Journal: Journal of cell science

Article Title: Mapping of the interface between leptin and the leptin receptor CRH2 domain.

doi: 10.1242/jcs.02386

Figure Lengend Snippet: Fig. 6. Molecular models of binding site II in mouse leptin. (A) Molecular surface map of leptin, coloured according to surface hydrophobicity (blue, hydrophilic; green, hydrophobic). A hydrophobic cleft is formed by residues L13, L86, L89 and F92. (B) Residues in binding site II that affect binding to CRH2 are coloured yellow. Residues in binding site II that affect both binding to CRH2 and LR activation are coloured orange. (C) Residues that become buried in the leptin/CRH2 interface, coloured according to the area that becomes buried (cyan, <25 Å2; blue, 25-50 Å2; green, >50 Å2).

Article Snippet: Expression was checked by anti-HA western blot analysis and quantified using an anti-mouse leptin ELISA kit (R&D systems) (Peelman et al., 2004b).

Techniques: Binding Assay, Activation Assay

Journal: Journal of cell science

Article Title: Mapping of the interface between leptin and the leptin receptor CRH2 domain.

doi: 10.1242/jcs.02386

Figure Lengend Snippet: Fig. 9. Model of the mouse leptin/CRH2 complex. The molecular surface of leptin is coloured according to the surface hydrophobicity (blue, hydrophilic; green, hydrophobic). The CRH2 model is presented as ribbons, the Cα atom and heavy side chain atoms of residues I501, F502, L503, L504S and D615 are displayed as white sticks. L504 of CRH2 fits into the hydrophobic cleft of leptin and interacts with L13 and L86 of leptin.

Article Snippet: Expression was checked by anti-HA western blot analysis and quantified using an anti-mouse leptin ELISA kit (R&D systems) (Peelman et al., 2004b).

Techniques:

Journal: Journal of cell science

Article Title: Mapping of the interface between leptin and the leptin receptor CRH2 domain.

doi: 10.1242/jcs.02386

Figure Lengend Snippet: Fig. 10. Molecular models of mouse LR CRH2. (A) CRH2 residues that become buried in the leptin/CRH2 interface, coloured according to the area that becomes buried (cyan, <25 Å2; blue, 25-50 Å2; green, >50 Å2). (B) CRH2 residues that lead to a drastic increase of leptin binding to CRH2 and LR activation upon mutation.

Article Snippet: Expression was checked by anti-HA western blot analysis and quantified using an anti-mouse leptin ELISA kit (R&D systems) (Peelman et al., 2004b).

Techniques: Binding Assay, Activation Assay, Mutagenesis

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Adipokine Profile in Patients with Type 2 Diabetes Depends on Degree of Obesity

doi: 10.12659/MSM.904318

Figure Lengend Snippet: Leptin/adiponectin ratio in control group and type 2 diabetic patients divided according to BMI value. K – control group, group I – type 2 diabetic patients with normal body weight, group II – type 2 diabetic patients with overweight, group III – type 2 diabetic patients with obesity and group IV – type 2 diabetic patients with severe obesity. * p<0.05 vs. group K; ** p<0.01 vs. group K; # p<0.05 vs. group I.

Article Snippet: The plasma concentrations of the studied adipokines were determined by immunoenzymatic methods using Human Leptin Quantikine ELISA Kit (R&D Systems, USA), Human Total Adiponectin/Acrp30 Quantikine ELISA (R&D Systems, USA), and Human Resistin Quantikine ELISA Kit (R&D Systems, USA), as well as Human Nicotinamide phosphoribosyltransferase, NAmPRTase ELISA Kit (Wuhan EIAab Science, China) for leptin, adiponectin, resistin, and visfatin, respectively.

