Journal: Nature Communications

Article Title: Adipocyte OGT governs diet-induced hyperphagia and obesity

doi: 10.1038/s41467-018-07461-x

Figure Lengend Snippet: Adipocyte OGT drives diet-induced hyperphagia and obesity. a Genetic cross scheme. b Growth curves and c body composition of adipocyte-specific OGT knockout mice (FKO) and littermate control mice (Con) on chow ( n = 8 (Con, dashed line with empty square/stripped bar) or 11 (FKO, blue line/bar)) or HFD ( n = 11 (Con, dashed line with empty circle/empty bar) or 13 (FKO, red line/bar)). d Circulating levels of leptin and e wet tissue weights in male Con ( n = 6) and FKO ( n = 6) mice fed HFD for 9 weeks, or age-matched Con ( n = 8) and FKO ( n = 8) mice fed chow. BAT, interscapular brown adipose tissue. White adipose tissues include depots from perigonadal (pg), subcutaneous (sc), and retroperitoneal (rp) regions. f Energy intake of male mice on HFD ( n = 7 per genotype) or Chow ( n = 5 (Con) or n = 6 (FKO)) for 13 weeks. g Fecal energy content. h Daily energy intake. Male Con ( n = 10) and FKO ( n = 10) mice fed HFD for 6 weeks. i Energy expenditure of 5-week HFD-fed male Con ( n = 7) and FKO ( n = 9) mice. j Energy intake in a two-choice diet preference test. Male Con ( n = 9) and FKO ( n = 10) mice have been fed HFD for 7 or 16 weeks. k Growth curves of HFD-fed mice. Con (AL, ad libitum, n = 11), Con (PF, pair-fed to littermate FKO, n = 7), FKO (AL, n = 7). Data were presented as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, post hoc Sidak’s tests ( b (HFD Con vs. HFD FKO), i , k (Con PF vs. FKO AL)), Tukey’s tests ( c , d , f ), or two-tailed Student’s t-test ( e , g , h , j )

Article Snippet: Recombinant mouse leptin protein (5 mg/kg, R&D Systems cat# 498-OB) was reconstituted at 1 mg/mL in sterile 20 mM Tris-HCl, pH 8.0.

Techniques: Knock-Out, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Adipocyte OGT governs diet-induced hyperphagia and obesity

doi: 10.1038/s41467-018-07461-x

Figure Lengend Snippet: Adipocyte OGT drives diet-induced hyperphagia through transactivation of lipid desaturation in adipose tissue. a Gene expression analysis of de novo lipid desaturation and synthesis in adipose tissue (Chow for 15 weeks, Con n = 8, FKO n = 8; age-matched HFD for 9 weeks, Con n = 5, FKO n = 6). b Protein levels of SCD and OGT in adipose tissue. c Western blotting analysis in adipose tissue from 9-week-HFD-fed FKO and Con mice (Con n = 5, FKO n = 6, two representative biological replicates were shown). d Growth curves and e energy intake of (m)HFD-fed Con and FKO mice ( n = 8–10 per group). mHFD denotes a mono-unsaturated fat-fortified high-fat diet that changes the saturated fat-rich soybean oil fraction into the mono-unsaturated fat-rich canola oil as illustrated in the diagram ( d ). f Contents of palmitate (C16:0) and palmitoleate (C16:1) DAG in adipose lipidome, and circulating levels of leptin ( g ), or of FGF21 ( h ) in mice fed HFD or mHFD for 15 weeks. Data were presented as mean ± s.e.m. n.s. not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, post hoc Sidak’s tests ( a , e – h ), or # P < 0.01 HFD Con vs. HFD FKO, two-tailed Student’s t-test ( a ). A dashed line in ( e – h ) indicates that data from HFD study and mHFD study are presented in a side-by-side manner, and inference may not be made between different diets

Article Snippet: Recombinant mouse leptin protein (5 mg/kg, R&D Systems cat# 498-OB) was reconstituted at 1 mg/mL in sterile 20 mM Tris-HCl, pH 8.0.

Techniques: Gene Expression, Western Blot, Two Tailed Test

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Leptin regulates gallbladder genes related to absorption and secretion.

doi: 10.1152/ajpgi.00389.2006

Figure Lengend Snippet: Fig. 1. Leptin administration decreased gallbladder volume, bile sodium concentration, and pH.

