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

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 14^th Ile (LEP^ΔI14) 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^ΔI14 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^ΔI14 and LEPTIN receptor (LEPR) suggests that the conformation of LEP^ΔI14 impairs its binding with LEPR, consistent with its inability to activate STAT3-binding element in the luciferase reporter assay. Phenotypic study demonstrated that Lep ^ΔI14 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^ΔI14/ΔI14 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.