leptin  (R&D Systems)

Journal: Nature communications

Article Title: Pharmacological and physiological activation of TGR5 in the NTS lowers food intake by enhancing leptin-STAT3 signaling.

doi: 10.1038/s41467-025-60331-1

Figure Lengend Snippet: Fig. 2 | Activation of TGR5 by CCDC enhances leptin-LepR-STAT3 signaling in the NTS to lower food intake. a Cumulative food intake of NTS CMC (n = 15; data replotted from Fig. 1b, h), leptin (n = 10; data replotted from Fig. 1h), CCDC (n = 10; data replotted from Fig. 1b) or CCDC+leptin (n = 11) HF rats 24 h after food was given back. b Representative western blot images of p-STAT3, t-STAT3, and GAPDH protein levels in NTS CCDC 5 min acute infusion, followed by a 3 h wait, then NTS CMC or leptin 5 min acute infusion, and a subsequent 30 min waiting time in HF rats. c Representative images (scale bar: 100 μm) of immunohistological staining for p-STAT3 with DAPI (top) and cFOS (bottom) in NTS CCDC 5 min acute infusion, followed by a 3 h wait, then NTS CMC or leptin 5 min acute infusion, and a sub- sequent 30 min waiting time in HF rats. d Relative NTS Tgr5 mRNA expression of NTS shMM (n = 6) or shTgr5 (n = 6) rats. e Relative NTS PKA activity of NTS CMC shMM (n = 6), CCDC shMM (n = 6), CMC shTgr5 (n = 6), or CCDC shTgr5 (n = 6) HF rats, measured 3 h after 5 min acute infusions. f Cumulative food intake of NTS CMC shMM (n = 5), CCDC+leptin shMM (n = 10), CMC shTgr5 (n = 5), or CCDC+leptin

Article Snippet: Leptin (497-LR, R&D Systems) was dissolved firstly in 0.9% saline and thenwith/withoutCCDC (final CCDCconcentration 100μM) in 1% CMC to the concentration of 153μM (equivalent to 0.5μg/site) as this amount of leptin infused into the hypothalamus or NTS lowers food intake23,64,65.

Techniques: Activation Assay, Western Blot, Staining, Expressing, Activity Assay

Journal: Nature communications

Article Title: Pharmacological and physiological activation of TGR5 in the NTS lowers food intake by enhancing leptin-STAT3 signaling.

doi: 10.1038/s41467-025-60331-1

Figure Lengend Snippet: Fig. 3 | Activation of TGR5 decreases inflammation and leptin signaling sup- pressor in the NTS. Relative NTS Cd68 (a), Il6 (b), Tnfa (c), LepRb (d), Ptp1b (e), and Socs3 (f) mRNA expression of NTS CMC (n = 8) or CCDC (n = 6) chow rats and NTS CMC (n = 8 for a–d; n = 7 for e, f) or CCDC (n = 8 for a–d; n = 7 for e, f) HF rats, measured 24 h after food was given back following 5 min acute infusion. g Representative images (scale bar: 100 μm; 25 μm for the digital zoomed-in ima- ges) showing FISH for Tgr5, Aif1, and DAPI or LepR, Aif1, and DAPI in the NTS of HF rats 30 min after CMC 5 min acute infusion. Digital zooms of the boxed regions are shown to the left. Arrowheads indicate cells co-expressing Tgr5 or LepR and Aif1. h Left: Proportion of Aif1+ microglia in the NTS that are Tgr5+ or Tgr5- and LepR+ or LepR- (pie chart). Right: Percentage of Aif1+ microglia expressing Tgr5or LepR in the NTS (n = 3 per group). i Representative images (scale bar: 100 μm; 25 μm for the digital zoomed-in images) showing FISH for Tgr5, Rbfox3, and DAPI or LepR, Rbfox3, and DAPI in the NTS of HF rats 30 min after CMC 5 min acute infusion.

Article Snippet: Leptin (497-LR, R&D Systems) was dissolved firstly in 0.9% saline and thenwith/withoutCCDC (final CCDCconcentration 100μM) in 1% CMC to the concentration of 153μM (equivalent to 0.5μg/site) as this amount of leptin infused into the hypothalamus or NTS lowers food intake23,64,65.

Techniques: Activation Assay, Expressing

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