Article Snippet: For leptin replacement, mice received daily intraperitoneal injections of either recombinant murine leptin (R&D Systems, Minneapolis, MN) at a dose of 5 g/g body wt or saline as a control for 4 wk.

Techniques: Concentration Assay

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG improved glucose tolerance in the absence of leptin in Lep ob mice and exhibited no effect in LepR-deficient Lepr db mice. ( A ) Body weight of Lep ob male mice in groups before and after treatment with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 26 days. Mice were on regular chow diet. Unpaired t -test, n = 7/group. ns, not significant. ( B , C ) Fat ( B ) and lean body ( C ) composition in same groups of mice at the end of the study was measured by Echo-MRI. Fat and lean mass are shown as % of the total weight (100%). ( D , E ) Glucose tolerance test (GTT) was performed in fasted Lep ob mice treated with PBS (Veh, open circles) or EREG (closed circles) ( n = 7 per group). GTT kinetics ( D ) and area under the curve (AUC) ( E ) are shown. Statistical significance was examined by ANOVA ( D ) and Student’s t -test ( E ). ( F ) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t -test. ( G ) Weight before and after treatment of Lepr db male mice with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 4 weeks ( n = 6 per treatment). Mice were on regular chow. Unpaired Student’s t -test, n = 6/group. ( H , I ) Fat ( H ) and lean body ( I ) composition (% of total weight) in the same groups of mice at the end of the study were measured by Echo-MRI. ( J , K ) GTT kinetics ( J ) and AUC ( K ) were obtained from Lepr db mice treated with PBS (Veh, open circles) or EREG (closed circles). ANOVA ( J ) and Student’s t -test ( K ). ( L ) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t -test.

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG regulated glucose uptake via binding with LepR in Lep ob mice. ( A ) EREG and insulin tolerance test in Lep ob mice ( n = 5 per group) treated with a single intraperitoneal injection of insulin (0.012 IU/g BW, triangle dashed line) or EREG (80 ng/g BW, closed circles. Asterisks, significant (* p < 0.05) compared to glucose levels before EREG treatment. # Hashtag, significant difference in glucose levels 30 min after treatment with EREG or insulin. Unpaired Student’s t -test. ( B ) Area under the curve (AUC) quantification of insulin (hatched bar) and EREG (black bar) tolerance tests. Unpaired Student’s t -test, ns . ( C ) GTT kinetics were measured in Lep ob mice ( n = 5 per treatment) treated with a single injection of PBS (Veh, open circles) or EREG (closed circles). Student’s t -test. * p < 0.05 from comparison between control and EREG treated mice at each time point. ( D ) Area under the curve (AUC) quantification of insulin (hatched bar) and EREG (black bar) tolerance tests. Unpaired Student’s t -test. ( E , F ). Immunoprecipitation of LepR was performed with anti-EREG antibody using homogenates from subcutaneous fat ( C ) and visceral fat ( D ). Fat tissue was isolated from non-treated Lep ob (Veh) as well as Lep ob mice 15 min after injection of EREG (50 ng/mL).

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Binding Assay, Injection, Comparison, Control, Immunoprecipitation, Isolation

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG-stimulated glucose uptake was dependent on LepR but independent of EGFR. ( A , B ) Fluorescently-labelled (FD) glucose uptake was measured in stromal vascular fraction (SVF) cells isolated from visceral tissues of Lepr db ( A ) or Lep ob mice ( B ). Cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), human insulin (Ins, 10 µg/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. For inhibition experiment, Lep ob SVF cells were pre-treated with EGFR inhibitor (EGFR-I, AST-1306, 10 µM) or vehicle (Veh, DMSO) for 40 min. Data are shown as a percentage of Veh-treated control (100%, n = 8 per treatment). Unpaired Student’s t -test. ( C – E ) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes. ( C ) Preadipocytes were treated with vehicle, human insulin (Ins, 10 µg/mL), and mouse EREG (50 ng/mL) for 30 min (mean ± SEM, n = 6, t -test). ( D ) Time-dependent uptake of FD-glucose in 3T3-L1 preadipocytes stimulated with human insulin (Ins, 10 µg/mL), mouse leptin (Lep, 200 ng/mL), and mouse EREG (50 ng/mL). Data are shown (mean ± SEM, n = 8, t -test) as % of glucose uptake compared to control cells at the same time point (Veh, 100%). ( E ) Concentration-dependent increase in FD-glucose uptake by 3T3-L1 preadipocytes stimulated with different concentrations of mouse EREG. Data are shown as a percentage of Veh-treated control (100%, n = 6 per concentration). * p < 0.05, significant differences compared to the vehicle group, one-way ANOVA). ( F ) NIH-3T3 preadipocytes were transiently transfected with pB- Glut4 -7myc-GFP and stimulated with vehicle, Ins (10 µg/mL), EREG (50 ng/mL) for 60 min. Data show representative fluorescent images of GFP-labeled GLUT4 selected from three independent experiments. 10× magnification. Yellow arrow shows GFP-labeled GLUT4 that was translocated to the cellular membrane. ( G ) Quantification of GFP was performed in adipocytes of similar size ( n = 10) in each group.

