Monitoring leptin activity using the chicken leptin receptor

Abstract

We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre-and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.

Leptin in farm animals: where are we and where can we go?

“…into hens and found no reduction in intake, whereas Dridi et al (2005b) infused re-ch.lep for 6 h into 3-week old broilers and reduced feed intake and ch-ObR expression. Hen et al (2008) transfected ch-ObR into human embryo kidney cells and developed a bioassay that could distinguish pre-partum from post-partum (PP) cows. Ironically, the same assay could not distinguish lean from fat birds.…”

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

“…However, we could not detect leptin-like activity in chicken serum. Other researchers had also failed to detect serum leptin activity in chicken and other birds using similar bioassay (Hen et al, 2008;Yosefi et al, 2010).…”

“…Recently it has been shown by us and others that chLEPR expressed in mammalian cells activates JAK -STAT signaling pathway Hen et al, 2008), indicating thereby that endogenous LEPR in bird is capable of processing leptin-like signals in a way similar to higher vertebrates. However, it has not been possible to detect leptin-like activity in the avian blood by the in vitro bioassay Hen et al, 2008;Yosefi et al, 2010). This may be due to plasma leptin levels being below the detection threshold of the quantitative bioassay using luciferase gene.…”

“…Phosphorylated STAT3 then translocates to the nucleus to modulate transcription of the target genes in mammals (Banks et al, 2000). Recently it has been shown by us and others that chLEPR expressed in mammalian cells activates JAK -STAT signaling pathway Hen et al, 2008), indicating thereby that endogenous LEPR in bird is capable of processing leptin-like signals in a way similar to higher vertebrates. However, it has not been possible to detect leptin-like activity in the avian blood by the in vitro bioassay Hen et al, 2008;Yosefi et al, 2010).…”

“…It is known that fluorescent protein fused STAT3 migrates into nucleus after cytokine stimulation (Kretzschmar et al, 2004;Pranada et al, 2004;Watanabe et al, 2004). In addition, leptin activates chLEPR which induces STAT dependent activation of luciferase gene Hen et al, 2008). Therefore, detection of translocation of GFP fused STAT3 into nuclei would be an alternate bioassay in detecting endogenous leptin-like activity in birds.…”