Differential effects of leptin on thermoregulation and uncoupling protein abundance in the neonatal lamb.

As the role of leptin in energy balance in neonate is unknown, we investigated the effect of acute (2 h) and chronic (7 days) administration of leptin (100 microg/day) on thermoregulation and mitochondrial protein abundance in adipose tissue. The concentration of uncoupling protein (UCP)1 and voltage-dependent anion channel (VDAC) located on the inner and outer mitochondrial membranes, respectively, were measured. Administration of leptin prevented the normal decline in colonic temperature over the first few hours and days after birth. It subsequently accelerated the loss of both mRNA and protein for UCP1 but had no effect on VDAC abundance. At seven days of age, colonic temperature was correlated strongly with both mRNA abundance and thermogenic potential of UCP1 in leptin-treated but not control lambs, indicating more effective use of UCP1 for heat production following leptin administration. Leptin had no effect on weight gain or adipose tissue deposition; at one day of age only, leptin mRNA was correlated positively with adipose tissue weight. In conclusion, leptin administration to neonatal lambs improves thermoregulation and promotes the loss of UCP1 in brown adipose tissue.

Divergent effects of leptin on luteinizing hormone and insulin secretion are dose dependent.

We have shown recently that fasting permits leptin to modulate both luteinizing hormone (LH) and insulin secretion in cows. In rodents, leptin causes divergent effects on LH and insulin release that are dose dependent. To test the hypothesis that leptin effects on LH and insulin secretion in fasted cows are dose related, we examined the effects of various doses of recombinant ovine leptin (oleptin) in mature cows. Twenty ovariectomized beef cows, each bearing an estradiol implant to maintain basal estradiol concentrations, were used. All cows were fasted for 60 hr with free access to water and were assigned randomly to one of four groups (n = 5/group): 1) saline control; 2) leptin, 0.2 microg/kg; 3) leptin, 2.0 microg/kg; and 4) leptin, 20 microg/kg body wt. Blood samples were collected at 10-min intervals for 6 hr on Days 0 and 2, with saline or oleptin injected intravenously immediately after the first intensive sample on Day 2 (54 hr). Leptin caused a dose-related increase (P < 0.001) in mean concentrations of circulating LH. Stimulation of LH release by leptin was significant at the lowest (141% of control) and middle (122% of control) doses used, but no increase was observed for the highest dose. Increased mean concentrations of LH appeared to result from an augmentation of basal secretion, as pulse characteristics were not affected. After 54 hr of fasting, plasma insulin concentrations were lowered (P < 0.01) in all treatment groups compared to Day 0. After leptin injections, plasma insulin concentrations increased (P < 0.01) and reached highest concentrations during the first hour of sampling. However, this increase was sustained for several hours only in the intermediate (2.0 microg/kg) dose group. Collectively, our results show that leptin has potent positive effects on both LH and insulin secretion in fasted cows, but the anterior pituitary and endocrine pancreas appear to become downregulated in the presence of excess ligand.

Comparison of R128Q mutations in human, ovine, and chicken leptins.

Human leptin and its R128Q mutant, as well as the R128Q mutants of ovine and chicken leptins, were prepared, expressed in Escherichia coli, refolded, and purified to homogeneity yielding electrophoretically pure, over 95% monomeric protein. R128Q mutations did not change the binding properties to BAF/3 cells stably transfected with the long form of human leptin receptor compared, respectively, to non-mutated human, ovine, and chicken leptins. In contrast, the biological activity tested in a proliferation assay in the same cells was drastically changed. Human leptin R128Q lost its activity and even became a weak antagonist, whereas the activities of ovine and chicken leptins were reduced 25- and 80-fold. If dimerization models were applicable leptin receptor activation, the present results would suggest that site 2 of the hormone was impaired. Two models, the human growth hormone:human growth hormone receptor (hGH:hGHR) (1:2) and the granulocyte-colony stimulating factor:granulocyte-colony stimulating factor receptor (GCSF:GCSFR) (2:2) complexes, were used for modeling. Superimposing the leptin structure on the hGH and GCSF models in the complex structures did not indicate any role for R128 in receptor binding. This made it impossible to correlate the results shown in the present work with the currently available models. Therefore, leptin may bind its receptors in a manner different than those proposed until now.

Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain.

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.