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Isolation, Inhibition, Control, Concentration Assay, Transfection, Labeling, Membrane

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG mediates glucose uptake via PI3K with transient activation of ERK. ( A ) FD-glucose uptake in 3T3-L3 preadipocytes treated with or without EREG (50 ng/mL) and in the presence of inhibitors for EGFR-I (AG1478, 10 µM), EGFR and ErbB2 (AST-1306 or CI-1033 10 µM), dual IR/IGF-1R inhibitor (BMS 536924, 1 µM), and SRC-I, AZM475271, 1 µM) for 30 min. Cells were starved for 90 min before stimulation. Dashed line shows glucose uptake in the presence of insulin (Ins, 10 µg/mL). ( B ) FD-glucose uptake was measured in mouse 3T3-L1 preadipocytes with or without EREG (50 ng/mL) and inhibitors of MEK1/2 and PI3K (MEK1/2-I, U0126 10 μM, and PI3K-I, wortmannin 200 nM). Data (mean ± SD, n = 6) are shown as a percentage of control (Veh 100%). Unpaired Student’s t -test. ( C ) 3T3-L1 preadipocytes were stimulated with EREG at different concentrations (0–100 ng/mL) for 5 or 15 min. The total and phosphorylated levels of AKT, STAT3, STAT5, and ERK were measured by Western blot in duplicates. Data are shown in a representative Western blot. ( D ) The kinetics of pERK expression was quantified based on the Western blots. pAKT, p-STAT3, and p-STAT5 analysis are described in . Pearson correlation analysis. ( E ) 3T3-L1 preadipocytes were stimulated with or without EREG or EGF (50 ng/mL, each) for 30 min in the presence and absence of EGFR inhibitor AST1306 (100 nM), and antibody against mouse LepR (Invitrogen, PA1-053, 10 μg/mL). For inhibition, cells were pre-treated 30 min before EREG and EGF stimulation. ( F ) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes pre-treated with either Veh (DMSO) or ERK inhibitors (U0126, SCH772984, or DEL 22379, each 10 µM in DMSO) for 40 min. Then, cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. Data are shown as a percentage of Veh-treated control (100%, n = 7 per group). Unpaired Student’s t -test. ns , not significant ( p > 0.05).

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Activation Assay, Control, Western Blot, Expressing, Inhibition

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: Kinetics of the changes in LepR film thickness in the presence of leptin ( A ) or EREG ( B ). Film thickness was measured using QCM and quantified based on the binding kinetics to a gold sensor.

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Binding Assay

Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: Evolutionary analysis of EREG binding to LepR. ( A – E ) EREG docking to LepR. Evolutionary analysis of 175 open The dependence of EREG-mediated glucose uptake on the ERK phosphorylation cascade was examined using (1) a specific inhibitor of ERK1/2 SCH772984 , (2) an inhibitor of ERK dimerization DEL-22379 , and (3) a selective inhibitor of MEK1 and MEK2 U0126 . All inhibitors increased basal glucose uptake, which was further increased by leptin ( F). The inhibition of ERK1/2 and MEK1/2 as well as ERK dimerization prevented stimulatory effect of EREG on FD-glucose uptake but did not decrease it beyond the levels seen in the control cells. Although transient ERK phosphorylation occurred in response to EREG stimulation, this pathway was dispensable for glucose uptake and dependent on PI3K and may be other pathways ( B and ). ( F ) Hypothetic mechanism suggesting EREG as an alternative ligand for both EGFR and LepR. The canonic leptin/LepR response can induce JAK/STAT3 signaling and required the long form of LepR. The alternative binding of EREG to LepR can induce ERK and PI3K activation increasing GLUT4 translocation and glucose uptake, but not the other canonic effects of leptin, including the regulation of appetite and energy expenditure.

