Preparation and characteristics of superactive rat leptin antagonist.

<p>(<b>A</b>) Inclusion bodies from 2.5 L fermentation culture were prepared and resuspended in 100 ml of DDW. Aliquots (corresponding to 0.8, 1.6, 3.2, 4.0, 8.0 and 12.0 µl per lane, from left to right) were separated by 15% SDS-PAGE in presence of β-mercaptoethanol. The molecular mass markers from the bottom up (last lane on the right) are (in kDa): 10, 15, 20, 25, 37, 50, 75, 100, 150 and 250. (<b>B</b>) SDS-PAGE (15%) of purified SRLA run after lyophilization in the absence (lanes 1–2 from the left) or presence (lanes 4–5 from the left) of β-mercaptoethanol (ME) at 2 concentrations: lanes 1 and 4–5 µg, lanes 2 and 5–10 µg. Lane 3– molecular weight markers (see above). (<b>C</b>) SDS-PAGE (10%) of purified PEG-SRLA run after lyophilization in the absence (lanes 1–2 from the left) or presence (lanes 4–5 from the left) of β-mercaptoethanol (ME) at 2 concentrations: lanes 1 and 4–5 µg, lanes 2 and 5–10 µg. Lane 3– molecular weight markers (see above); (<b>D</b>) Gel-filtration analysis of the purified SRLA on analytical Superdex 75 column pre-equilibrated with TN buffer, pH 8. The main peak with retention time of 15.93 min corresponds to monomer and the preceding small shoulder to dimer. (<b>E</b>) Gel-filtration analysis of the purified PEG-SRLA on analytical Superdex 200 column pre-equilibrated with TN buffer, pH 8. The main peak with retention time of 14.93 min corresponds to mono-pegylated PEG-SRLA and the preceding small shoulder to double-pegylated PEG-SRLA. To estimate the molecular mass shown in (D) and (E) the columns were calibrated with BSA (66 kDa), rat CNTF (22 kDa) and human leptin (16 kD); (<b>F</b>) Competitive non-radioactive receptor-binding assay of SRLA and PEG-SRLA. Binding of biotinylated human leptin to immobilized human leptin binding domain (hLBD) consisting of the amino acids 428–635 of human leptin receptor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086744#pone.0086744-Sandowski1" target="_blank">[51]</a> was performed in the presence of the indicated protein SRLA or PEG-SRLA concentrations. The experiment was carried out in triplicates and the results are presented as mean ± SEM. As the variations in this assay was very small the error bars are not seen; (<b>G</b>) Biological activity of SRLA and PEG-SRLA. The experiment was performed in BAF/3 cells stably transfected with the chimeric leptin receptor construct consisting of the extra-cellular and transmembrane domain of the murine leptin receptor with the intracellular domain of the human βc receptor. Synchronized cells were grown for 48 h in the presence of rat leptin (50 pg/well) and various concentrations of SRLA or PEG-SRLA. The number of cells was then determined by the MTT method. In both bioassays the experiment was carried out in triplicates and the results are presented as mean ± SEM. Detailed description of the binding experiments and the bioassay is provided in our former paper <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086744#pone.0086744-Shpilman1" target="_blank">[28]</a>.</p

Diets and treatments applied in different experimental groups.

<p>Diets and treatments applied in different experimental groups.</p

Leptin-induced relaxation of phenylephrine-preconstricted mesenteric artery rings in different experimental groups.

<p>NO-dependent vasorelaxation was measured in the presence of apamin (5 µM), TRAM-34 (1 µM) and indomethacin (10 µM), and EDHF-dependent vasorelaxation was examined in the presence of L-NAME (100 µM) plus indomethacin (10 µM). Maximal relaxation (R_max) was calculated as the leptin-induced percent decrease in tension developed in response to phenylephrine. EC₅₀– leptin concentration (ng/ml) which induced a half-maximal relaxation of PE-preconstricted segments. R_max and EC₅₀ values were calculated for each individual vascular preparation and data presented in the table are mean ± SD from 6 animals per group.</p>*<p>p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001 vs. control group (ANOVA and Tukey test).</p

GYY4137-induced relaxation of phenylephrine-preconstricted mesenteric artery rings.