Techniques: Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 4. Inhibition of mTOR with rapamycin inhibits the expression of leptin and LepR in Teffs and at the systemic level. (A) Proliferation of hu- man Teffs, pretreated or not with rapamycin or anti- leptin neutralizing mAb or both, before anti-CD3/ CD28 stimulation. Data are mean 6 SD (n = 20). *p , 0.005, **p , 0.0001. (B) Proliferation of Teffs, pretreated or not with rapamycin before treatment with leptin (100 ng/ml), before anti-CD3/ CD28 stimulation. Data are mean 6 SD (n = 10). *p , 0.005, **p , 0.0001. (C) Confocal micros- copy of human Teffs, pretreated or not with rapamycin in the presence of anti-CD3/CD28 stimulation, and stained for leptin (green) and LepR (red). Representative of three independent experi- ments. Immunofluorescence images were acquired in the green, red, and blue channels at a resolution of 1024 3 1024 pixels. (D) Real-time PCR for leptin in human Teffs, pretreated or not with rapa- mycin, in the presence of anti-CD3/CD28 stimu- lation. Data are mean 6 SD (n = 5). *p , 0.001. (E) Serum leptin levels in patients with acquired cystic kidney disease, chronically treated with rapamycin, analyzed after 6 and 12 mo of mTOR inhibition treatment (black line). BMI in all of the treated patients at the different time points is represented by the gray bars. Data are mean 6 SD (n = 15 patients/group). *p , 0.05. (F) Serum leptin levels in vehicle or rapamycin-treated mice analyzed at different time points. Data are mean 6 SD (n = 5 mice/group). *p , 0.05.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Inhibition, Expressing, Staining, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 5. Leptin activates the mTOR pathway and controls Teff pro- liferation. (A) Immunoblot for p-STAT3, p-AKT, and p-S6 on Baf/3 cells stably transfected with the long form of human leptin receptor, treated or not with leptin for 30 or 60 min, in the presence or absence of LY294002 or rapamycin. One representative of three independent experiments. (B) Im- munoblot for p-mTOR, p-p70S6K, p-S6, and p-STAT3 on unstimulated Teffs treated or not with recombinant leptin for 1 h. One representative of five independent experiments. (C) Immunoblot for p-mTOR, p-p70S6K, p-S6, and p-STAT3 on Teffs in the presence or absence of anti-CD3/28 stimula- tion and treated or not with recombinant leptin or leptin-neutralizing mAb for 1 h. One representative of five independent experiments. (D) Ex vivo p-S6 expression in Teffs from db/+ mice or db/db mice (n = 3 mice/ group, representative of three independent experiments). (E) Ex vivo p-S6 expression in Teffs from ob/ob mice treated or not with recombinant leptin after 2 h from i.p. injection (n = 3 mice/group, representative of three independent experiments). (F) Flow cytometry for BrdU incorpo- ration in Teffs from the lymph nodes of ad libitum fed, 48-h fasted, and 48-h fasted + leptin eGFP-Foxp3 mice. Representative of two independent experiments (n = 3). The gray shaded graph represents the isotype-matched negative control. *p , 0.05 versus 48-h fasted mice, **p , 0.001 versus ad