Article Snippet: Mouse recombinant leptin receptor (LepR) protein (1.6 pM in PBS; R&D systems, 497-LR/CF) was added to establish a monolayer, followed by the addition of mouse leptin (1.6 fM in PBS; Crystal Chem, Elk Grove Village, IL, USA, 90030-B) or mouse EREG (1.6 fM).

Techniques: Binding Assay, Phospho-proteomics, Inhibition, Control, Activation Assay, Translocation Assay

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 1. Preparation of recombinant murine leptin and its PASylated fusion proteins. (A) Expression plasmids are based on the generic pASK75 vector comprising the chemically inducible tetp/o promoter, an OmpA signal sequence for periplasmic secretion, a His6-tag for affinity purification, a short linker allowing insertion of the PAS gene cassette, and the coding region for mature murine leptin. The preparation of PASylated leptin versions from E. coli in a functional monomeric form was confirmed by SDS−PAGE (B) and ESI-TOF mass spectrometry (C). (D) The correct fold of the leptin moiety and the secondary structure of the attached PAS polypeptide were investigated by CD spectroscopy.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Recombinant, Expressing, Plasmid Preparation, Sequencing, Functional Assay, SDS Page, Mass Spectrometry, Circular Dichroism

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 2. Size measurements for PASylated leptin versions. The effect of the genetic fusion of leptin with the conformationally disordered PAS polypeptide was investigated by analytical size exclusion chromatography (SEC) and dynamic light scattering (DLS). (A) SEC in the presence of phosphate-buffered saline (PBS) resulted in a single peak with decreasing elution volume for PAS fusions with increasing number of amino acid residues. Comparison with a half- logarithmic calibration curve (inset) led to the apparent molecular masses listed in Table 1. (B) Absolute and relative molecular sizes determined by both SEC and DLS measurements in the presence of PBS vs the length of the PAS tag.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Size-exclusion Chromatography, Saline, Comparison

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 3. Investigation of receptor binding activity for PASylated leptin by real-time SPR analysis and in a cell culture reporter assay. (A) Dilution series of leptin versions from 64 to 1 nM were applied in PBS/T to a sensor chip charged with a murine LepRb-Fc fusion protein. Biacore sensorgrams were fitted to a 1:1 Langmuir binding model, and resulting dissociation constants and kinetic parameters are listed in Table 1. (B) Visualization of the parameters from plot A in a kon/koff plot. (C) Leptin versions were applied to HEK cells transiently co-transfected with an LepRb-expression vector and a STAT3- responsive Photinus luciferase gene. The leptin versions were applied in a dilution series in duplicate, and after incubation for 18 h luciferase activity was assayed (N = 4). Luminescence values (normalized against constitutively coexpressed Renilla luciferase) for each test protein were fitted to a sigmoidal dose−response curve; resulting EC50 values are listed in Table 1.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Binding Assay, Activity Assay, Cell Culture, Reporter Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Incubation

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 4. PK of recombinant murine leptin and its PASylated versions in mouse plasma. C57BL6/J mice (N = 9) were injected ip with a protein dose of 287 nmol·kg−1 bw. The leptin concentration in plasma was quantified in a sandwich ELISA with antibodies directed against the adipokine, using appropriate calibration curves, and data were plotted in a semilogarithmic fashion against the time of sampling post injection. The PK profiles show distinct resorption and elimination phases; deduced terminal half-lives, clearance, and AUC parameters are listed in Table 1. Plot of the same data in a linear fashion (inset) illustrates the strongly increased AUC, indicating high bioavailability of PASylated leptins.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Recombinant, Clinical Proteomics, Injection, Concentration Assay, Sandwich ELISA, Sampling

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 5. Hypothalamic receptor signaling of PASylated murine leptin versions. The PD of unmodified recombinant leptin and its PASylated versions was investigated in lean C57BL/6J mice by quantifying STAT3 phosphorylation in the hypothalamus after ip injection of 287 nmol·kg−1 bw for each test protein. (A) Downstream signaling in neurons of the hypothalamus was examined by immunostaining of phosphorylated (pSTAT3) and total STAT3 (tSTAT3) on a Western blot. For determination of basal STAT3 phosphorylation, a group of mice that had only received PBS injections was included (samples from these mice are labeled on the Western blot as “0”). The bands were quantified by densitometry (N = 3), and the change in the pSTAT3/tSTAT3 ratio relative to the mock samples is shown in the lower panel. (B) The area under the curve from the test proteins shown in panel A as well as some other PASylated leptin versions (see Table 1) was plotted against the corresponding terminal plasma half- life (N = 3), revealing a linear correlation.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Recombinant, Phospho-proteomics, Injection, Immunostaining, Western Blot, Labeling, Clinical Proteomics

