Effects of PEG-SMLA treatment on food intake and adipose tissues mass.

<p>(A) Food consumption (g) is calculated reported to body weight of each mouse on first or second week of treatment. PEG-SMLA injections are represented within hatched bars. b, p<0.001 related to Control WT mice. (B) At the end of treatment, the intact abdominal fat depot (delimited lines) is shown on both genotypes. (C) Ratio of adipose depot mass per body weight. b, p<0.001 and c, p<0.01 related to Control WT mice. (n = 7–10/group).</p

Effect of PEG-SMLA treatment on weight gain of WT and PRLR^−/− mice.

<p>(A) Representative dorsal view of male mice after 20 days of saline (control) or PEG-SMLA injections. (B) Comparison of PEG-SMLA effects on weight gain of WT (open squares) or PRLR^−/− (black squares) male mice. PEG-SMLA was injected daily at 6 mg/kg. <i>***, p<0.005</i> between non-treated (Cont) and PEG-SMLA treated groups.</p

Trabecular bone parameters of third and fourth lumbar vertebrae (LV3 and LV4) of PEG-SMLA <i>vs</i> saline-injected PRLR^−/− and WT mice.

<p>Trabecular bone volume (BV)/tissue volume (TV) (%), trabecular number (Tb.N) (1/μm), trabecular thickness (Tb-Th) (μm), and trabecular separation (Tb.Sp) (μm). Data are mean ± SEM. Results shown in each row that are not designed with the same letter are statistically different, p<i><</i>0.05, n = 6.</p

Effects of PEG-SMLA treatment on food intake and adipose tissues mass.

<p>(A) Food consumption (g) is calculated reported to body weight of each mouse on first or second week of treatment. PEG-SMLA injections are represented within hatched bars. b, p<0.001 related to Control WT mice. (B) At the end of treatment, the intact abdominal fat depot (delimited lines) is shown on both genotypes. (C) Ratio of adipose depot mass per body weight. b, p<0.001 and c, p<0.01 related to Control WT mice. (n = 7–10/group).</p

Metabolic parameters in WT and PRLR^−/− mice after saline or PEG-SMLA treatment.

<p>Number of mice appears in brackets. Data are represented as mean ± SEM. Different letters within each line indicate statistically different groups.Statistical analyses were performed between the all groups. b, p<0.001; c, p<0.05.</p

Expression of key adipose genes.

<p>(A–D) <i>Leptin, Zfp423, PPAR</i> and <i>aP2</i> gene expression was quantified by qPCR in each gonadal, peri-renal, inguinal adipose depot of WT and PRLR^−/− non-treated mice (Control) and treated animals (PEG-SMLA, hatched bars) respectively (n = 5–8/group) b, p<0.01. (E) Distribution of adipocyte surfaces in inguinal adipose tissue in untreated WT and PRLR^−/− (Control) and treated animals (PEG-SMLA, hatched bars). Statistical analysis was performed. b, p<0.01, c, p<0.001.</p

PEG-SMLA treatment induced glucose intolerance and insulin resistance on WT and PRLR−/− animals.

<p>(A) Fasting glycemia in saline treated and PEG-SMLA treated mice. b, *p<0.05. (B) Oral glucose tolerance test (2g/kg) was performed in WT and PRLR^−/− mice PEG-SMLA treated or not. (C) Fasting (left panel) and 30 minutes after glucose gavage (right panel) plasma insulin levels. (D) HOMA-IR index reflecting insulin resistance is calculated as follows: fasting plasma glucose (mg/dl) × fasting insulin (mU/L)/405.</p

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

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