,Guohui Ding,Shaorong Gao,Guizhen Du,Hong Li,Youjin Hu,Lina Lu,Jiayu Chen,Yixue Li,Di He,Youtian Hu,Xianmin Zhu

doi:10.1038/srep28508

Abstract

LEPTIN (LEP) is a circulating hormone released primarily from white adipocytes and is crucial for regulating satiety and energy homeostasis in humans and animals. Using the CRISPR technology, we created a set of Lep mutant rats that carry either null mutations or a deletion of the 14th Ile (LEP∆I14) in the mature LEP protein. We examined the potential off-target sites (OTS) by whole-genome high-throughput sequencing and/or Sanger-sequencing analysis and found no OTS in mutant rats. Mature LEP∆I14 is incessantly produced and released to blood at a much elevated level due to the feedback loop. Structure modeling of binding conformation between mutant LEP∆I14 and LEPTIN receptor (LEPR) suggests that the conformation of LEP∆I14 impairs its binding with LEPR, consistent with its inability to activate STAT3-binding element in the luciferase reporter assay. Phenotypic study demonstrated that Lep∆I14 rats recapitulate phenotypes of Lep-null mutant rats including obesity, hyperinsulinemia, hepatic steatosis, nephropathy, and infertility. Compared to the existing ob/ob mouse models, this Lep∆I14/∆I14 rat strain provides a robust tool for further dissecting the roles of LEP in the diabetes related kidney disease and reproduction problem, beyond its well established function in regulating energy homeostasis.

Highlights

LEPTIN (LEP), a secreted peptide by white adipocyte tissues (WAT), is one of the most widely studied adipokines that regulate mammalian body weight and maintain energy balance
We found that Lep mutant rats carrying homozygous deletions of 3 nucleotides (ATC) encoding isoleucine at position 14 (I14) in the mature LEP protein exhibited similar mutant phenotypes to LEP- and LEP and its receptor (LEPR)- null rats
We found that each targeting sequence results in mutations so that the mismatched PCR products were cleaved by T7 Endonuclease I (T7EI) as expected (Supplementary Materials, Fig. S1)

Summary

Introduction

LEPTIN (LEP), a secreted peptide by white adipocyte tissues (WAT), is one of the most widely studied adipokines that regulate mammalian body weight and maintain energy balance. The Pakistani cousins had homozygous deletion of guanosine at Codon 133 (∆133G) on LEP which resulted in premature stop of translation via frame shift. Another Pakistani child was reported to have the same ∆133G mutation inherited from their heterozygous parents originally from the same geometrical area, the different families were not genetically related for at least 4 generations[6]. Similar symptoms were reported in different families with the homozygous missense mutations on LEP, i.e., R105W7, N103K8, L72S9, Q55X10, Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Our molecular analyses suggest that I14 is of great importance for the interaction between LEP and LEPR and the down streaming JAK2-STAT3 pathways

Methods

Results

Conclusion