Journal: Molecular pharmaceutics

Article Title: PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

doi: 10.1021/mp5007147

Figure Lengend Snippet: Figure 6. Physiological effects of PASylated murine leptin versions. The PD of unmodified recombinant leptin, as well as the superactive mouse leptin antagonist (SMLA), and its PASylated versions was investigated in mice with regard to daily food consumption (A) and body weight change (B) using a phenotyping device (N = 8). Lean C57BL/6J mice were injected with a single ip dose of 287 nmol·kg−1 bw test protein on day 0.

Article Snippet: To measure the biochemical activity of leptin as well as its PASylated versions, a mouse leptin receptor Fc chimera (R&D Systems, Minneapolis, MN) was immobilized in 10 mM Na-acetate pH 5.5 at a concentration of 5 μg/mL on a CMD 2D sensorchip (Xantec, DOI: 10.1021/mp5007147 Mol.

Techniques: Recombinant, Injection

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Pluronic modified leptin with increased systemic circulation, brain uptake and efficacy for treatment of obesity.

doi: 10.1016/j.jconrel.2014.05.044

Figure Lengend Snippet: Fig. 3. Purification and characterization of leptin–P85 conjugates. (A) Lep(ss)–P85 conjugate contained unmodified leptin and leptin modified with different numbers of P85 chains. SEC elution profile in TSKgel G2000SW column showed separation of leptin–P85 conjugates from unmodified leptin. (B) SDS-PAGE and (C) MALDI-TOF spectra further characterized the col- lected fractions at 8.8 min and 9.5 min as leptin conjugates modified with multiple P85 chains (Lep(ss)–P85(H)) and single P85 chain (Lep(ss)–P85(L)), respectively.

Article Snippet: Mouse recombinant leptin (Lep) and a chimera leptin receptor (ObRFc) were purchased from R&D Systems (Minneapolis, MN).

Techniques: SDS Page

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Pluronic modified leptin with increased systemic circulation, brain uptake and efficacy for treatment of obesity.

doi: 10.1016/j.jconrel.2014.05.044

Figure Lengend Snippet: Fig. 5. Disulfide bond stability in Lep(ss)–P85 conjugate upon its exposure to serum. Western blot analysis shows that leptin or Lep(ss)–P85 remained stable after incubating with serum for up to 24 hr.

Article Snippet: Mouse recombinant leptin (Lep) and a chimera leptin receptor (ObRFc) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Western Blot

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Pluronic modified leptin with increased systemic circulation, brain uptake and efficacy for treatment of obesity.

doi: 10.1016/j.jconrel.2014.05.044

Figure Lengend Snippet: Fig. 7. Serum clearance of leptin–pluronic conjugates during shorter (A and B) and longer time periods of study (C and D). The two lines in panels A and B were significantly different (p b 0.05). Both 125I-Lep(ss)–P85(L) and 125I-Lep(ss)–P85(H) showed significantly longer circulation time than that of co-injected 131I-leptin. The serum disappearance (T1/2) was 40.75 min for 125I-Lep(ss)–P85(L) (r = 0.75, p b 0.001; n = 1 ~ 2 mice/time point), 75.80 min for 125I-Lep(ss)–P85(H) (r = 0.73, p b 0.0005; n = 1 ~ 2 mice/time point) and 11.98 min for 131I-Lep (r = 0.64, p p b 0.005; n = 1 ~ 2 mice/time point). The vascular volume distribution (Vi), as shown by the y intercept was not significantly different. Panels C and D further showed that initial serum clearance was a linear distribution for both of leptin analogs followed by a plateau phase.

Article Snippet: Mouse recombinant leptin (Lep) and a chimera leptin receptor (ObRFc) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Injection

Journal: Degenerative Neurological and Neuromuscular Disease

Article Title: Neuroprotective Effects of Leptin on the APP/PS1 Alzheimer’s Disease Mouse Model: Role of Microglial and Neuroinflammation

doi: 10.2147/DNND.S427781

Figure Lengend Snippet: Influence of leptin on the levels of Aβ1-40 (Left) and Aβ1-42 (Right) in the adult + leptin group, adult + saline group, aged + leptin group and aged + saline group.

