Leptin pharmacology and endocrinology
Creators: name: Margetic, Senka
Publisher: name: CQUniversity
Year: 2023
Subjects: Veterinary pharmacology
Endocrinology

description: The aims of the present study were to investigate leptin pharmacology, endocrinology and the effects of leptin on different peripheral tissues of rats and cattle. The pharmacokinetics and tissue distribution of radiolabelled leptin was investigated in rats. A two pool model described the pharmacokinetics of leptin, an initial fast decaying pool (t<sub>1/2</sub> 3.4 min) and a slower decaying pool (t<sub>1/2</sub> 71 min) with an overall clearance rate of 6.16 ml/min/kg. Size exclusion chromatography showed a consistent broad peak (all time-points tested) of <sup>125</sup>I-leptin resolving at 66 kDa. The size of the free <sup>125</sup>I-leptin peak became diminished or absent in later time-point plasma samples. Tissue distribution of radiolabelled leptin at 60 min and 180 min time-points showed that small intestine contained the highest concentration of leptin, almost four times the level found in kidneys, liver, stomach and lungs. The effect of 7 days dietary restriction on plasma leptin concentration and tissue distribution in female rats were studied. There was no significant difference in plasma leptin levels and tissue distribution of <sup>125</sup>I-leptin between restricted diet group and control ad libitum group. Tissue distribution of radiolabelled leptin at 60 min time-point in both groups, showed that small intestine contained the highest concentration of <sup>125</sup>I-leptin. Using size exclusion chromatography, evidence that leptin circulates in multiple molecular mass protein complexes in bovine serum was provided. Putative bovine leptin binding proteins appear to have molecular masses of 50, 200 and 650 kDa. Immunoblot analysis showed that anti-leptin antibodies (rabbit anti-mouse and sheep anti-bovine leptin antibodies) cross-reacted with bovine and mouse leptin, presumably because of the high homology between bovine and mouse leptin. Also, endogenous leptin from bovine serum was detected using a highly sensitive enhanced chemiluminescence (ECL) method. Characterisation of leptin receptors in bovine kidney membranes was also investigated. Binding of <sup>125</sup>I-leptin to bovine kidney membranes was specific, time dependent, and reached an apparent equilibrium by 60 min at 4°C. A biphasic pattern was observed with k<sub>obsl</sub>= 0.315 ± 0.06 min<sup>-1</sup> (high affinity site) and k<sub>obs2</sub> = 0.030 ± 0.008 min<sup>-1 </sup>(low affinity binding site). Leptin dissociation from the receptor was described by a single dissociation constant (k<sub>off</sub>) value of 0.0036 ± 0.0004 min -1. Using 125I-leptin with high specific activity, one high affinity binding site was detected with an apparent K<sub>d</sub> of 0.976 ± 0.107 nM and B<sub>max</sub> of 110.88 ± 13.81 fmol/mg<sub>p</sub>. Using <sup>125</sup>I-leptin with reduced activity, two binding sites were characterised. A high affinity/low density binding site (K<sub>d</sub> = 0.098 ± 0.0009 nM and B<sub>max</sub> of 46.18 ± 1.68 fmol/mg<sub>p</sub>) and low affinity/high density (K<sub>d</sub> = 174.67 ± 5.89 nM, B<sub>max</sub> = 5743 ± 203.6 fmol/mg<sub>p</sub>). <sup>125</sup>I-leptin binding was inhibited in a dose dependent manner by unlabelled mouse and human leptin and by polyclonal anti-bovine leptin antibodies. Specificity of binding was characterised, as bound <sup>125</sup>I leptin was not displaced by insulin or control antibodies. Several protein bands were identified when bovine kidney membrane preparations were cross-linked with Bis (sulfosuccinimidyl) suberate (BS<sup>3</sup>), with molecular masses of approximately 45, 97 and &gt; 220 kDa. These bands were displaced through addition of unlabelled leptin (0.1 and 1.0 μM respectively). In rat small intestine, binding studies suggested the presence of a dense population of leptin receptors. Results from saturation assays at the low concentration range of <sup>125</sup>I-leptin, showed the presence of a single class of binding sites, with a K<sub>d</sub> of 1.76 ± 0.44 nM and B<sub>max</sub> of 205.7 ± 43.7 fmol/mgp. Also, the physiological role of leptin in rat isolated ileum was investigated using organ bath studies. Leptin substantially inhibited nerve-mediated contractions evoked by 5-hydroxytryptamine (5-HT) in comparison to the control, but had no effect on 5-HT contractions after nerve transmission was blocked with tetrodotoxin (TTX). This suggests that the effect of leptin was due to an interaction of the hormone with myenteric neurons, rather than through a direct mechanism via the muscle layer or muscle receptors. The effect of mouse, human and bovine leptin on angiogenesis was investigated using the chicken chorioallantoic membrane (CAM) model. Mouse leptin induced angiogenesis in a dose-dependant manner. Discs that contained 0.1, 0.5 and 1.0 ug mouse leptin promoted angiogenesis, while discs that contained 3.0 and 10 lig leptin produced both angiogenesis and vascular leakage with associated avascular zones. Surprisingly, the human and bovine leptin showed less angiogenic effect on CAM than mouse leptin. This may be a result of different post translational folding of leptin. The physiological actions of leptin in peripheral tissues appear to be complex. The roles of other proteins which act as specific binding proteins and other non-specific (hydrophobic) interactions of leptin require further investigations. Further work would be needed to: (1) isolate and purify leptin binding protein genes; (2) isolate and determine amino acid sequences of leptin receptor(s) in bovine kidney; (3) further characterise hydrophobic interactions of leptin with other proteins.