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Western Blot, Stable Transfection, Transfection, Recombinant, Ex Vivo, Expressing, Injection, Flow Cytometry, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 6. In vivo leptin enhances Teff proliferation through mTOR activation. (A) Schematic model of the experimental design. Briefly, mice were treated daily with BrdU in basal conditions and upon Ag immunization with CFA; they were injected with a single dose of rapamycin or vehicle or leptin or rapamycin plus leptin 12 h before CFA priming, and the proliferation of Teffs was followed over time. Blood samples were obtained at day 5, and draining lymph nodes were harvested at days 8 and 12. Percentage (B) and absolute number (C) of Teffs gated on CD4+ cells in the lymph nodes from all of the groups of mice immunized with CFA. Data are mean 6 SD (n = 6). *p , 0.05, **p , 0.001. (D) Flow cytometry for BrdU incorporation in Teffs from the lymph nodes of mice pretreated in vivo with vehicle, rapamycin (RAPA), leptin, or RAPA and leptin 12 d after immunization with CFA. Representative of three independent experiments (n = 3 mice/group). *p , 0.005 versus RAPA pretreatment, **p , 0.001 versus vehicle. No significant difference between vehicle versus leptin or vehicle versus RAPA + leptin was detected. The gray shaded graph represents the isotype-matched negative control. (E) p-S6 expression in Teffs from lymph nodes of all groups of mice. Representative of three independent experiments (n = 3 mice/group). *p , 0.005 versus RAPA pretreatment, **p , 0.001 versus vehicle.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry, BrdU Incorporation Assay, Negative Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 8. Schematic model of leptin-induced mTOR activation in CD4+CD252FOXP32 Teffs. Under normal conditions (left panel), leptin, by binding its receptor (LepR), activates the mTOR pathway in CD4+CD252FOXP32 Teffs, resulting in an increase in T cell proliferation, Th1/Th17 cytokine se- cretion, and TCR signaling activation. The mTOR pathway itself is responsible for the autocrine leptin secretion by Teffs, which, in turn, sustains their own proliferation. Rapamycin pretreatment (middle) inhibits activation of the leptin-mediated mTOR pathway, resulting in the inhibition of Teff proliferation, a decrease in Th1/Th17 cytokine production, and TCR activation. Moreover, mTOR inhibition deceases leptin production and secretion by Teffs. Similar results can be detected in LepR-deficient Teffs, in which mTOR-pathway activity is impaired because of a lack of proper leptin signaling. These data sustain the hypothesis that rapamycin pretreatment and LepR deficiency share a common cellular, biochemical, and gene-expression profile, suggesting the presence of a convergence between leptin and mTOR at the signaling-pathway level to drive and control Teff responses.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Activation Assay, Binding Assay, Inhibition, Activity Assay, Gene Expression, Control

Journal: European Journal of Inflammation

Article Title: Lipopolysaccharide influence on leptin hormone and tumor necrosis factor-alpha release from human adipose tissue

doi: 10.1177/2058739218774975

Figure Lengend Snippet: Figure 4. LPS influence on TNF-α secretion. SAT explants derived from each patient were incubated in triplicate with or without 10 µg/mL of LPS for (a) 2 h and (b) 20 h. Secreted quantities of leptin in media were determined by ELISA. Results were depicted as relative quantities (RQs) compared to the control (without LPS; C). ****P < 0.0001 versus control. Error bars, SEM (N = 8).

Article Snippet: Human leptin DuoSet enzymelinked immunosorbent assay (ELISA) kit, substrate solution were purchased from R&D systems (USA), and ELISA plates were purchased from Greiner Bio-One (Germany).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: European Journal of Inflammation

Article Title: Lipopolysaccharide influence on leptin hormone and tumor necrosis factor-alpha release from human adipose tissue

doi: 10.1177/2058739218774975

Figure Lengend Snippet: Figure 2. Role of BMI in leptin response. SAT explants derived from each patient were incubated in triplicate for 20 h with or without 10 µg/mL of LPS. (a) The correlation between BMI and leptin response to LPS was determined employing Pearson correlation test. Pearson correlation coefficient was 0.8 and *P < 0.05. (b) Explants derived from low BMI patients (BMI ⩽ 25) responded to LPS treatment, while explants derived from high BMI patients (BMI > 25) did not respond. Secreted quantities of leptin in media were determined by ELISA. Results were depicted as relative quantities (RQs) compared to the control (without LPS; C). ****P < 0.0001 versus control. #P < 0.0001 versus BMI ⩽ 25. Error bars, SEM (N = 9).

Article Snippet: Human leptin DuoSet enzymelinked immunosorbent assay (ELISA) kit, substrate solution were purchased from R&D systems (USA), and ELISA plates were purchased from Greiner Bio-One (Germany).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: European Journal of Inflammation

Article Title: Lipopolysaccharide influence on leptin hormone and tumor necrosis factor-alpha release from human adipose tissue

doi: 10.1177/2058739218774975

Figure Lengend Snippet: Figure 3. Role of (a) age and (b) gender in leptin response. SAT explants derived from each patient were incubated in triplicate for 20 h with or without 10 µg/mL of LPS. (a) Role of age, the correlation between age and leptin response to LPS was determined employing Pearson correlation test. Pearson correlation coefficient was 0.3 (P = 0.46). (b) Role of gender: patients were divided into two groups according to their gender. Secreted quantities of leptin in media were determined by ELISA. Results were depicted as relative quantities (RQs) compared to the control (without LPS; C, N = 9).

