Structural And Functional Studies Of Leptins From Hibernating And Non-hibernating Bats

Leptin, a 16-kDa hormone produced by mature adipocytes, acts via leptin receptor of hypothalamus to regulate the food intake, energy expenditure and adiposity. Leptin receptor (OB-R) mainly initiates signals by activating Janus kinases (JAK) and signal transducers and activators of transcription (STAT) on numerous tyrosine residues of OB-R during leptin stimulation. Leptin has been shown to play an important role in hibernating regulation of mammals. In this study, the leptin gene sequences of hibernating (Rhinolophus ferrumequinum) and non-hibernating (Rousettus leschenaultii) bats were determined, and the leptin proteins from these two different species of bats were expressed in E. coli for the first time. After purification, the GST-leptins were confirmed by SDS-PAGE and Western blot. The fact that recombinant leptin protein decreased cell viability of 3T3-L1 preadipocytes indicated its functional activity. Sequences analysis showed that the amino acid sequence of the leptin protein from hibernating bats (Rhinolophus ferrumequinum, leptin-Rh) had a lower degree of identity than that from non-hibernating bats (Rousettus leschenaultii, leptin-Ro) to those of several non-hibernating mammals. The leptin protein of hibernating bats (leptin-Rh) had a stronger growth inhibitory effect on 3T3-L1 cells than that of the non-hibernating bats (leptin-Ro). Structural modeling revealed that the structures of the leptin receptor binding siteâ…¢(helices C to D), which is critical for signal transduction of JAK/STAT pathway, of the two bat leptins were very different. Furthermore, the hydrophilic amino acid threonine 121 located in the binding site III of both human leptin and leptin-Rh was substituted by the hydrophobic methionine in leptin-Ro. Similar to the human leptin, the leptin protein of non-hibernating bats was predicted to have a random loop, whereas that of hibernating bats had a helical structure in this region. This observation provided a clue as to the differential effects of the two different leptins on 3T3-L1 cells. Our study clearly demonstrated that the amino acid sequences, protein structures and biological activities of the two leptin proteins were different. This article provides some clues for the research about the role that leptin plays in hibernation.