Article Snippet: Recombinant mouse leptin (Elabscience.

Techniques: Saline

Journal: Degenerative Neurological and Neuromuscular Disease

Article Title: Neuroprotective Effects of Leptin on the APP/PS1 Alzheimer’s Disease Mouse Model: Role of Microglial and Neuroinflammation

doi: 10.2147/DNND.S427781

Figure Lengend Snippet: Western blotting analysis of synaptophysin expression in the adult + leptin group, adult + saline group, aged + leptin group and aged + saline group.

Article Snippet: Recombinant mouse leptin (Elabscience.

Techniques: Western Blot, Expressing, Saline

Journal: Degenerative Neurological and Neuromuscular Disease

Article Title: Neuroprotective Effects of Leptin on the APP/PS1 Alzheimer’s Disease Mouse Model: Role of Microglial and Neuroinflammation

doi: 10.2147/DNND.S427781

Figure Lengend Snippet: Semi-quantitative analysis of synaptophysin expression in the adult + leptin group, adult + saline group, aged + leptin group and aged + saline group.

Article Snippet: Recombinant mouse leptin (Elabscience.

Techniques: Expressing, Saline

Journal: Degenerative Neurological and Neuromuscular Disease

Article Title: Neuroprotective Effects of Leptin on the APP/PS1 Alzheimer’s Disease Mouse Model: Role of Microglial and Neuroinflammation

doi: 10.2147/DNND.S427781

Figure Lengend Snippet: Leptin increased microglial cell number in the hippocampus of adult mice, but not aged mice. ( A – D ) Representative images of Iba-1+ cells, in the hippocampus sections of the mice. ( E – G ) Quantitative analysis of Iba-1+ stained cells.

Article Snippet: Recombinant mouse leptin (Elabscience.

Techniques: Staining

Journal: Degenerative Neurological and Neuromuscular Disease

Article Title: Neuroprotective Effects of Leptin on the APP/PS1 Alzheimer’s Disease Mouse Model: Role of Microglial and Neuroinflammation

doi: 10.2147/DNND.S427781

Figure Lengend Snippet: Influence of leptin on the levels of IL-1β ( Left ), IL-6 ( Right ) in the adult + leptin group, adult + saline group, aged + leptin group and aged + saline group.

Article Snippet: Recombinant mouse leptin (Elabscience.

Techniques: Saline

Journal: Degenerative Neurological and Neuromuscular Disease

Article Title: Neuroprotective Effects of Leptin on the APP/PS1 Alzheimer’s Disease Mouse Model: Role of Microglial and Neuroinflammation

doi: 10.2147/DNND.S427781

Figure Lengend Snippet: Western blotting analysis of leptin receptor expression in the adult + leptin group, adult + saline group, aged + leptin group and aged + saline group.

Article Snippet: Recombinant mouse leptin (Elabscience.

Techniques: Western Blot, Expressing, Saline

Journal: Degenerative Neurological and Neuromuscular Disease

Article Title: Neuroprotective Effects of Leptin on the APP/PS1 Alzheimer’s Disease Mouse Model: Role of Microglial and Neuroinflammation

doi: 10.2147/DNND.S427781

Figure Lengend Snippet: Semi-quantitative analysis of leptin receptor expression in the adult + leptin group, adult + saline group, aged + leptin group and aged + saline group.

Article Snippet: Recombinant mouse leptin (Elabscience.

Techniques: Expressing, Saline

Journal: European journal of immunology

Article Title: Involvement of leptin signaling in the survival and maturation of bone marrow-derived dendritic cells.

doi: 10.1002/eji.200636602

Figure Lengend Snippet: Figure 1. Expression of Ob-R in primary and BM-derived DC. (A) Expression of endogenous Ob-Rb (413 bp) and the longer insertion product (519 bp) was detected by semi-quantitative RT-PCR in both spleen- and BM-derived DC (sorted for CD11c+MHC-II+) from WT control mice and db/db mice, respectively. (B) Staining of surface Ob-R on CD11c+ DC derived from BM culture of control mice. Representative results from three independent experiments are shown. (C) Analysis of leptin transcript expression in BM-derived immature (iDC) and LPS-matured DC (mDC) from days 7 and 9, respectively, of BM culture using semi-quantitative RT-PCR analysis. Adipocytes served as a positive control for leptin transcript expression. (D) BM-derived DC were starved without serum and incubated in the absence or presence of recombinant leptin at the indicated doses for 30 min. To test the specificity of the Ob-R chimera, 5000 ng/mL Ob-R:Fc was used to pre-treat DC for 30 min before incubation with recombinant leptin as shown in the last sample lane. Protein lysates were subjected to immunoblotting with anti-phospho-JAK2Tyrosine1007/1008. The same blot was stripped and re-probed with anti-JAK2. GAPDH protein detection was used as a loading control.