<p>Effect of increasing concentrations of GYY4137 on mesenteric artery segments with intact or removed endothelium was examined in the absence or in the presence of K⁺ channel inhibitors and R_max and EC₅₀ values were calculated for each preparation (n = 6/group).</p>***<p>p<0.001 vs. preparation without K⁺ channel inhibitors (Student t-test for related variables),</p>†††<p>p<0.001 vs. segments with intact endothelium (Student t-test for unrelated variables). Because glibenclamide almost completely abolished the effect of GYY4137 in endothelium-denuded segments, R_max and EC₅₀ could not be calculated.</p

EDHF-mediated leptin-induced relaxation of phenylephrine-preconstricted mesenteric artery rings in the absence and in the presence of stigmatellin.

*<p>p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001 vs. respective segment not treated with stigmatellin (Student t-test for related variables),</p>†<p>p<0.05,</p>††<p>p<0.001,</p>†††<p>p<0.001 vs. control group (ANOVA and Tukey post-hoc test).</p

Effect of inhibitors of different EDHF mechanisms on leptin-induced vasorelaxation.

<p><b>A:</b> Mesenteric artery segments of control rats were preconstricted with phenylephrine, and the vasodilating effect of leptin (200 ng/ml) was examined in the presence of L-NAME and indomethacin without other inhibitors or after addition of cytochrome P450 inhibitor SKF 525A (SKF, 10 µM), lipoxygenase inhibitor nondihydroguaiateric acid (NDGA, 20 µM), H₂O₂ scavenger PEG-catalase (CAT, 250 U/ml), heme oxygenase inhibitor Cr(III) mesoporphyrin IX (CrMP, 10 µM), protein kinase G inhibitor KT5823 (KT, 1 µM), H₂S scavenger bismuth (III) subsalicylate (BSS, 10 µM) or cystathionine γ-lyase inhibitor D,L-propargylglycine (PAG, 1 mM). ***p<0.001 vs. preparation treated only with L-NAME and indomethacin (Student t-test for related variables) <b>B:</b> Effect of leptin (200 ng/ml) on PE-preconstricted mesenteric artery segments was examined in different experimental groups in the presence of L-NAME and indomethacin (black bars), L-NAME, indomethacin and BSS (white bars) or L-NAME, indomethacin and PAG (grey bars). **p<0.01 vs. control group, †p<0.001 vs. respective group not treated with either PAG or BSS.</p

Effect of SQR inhibitor, stigmatellin, on EDHF-mediated leptin-induced relaxation of mesenteric artery segments.

<p>Concentration-dependent effect of leptin on PE-preconstricted rings from control (A), leptin-treated (B) and obese (C) rats was examined in the presence of L-NAME+indomethacin. Segments were examined in the absence (black squares, continuous line) or in the presence of 3 µM stigmatellin (white squares, broken line).</p

GYY4137-induced relaxation of phenylephrine-preconstricted mesenteric artery rings in control, leptin-treated and obese rats.

<p>Concentration-dependent effect of GYY4137 was examined in the presence of apamin and TRAM-34 (K_ATP channel-dependent relaxation) or glibenclamide (SK_Ca and IK_Ca channel-dependent relaxation).</p>***<p>p<0.001 vs. control group (ANOVA and Tukey test).</p

Leptin-induced relaxation of phenylephrine (PE)-preconstricted mesenteric artery rings of control rats with intact endothelium in the absence of inhibitors and in the presence of NO synthase inhibitor, L-NAME, or inhibitors of small and intermediate-conductance Ca²⁺-activated K⁺ channels, apamin (Ap) and TRAM-34 (TRAM), respectively, which together block EDHF-dependent response.

<p>Leptin-induced relaxation of phenylephrine (PE)-preconstricted mesenteric artery rings of control rats with intact endothelium in the absence of inhibitors and in the presence of NO synthase inhibitor, L-NAME, or inhibitors of small and intermediate-conductance Ca²⁺-activated K⁺ channels, apamin (Ap) and TRAM-34 (TRAM), respectively, which together block EDHF-dependent response.</p

Metabolic characteristics of animals in different experimental groups.

***<p>p<0.001 vs. control group (ANOVA and Tukey test).</p