Article Snippet: Human leptin DuoSet enzymelinked immunosorbent assay (ELISA) kit, substrate solution were purchased from R&D systems (USA), and ELISA plates were purchased from Greiner Bio-One (Germany).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: European Journal of Inflammation

Article Title: Lipopolysaccharide influence on leptin hormone and tumor necrosis factor-alpha release from human adipose tissue

doi: 10.1177/2058739218774975

Figure Lengend Snippet: Figure 1. LPS influence on leptin secretion. SAT explants derived from each patient were incubated in triplicate with or without 10 µg/mL of LPS for (a) 2 h (N = 10) and (b) 20 h (N = 9). Secreted quantities of leptin in media were determined by ELISA. Results were depicted as relative quantities (RQs) compared to the control (without LPS; C). **P < 0.01 versus control. Error bars, SEM.

Article Snippet: Human leptin DuoSet enzymelinked immunosorbent assay (ELISA) kit, substrate solution were purchased from R&D systems (USA), and ELISA plates were purchased from Greiner Bio-One (Germany).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: European Journal of Inflammation

Article Title: Lipopolysaccharide influence on leptin hormone and tumor necrosis factor-alpha release from human adipose tissue

doi: 10.1177/2058739218774975

Figure Lengend Snippet: Figure 5. Role of (a) BMI and (b) age in TNF-α response. SAT explants derived from each patient were incubated in triplicate for 20 h with or without 10 µg/mL of LPS. Correlation between BMI and age on one hand and TNF-α response to LPS on the other hand was determined employing Pearson correlation test. Pearson correlation coefficients were −0.4 (P = 0.32) and 0.2 (P = 0.62) for both BMI and age, respectively. Secreted quantities of leptin in media were determined by ELISA. Results were depicted as relative quantities (RQs) compared to the control (without LPS; C, N = 8).

Article Snippet: Human leptin DuoSet enzymelinked immunosorbent assay (ELISA) kit, substrate solution were purchased from R&D systems (USA), and ELISA plates were purchased from Greiner Bio-One (Germany).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Protein engineering, design & selection : PEDS

Article Title: Engineering a pharmacologically superior form of leptin for the treatment of obesity.

doi: 10.1093/protein/gzh102

Figure Lengend Snippet: Fig. 1. Transient expression analysis of leptin fusion proteins. (a) Expression vectors for (1) muFc (control), (2) muFc–muLeptin, (3) muLeptin–muFc and (4) muLeptin linker-muFc were used to transfect 293 cells. Total cell lysates (C) and supernatants (S) were analyzed by western blot analysis using horseradish peroxidase (HRP)-conjugated anti-mouse Fc-g antisera (upper gel) and biotinylated anti-mouse leptin antibody (R&D Systems) (lower gel). Under reducing conditions, muFc and muFc–muLeptin have apparent molecular weights of 33 and 48 kDa, respectively. (b) Non-reducing SDS-PAGE comparison of the soluble fractions obtained from the expression of (1) muFc–muLeptin, (2) huFcg1–huLeptin, (3) huFcg2h–huLeptin and (4) huFcg2–huLeptin. The misfolding and aggregation of the huFcg2–huLeptin can clearly be seen, resulting from intermolecular cross-linking of the four disulfide bridges present in the human Fcg2 domain. By contrast, the modified Fcg2h variant with only two disulfide bridges shows the uniform and high-yield expression of a single molecular species.

Article Snippet: Total cell lysates (C) and supernatants (S) were analyzed by western blot analysis using horseradish peroxidase (HRP)-conjugated anti-mouse Fc-g antisera (upper gel) and biotinylated anti-mouse leptin antibody (R&D Systems) (lower gel).

