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Leptin (LEP) is a cytokine-like hormone proven to be involved in diverse biological processes. In livestock, it regulates feed intake, BW homeostasis, and energy balance, among other traits. Natural nonsynonymous genetic polymorphisms in the ovine leptin (oLEP) alter the biochemical and physiological characteristics of its gene products. Here we studied in vitro and in vivo the biochemical and physiological characteristics of recombinant hormones representing the oLEP and bovine leptin (bLEP) reference sequences of wild-type (WT) leptins (GenBank accession No. U84247 and U50365, respectively), oLEP and bLEP recombinant muteins carrying the R4C mutation, and oLEP recombinant hormones carrying the A59V and Q62R mutations, which were detected in bLEP. All proteins were purified to homogeneity as monomers and formed 1:1 molar ratio complexes with the chicken leptin-binding domain (LBD). Surface plasmon resonance experiments revealed that all protein variants exhibit reduced (P < 0.05) affinity to chicken (ch) and human (h) LBD compared with the WT oLEP and bLEP recombinant proteins. The ovine and bovine R4C muteins exhibited significantly (P < 0.05) greater induction of cell proliferation in a Baf/3 cell line bioassay, despite lower affinity toward both hLBD and chLBD. Intra-third cerebral ventricle infusion of oLEP and its 3 muteins in sheep resulted in reduced feed intake. However, the 3 tested muteins had a decreased (P < 0.05) inhibitory effect than the WT LEP. It was concluded that natural genetic polymorphisms in the bLEP are associated with variation in the biochemical and physiological properties of the protein.

The hormone leptin is involved in diverse biological processes, including regulation of food intake, body-weight homeostasis and energy balance. Sequence variation in the bovine leptin gene has been found to be associated with variations in carcass fat content and average daily gain, as well as in milk yield, milk somatic cell count and several traits governing reproduction. We sequenced genomic DNA and cDNA samples of individuals from three divergent sheep breeds and revealed synonymous as well as novel non-synonymous allelic variation at the third exon of the ovine leptin gene (oLEP) as compared to the sequence published at Accession No. U84247 (reference sequence). In addition, two alternatively spliced oLEP transcripts were found in the abdominal fat tissue. The biochemical and the in vitro biological significance of the sequence variation in the oLEP was examined by generating recombinant oLEP-protein variants namely: p.Q28del, p.N78S, p.R84Q, p.P99Q, p.V123L and p.R138Q, carrying the corresponding sequence variation. Surface plasmon resonance experiments revealed, in most cases, reduced affinity of the oLEP protein variants examined, to human leptin-binding domain (hLBD), relative to the reference variant, being 0.75, 0.60, 0.60, 0.89, 0.92 and 1.03, respectively. In competitive binding assays between biotinylated oLEP and the recombinant leptin protein variants, p.N78S and p.R84Q variants exhibited the lowest affinity to hLBD (0.18 and 0.41, respectively) as compared to the reference hormone. We then tested the protein variants' ability to induce proliferation in Baf-3 cells stably expressing the long form of the human leptin receptor: significant differences in proliferative activity were only found for p.N78S (1.8-fold higher) and p.R138Q (4.2-fold lower) relative to the reference oLEP variant., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Leptin, a pleiotropic adipokine, is a central regulator of appetite and weight and a key immunomodulatory protein. Although inborn leptin deficiency causes weight gain, it is unclear whether induced leptin deficiency in adult wild-type animals would be orexigenic. Previous work with a potent competitive leptin antagonist did not induce a true metabolic state of leptin deficiency in mice because of a short circulating half-life. In this study, we increased the half-life of the leptin antagonist by pegylation, which resulted in significantly increased bioavailability and retaining of antagonistic activity. Mice administered the pegylated antagonist showed a rapid and dramatic increase in food intake with weight gain. Resulting fat was confined to the mesenteric region with no accumulation in the liver. Serum cholesterol, triglyceride, and hepatic aminotransferases remained unaffected. Weight changes were reversible on cessation of leptin antagonist treatment. The mechanism of severe central leptin deficiency was found to be primarily caused by blockade of transport of circulating leptin across the blood-brain barrier with antagonisms at the arcuate nucleus playing a more minor role. Altogether we introduce a novel compound that induces central and peripheral leptin deficiency. This compound should be useful in exploring the involvement of leptin in metabolic and immune processes and could serve as a therapeutic for the treatment of cachexia.