Article Snippet: Recombinant mouse leptin R/Fc chimera (Ob-R:Fc) was purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Control, Staining, Positive Control, Incubation, Recombinant, Western Blot

Journal: European journal of immunology

Article Title: Involvement of leptin signaling in the survival and maturation of bone marrow-derived dendritic cells.

doi: 10.1002/eji.200636602

Figure Lengend Snippet: Figure 4. Reduced survival capacity of db/db DC. (A) BM-derived DC cultures were stained with anti-Annexin V-FITC, anti-CD11c-PE and anti-MHC-II-Cy5 for the detection of apoptosis among immature DC (iDC) and LPS-matured DC (mDC) by flow cytometry on days 7 and 9 of BM culture, respectively. The black line represents Annexin V staining gated on CD11c+MHC-II+ cells, and the grey line indicates autofluorescence. Representative results from one of four separate experiments are shown. (B) The proliferative capacity of BM-derived DC from db/db and control mice was determined by [3H]-thymidine incorporation. DC cultures of control BM were treated with the Ob-R blocker Ob-R:Fc for 24 h prior to addition of [3H]-thymidine. Levels of [3H]-incorporation (cpm) were similar in cells from db/db and control mice. (C) CFSE-labeled DC were cultured for 2 days before staining with anti-CD11c-PE and anti-MHC-II-Cy5. Cells were gated on CD11c+MHC-II+ populations for the assessment of CFSE profiles of db/db and control DC (thick grey and thin black line, respectively; left) or control DC in the presence of leptin blocker or control Ig (thick black and thin grey line, respectively; right). One representative experiment of four is shown. (D) LPS-matured CD11c+MHC-IIhigh DC (1 106 cells) were purified by sorting on day 9 and cultured with complete medium supplemented with 20 ng/mL GM-CSF with or without Ob- R:Fc treatment. Viable cell counts, determined by Trypan blue staining for the exclusion of dead cells, are shown at the indicated days post-sorting. Data are from three separate experiments (mean SEM, **p<0.01).

Article Snippet: Recombinant mouse leptin R/Fc chimera (Ob-R:Fc) was purchased from R&D systems (Minneapolis, MN).

Techniques: Derivative Assay, Staining, Flow Cytometry, Control, Labeling, Cell Culture, Purification

Journal: European journal of immunology

Article Title: Involvement of leptin signaling in the survival and maturation of bone marrow-derived dendritic cells.

doi: 10.1002/eji.200636602

Figure Lengend Snippet: Figure 6. Impaired immunostimulatory capacity of both primary and in vitro-derived db/db DC in inducing T cell proliferation. (A) Proliferation of allogeneic responder T cells was used to determine the immunostimulatory capacity of DC. Both db/db and control BM-derived CD11c+MHC-IIhigh LPS-matured DC (day 9 of culture) were sorting-purified and co-cultured with allogeneic T cells for 3 days in the presence or absence of the leptin blocker Ob-R:Fc. T cell proliferation was determined by [3H]-incorporation. Data are the mean values of one representative set of triplicate samples from the MLR. (B) Sorting-purified primary CD11c+MHC-II+ splenic DC from db/db and control mice and CFSE-labeled CD4+ T cells were co-cultured for MLR. Cells were incubated for 3, 4 and 5 days before the proliferation of T cells was determined by flow cytometry. The numbers indicated in the histograms represent the percentages of dividing cells among CFSE-labeled CD4+ T cells. Representative histograms from each triplicate set of cultures after 3 and 5 days of co-culture are shown.

Article Snippet: Recombinant mouse leptin R/Fc chimera (Ob-R:Fc) was purchased from R&D systems (Minneapolis, MN).

Techniques: In Vitro, Derivative Assay, Control, Purification, Cell Culture, Labeling, Incubation, Flow Cytometry, Co-Culture Assay