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: a Relative Tgr5 mRNA expression in the NTS and AP of chow (NTS n = 9; AP n = 7) or HF (NTS n = 6; AP n = 6) rats. b Cumulative food intake of NTS CMC (n = 12) or CCDC (n = 10) HF rats 24 h after food was given back. c Percentage saccharin preference in HF rats following IP saline (n = 5) vs. IP LiCl (n = 5) and NTS CMC (n = 5) vs. NTS CCDC (n = 5) infusion. d Relative PKA activity in the NTS CMC (n = 5) or CCDC (n = 5) HF rats 3 h after infusion. e Cumulative food intake of AP CMC (n = 10) or CCDC (n = 10) HF rats 24 h after food was given back. f Relative LepRb mRNA expression in the NTS (n = 9) and AP (n = 7) chow or NTS (n = 6) and AP (n = 5) HF rats. g Cumulative food intake in NTS CMC (n = 11), STAT3-PI (n = 5), leptin (n = 11), leptin+STAT3-PI (n = 15) chow rats 24 h after food was given back. h Cumulative food intake of NTS CMC (n = 6) or leptin (n = 10) HF rats 24 h after food was given back. i Cumulative food intake of AP CMC (n = 6) or leptin (n = 10) chow rats 24 h after food was given back. j Representative western blot images of p-STAT3, t-STAT3, and GAPDH protein levels in the NTS of chow rats 30 min after CMC, leptin, or leptin+STAT3-PI 5 min acute infusion. k Schematic representation of brainstem tissue section for immunohistological and FISH images. l Representative images (scale bar: 100 μm) of immunohistological staining for p-STAT3 with DAPI (top) and cFOS (bottom) in the NTS of chow rats 30 min after CMC or leptin 5 min acute infusion. m Representative western blot images of p-STAT3, t-STAT3, and GAPDH protein levels in the NTS of HF rats 30 min after CMC or leptin 5 min acute infusion. n Representative images (scale bar: 100 μm) of immunohistological staining for p-STAT3 with DAPI (top) and cFOS (bottom) in the NTS of HF rats 30 min after CMC or leptin 5 min acute infusion. a–f , h , i P values were calculated by two-tailed t tests. g P values were calculated by one-way ANOVA with Tukey’s multiple comparison. Data presented as mean ± SEM. IP intraperitoneal.

Article Snippet: Leptin (497-LR, R&D Systems) was dissolved firstly in 0.9% saline and then with/without CCDC (final CCDC concentration 100 μM) in 1% CMC to the concentration of 153 μM (equivalent to 0.5 μg/site) as this amount of leptin infused into the hypothalamus or NTS lowers food intake , , .

Techniques: Expressing, Saline, Activity Assay, Western Blot, Staining, Two Tailed Test, Comparison

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: a Cumulative food intake of NTS CMC (n = 15; data replotted from Fig. ), leptin (n = 10; data replotted from Fig. ), CCDC (n = 10; data replotted from Fig. ) 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 subsequent 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 shTgr5 (n = 8) HF rats 24 h after food was given back. g Relative NTS LepRb mRNA expression of NTS shMM (n = 6) or shLepR (n = 6) rats. h Cumulative food intake of NTS CMC shMM (n = 7), leptin shMM (n = 7), CMC shLepR (n = 7), leptin shLepR (n = 7) chow rats 24 h after food was given back. i Representative western blot images of p-STAT3, t-STAT3, and GAPDH protein levels in the NTS of NTS shMM or shLepR HF rats receiving a NTS CCDC 5 min acute infusion, followed by a 3 h wait, then NTS leptin 5 min acute infusion, and a subsequent 30 min waiting time. j Cumulative food intake of NTS CCDC+leptin shMM (n = 8) or CCDC+leptin shLepR (n = 8) HF rats 24 h after food was given back. k Cumulative food intake of NTS CMC (n = 15, data replotted from Fig. ), STAT3-PI (n = 7), CCDC + STAT3-PI (n = 11), CCDC+leptin+STAT3-PI (n = 6), or CCDC+leptin (n = 11, data replotted from Fig. 2a) HF rats 24 h after food was given back. a , e , f , h , k P values were calculated by one-way ANOVA with Tukey’s multiple comparison. d , g , j P values were calculated by two-tailed t tests. Data presented as mean ± SEM.

Article Snippet: Leptin (497-LR, R&D Systems) was dissolved firstly in 0.9% saline and then with/without CCDC (final CCDC concentration 100 μM) in 1% CMC to the concentration of 153 μM (equivalent to 0.5 μg/site) as this amount of leptin infused into the hypothalamus or NTS lowers food intake , , .

Techniques: Western Blot, Staining, Expressing, Activity Assay, Comparison, Two Tailed Test

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: a Total bile acid levels in the plasma of chow rats during fasting (n = 5) or at 0.5 h after refed (n = 5). b Unconjugated bile acid levels in the plasma of chow rats during fasting (n = 5) or at 0.5 h after refed (n = 5). c Total bile acid levels in the plasma of HF rats during fasting (n = 6) or at 0.5 h after refed (n = 5). d Unconjugated bile acid levels in the plasma of HF rats during fasting (n = 6) or at 0.5 h after refed (n = 5). e DCA levels in the plasma of chow and HF rats during fasting (chow n = 5; HF n = 6) or at 0.5 h after refed (chow n = 5; HF n = 5). f Unconjugated bile acid levels in the NTS (pmol/g of tissue) of chow rats at 0.5 h (n = 6) or 2 h (n = 5) after refed. g Total bile acid levels in the NTS (pmol/g of tissue) of chow rats at 0.5 h (n = 6) or 2 h (n = 5) after refed. h Unconjugated bile acid levels in the NTS (pmol/g of tissue) of HF rats at 0.5 h (n = 5) or 2 h (n = 6) after refed. i Total bile acid levels in the NTS (pmol/g of tissue) of HF rats at 0.5 h (n = 5) or 2 h (n = 6) after refed. j DCA levels in the NTS (pmol/g of tissue) of chow and HF rats at 0.5 h (chow n = 6; HF n = 5) or 2 h (chow n = 5; HF n = 6) after refed. k Cumulative food intake of NTS saline shMM (n = 8), DCA shMM (n = 8), saline shTgr5 (n = 7), DCA shTgr5 (n = 8) HF rats 24 h after food was given back. l Representative western blot images of p-STAT3, t-STAT3, and GAPDH protein levels in the NTS of HF rats receiving a NTS DCA 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. m Illustration of TGR5 and leptin-STAT3 signaling in feeding regulation and associated experimental approaches. a–d , f–i P values were calculated by two-tailed t tests; e , j , k P values were calculated by one-way ANOVA with Tukey’s multiple comparison. Data presented as mean ± SEM. DCA deoxycholic acid, HDCA hyodeoxycholic acid, CDCA chenodeoxycholic acid, CA cholic acid, UDCA ursodeoxycholic acid, α-MCA α-muricholic acid, β-MCA β-muricholic acid.

Article Snippet: Leptin (497-LR, R&D Systems) was dissolved firstly in 0.9% saline and then with/without CCDC (final CCDC concentration 100 μM) in 1% CMC to the concentration of 153 μM (equivalent to 0.5 μg/site) as this amount of leptin infused into the hypothalamus or NTS lowers food intake , , .

Techniques: Clinical Proteomics, Saline, Western Blot, Two Tailed Test, Comparison

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: 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