Detection of Leptin Activity in Living Cells Expressing Chicken Leptin Receptor and STAT3

Abstract

The chicken leptin receptor (chLEPR) activates Janus kinase (JAK) -signal transducers and activator of transcription (STAT) signaling pathway after leptin stimulation. We have previously developed a bioassay using leptin inducible reporter gene in cultured cells. However, we failed to detect leptin-like activity in chicken blood. In the present study, we expressed green fluorescent protein (GFP) fused chSTAT3 (GFP-chSTAT3) in cells expressing chLEPR and analyzed leptin dependent activation of the chSTAT3. Leptin phosphorylated GFP-chSTAT3 and by which fluorescent signal translocated into nuclei in COS-7 cells transiently expressing GFP-chSTAT3 with chLEPR. Furthermore, we established CHO-K1 cells stably expressing chLEPR and chSTAT3 (CHO-chLEPR/chSTAT3), and in which detected time-and dose-dependent activation of chSTAT3 by leptin. Therefore, the CHO-chLEPR/STAT3 cells would be an excellent tool to detect and monitor leptin-like activity in avian tissues.

Discovery of the Elusive Leptin in Birds: Identification of Several ‘Missing Links’ in the Evolution of Leptin and Its Receptor

“…This observation suggests conserved binding sites between leptin and its receptor across vertebrate classes. The intracellular (N-terminal) domain of bird leptin is less homologous to human and other mammals, suggesting a bird-specific JAK/STAT activation, which may contribute to the difficulty of addressing chicken STAT3 activation using mammalian cell culture [30] .…”

“…Our description of the structure and evolution of the first bona fide avian leptin gene and its encoded leptin protein represents a critical first step toward understanding the distribution and function of leptin among birds. We suggest and provide the tools for further in vitro and in vivo validations of the bird leptin, such as additional luciferase assays using the newly designed bird specific STAT3 assay [30] . In this paper we have performed a genome analysis followed by gene sequence identification and protein structure to protein function analysis (genome-to-sequence-to-structure-to-function, or G-S-S-F for short) to validate a potential gene of interest to a research community.…”

Leptin activates chicken growth hormone promoter without chicken STAT3 in vitro

“…CHO-K1 cells stably expressing chLEPR (CHO-chLEPR) or GFP-fused chicken STAT3 (CHO-chSTAT3) (Adachi et al, 2008(Adachi et al, , 2012 were maintained and transfected in Ham's F12 medium (WakoPure Chemical Industries, Tokyo, Japan) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in an atmosphere of 5% CO 2 at 37°C. Transfection was conducted as follows unless otherwise stated.…”

Leptin Modulates the mRNA Expression of Follicle Development Markers in Post-hatch Chicks in an Age-Dependent Manner

“…For a leptin administration study, recombinant mouse leptin (rmleptin; Sigma–Aldrich Japan, Tokyo, Japan) was used in this study, because genuine recombinant chicken leptin derived from recently cloned cDNA (Farkašová et al, 2016 ; Seroussi et al, 2016 ) may not be available. On the other hand, rmleptin remains biologically active in chicken and other birds and is used in ovarian function and other studies (Bungo et al, 1999 ; Máčajová et al, 2002 , 2003 ; Lamošová et al, 2003 ; Song et al, 2009 ; Adachi et al, 2012 ; Hu et al, 2012 ; Niu et al, 2019 ). The rmleptin was dissolved in phosphate-buffered saline [PBS (–)] and injected intraperitoneally (i.p.)…”