Techniques: Expressing, Control, Western Blot, SDS Page, Comparison, Modification, Variant Assay

Journal: Food & Nutrition Research

Article Title: Cudrania tricuspidata water extract improved obesity-induced hepatic insulin resistance in db/db mice by suppressing ER stress and inflammation

doi: 10.3402/fnr.v59.29165

Figure Lengend Snippet: Levels of glucose, insulin, glucagon, and leptin in the C57BL/6J- db/db mice with dietary supplementation of Cudrania tricuspidata water extract

Article Snippet: Serum insulin, glucagon, and leptin were respectively measured with an Ultra Sensitive Mouse Insulin ELISA kit (Crystal Chem, Downers Grove, IL, USA), Glucagon Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA), and Mouse/Rat Leptin Quantikine ELISA kit (R&D Systems) according to the manufacturer's protocols.

Techniques: Control

Journal: Journal of Pediatric Gastroenterology & Nutrition

Article Title: Hyperadiponectinemia During Infliximab Induction Therapy in Pediatric Crohn Disease

doi: 10.1097/mpg.0000000000001876

Figure Lengend Snippet: FIGURE 1. Serum adipokine levels according to sex in healthy controls and in CD patients at baseline (week 0) and under infliximab therapy (weeks 2 and 14) displaying each patient individually and the median lines (n ¼ 18). (A) Resistin levels were higher in CD at baseline, decreased under therapy and showed no sex differences. (B) Leptin was significantly higher in girls at all time points and increased under IFX. (C) Adiponectin tended to be higher in girls, peaked 2 weeks after first IFX infusion and thereafter decreased. Differences were analyzed with Wilcoxon matched- pairs signed rank test. P < 0.05, P < 0.01, P < 0.001, P < 0.0001. CD ¼ Crohn disease, HC ¼ healthy controls, IFX ¼ infliximab.

Article Snippet: Resistin, leptin (Human Leptin and Resistin Quantikine ELISA Kits, R&D Systems, Minneapolis, MN) and total adiponectin (Total Adiponectin ELISA Kit, ALPCO Diagnostics, Salem, NH) serum levels were assessed with commercially available enzyme-linked immunosorbent assays (ELISA) according to the manufacturer’s instructions.

Techniques:

Journal: Molecular human reproduction

Article Title: Modulation of placental vascular endothelial growth factor by leptin and hCG.

doi: 10.1093/molehr/gag053

Figure Lengend Snippet: Figure 1. Release of hCG (% of controls) at 4 h from cytotrophoblastic cells incubated for 4 h with a range of concentrations of recombinant human leptin. Statistics were performed by analysis of variance and P values refer to differences compared with control (Leptin, 0). Error bars are SEM (n = 4 experiments in duplicate).

Article Snippet: Recombinant human leptin (rhLept) was purchased from R&D Systems (BuÈhlmann Laboratories, Switzerland). hCG (Profasi) was purchased from Ares±Serono (Switzerland).

Techniques: Incubation, Recombinant, Control

Journal: Molecular human reproduction

Article Title: Modulation of placental vascular endothelial growth factor by leptin and hCG.

doi: 10.1093/molehr/gag053

Figure Lengend Snippet: Figure 2. Release of vascular endothelial growth factor (VEGF) (% of controls) at 4 h from cytotrophoblastic cells incubated for 4 h with a range of concentrations of recombinant human leptin. Statistics were performed by analysis of variance and P values refer to differences compared with controls (Leptin, 0). Error bars are SEM (n = 4 experiments in duplicate).

Article Snippet: Recombinant human leptin (rhLept) was purchased from R&D Systems (BuÈhlmann Laboratories, Switzerland). hCG (Profasi) was purchased from Ares±Serono (Switzerland).

Techniques: Incubation, Recombinant

Journal: Molecular human reproduction

Article Title: Modulation of placental vascular endothelial growth factor by leptin and hCG.

doi: 10.1093/molehr/gag053

Figure Lengend Snippet: Figure 3. Release of leptin (% of controls) at 4 h from cytotrophoblastic cells incubated for 4 h with a range of concentrations of hCG (Profasi). Statistics were performed by analysis of variance and P values refer to differences compared with control (hCG, 0). Error bars are SEM (n = 4 experiments in duplicate).

Article Snippet: Recombinant human leptin (rhLept) was purchased from R&D Systems (BuÈhlmann Laboratories, Switzerland). hCG (Profasi) was purchased from Ares±Serono (Switzerland).

Techniques: Incubation, Control