Journal: The Journal of nutrition

Article Title: Mango Supplementation Modulates Gut Microbial Dysbiosis and Short-Chain Fatty Acid Production Independent of Body Weight Reduction in C57BL/6 Mice Fed a High-Fat Diet.

doi: 10.3945/jn.115.226688

Figure Lengend Snippet: FIGURE 2 Protein expression of pancreatic in- cretin receptors in C57BL/6 mice fed a control diet or an HF diet supplemented with 0%, 1%, or 10% mango for 12 wk. GLP1R (A) and GIPR (B) protein expression were determined via immunoblot anal- ysis (normalized to b-actin) in the pancreas of mice after 12 wk of dietary treatment. Data are means 6 SEMs (n = 5 mice/group). Labeled means without a common letter differ, P # 0.05. C, control; GIPR, gastric inhibitory peptide receptor; GLP1R, glucagon- like peptide 1 receptor; HF, high fat; HF+1%M, high fat + 1% mango; HF+10%M, high fat + 1% mango.

Article Snippet: Total protein (20 mg) was electrophoretically separated by use of SDS-PAGE and transferred to PVDF membranes (ThermoScientific), and membranes were blocked with 5% nonfat dried milk (Nestle) followed by overnight incubation at 4 C with the following rabbit polyclonal antibodies: GLP1R and GIPR (Santa Cruz Biotechnology) and b-actin (Cell Signaling Technology).

Techniques: Expressing, Control, Western Blot, Labeling

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Beta cell GLP-1R vs GIPR trafficking patterns. (A) Internalization AUC dose response curves from DERET assays in INS-1 832/3 SNAP-GLP-1R vs SNAP-GIPR cells stimulated with the indicated concentrations of GLP-1 or GIP, respectively. Results were fitted to a 3-parameter dose response curve to obtain Emax and logEC50 for both receptors, with comparisons between these parameters included; n = 5. (B) Rates of GLP-1R vs GIPR internalization (k values) derived from (A) by one-phase association (with Y0 = 0) of baseline-deleted DERET data; n = 5. (C) GLP-1R vs GIPR internalization dose response curves measured by high-content microscopy assay (HCA) in the same cells as above. Results fitted and Emax and logEC50 comparisons included as above; n = 5. (D) Confocal microscopy analysis of SNAP-GLP-1R vs SNAP-GIPR (red, middle panels) localization following 30 minutes stimulation with 100 nM GLP-1-FITC or GIP-FITC (green, left panels), respectively, in the same cells as above. (E) Confocal microscopy analysis of isolated intact mouse islets stimulated with fluorescently labeled agonists as indicated: WT islets were imaged following stimulation with 100 nM GLP-1-TMR, while both WT and GIPR −/− (KO) islets were imaged following stimulation with 1 µM GIP-TMR. (F) Quantification of surface GLP-1R vs GIPR levels in WT mouse islets using fluorescent agonist uptake data from (E); data corrected for binding affinity differences between both agonists; n = 4. (G) GLP-1R vs GIPR recycling dose response curves measured by high-content microscopy assay (HCA) in cells from (C); n = 5. (H) Confocal microscopy analysis of SNAP-GLP-1R vs SNAP-GIPR (red, middle panels) colocalization with SNX27-GFP (green, left panels) following 3 hours stimulation with 100 nM GLP-1 or GIP, respectively, in the same cells as above. Nuclei (DAPI), blue. Data are mean ± SEM, compared by paired or unpaired t tests, or two-way ANOVA with Sidak's post hoc test; * P < .05, ** P < .01, *** P < .001; size bars: 10 µm.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Derivative Assay, Microscopy, Confocal Microscopy, Isolation, Labeling, Binding Assay

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Endosomal vs plasma membrane localization of GLP-1R compared with GIPR in beta cells. (A) GLP-1R vs GIPR plasma membrane localization dose response curves from NanoBRET assays performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing KRAS-Venus and GLP-1R- or GIPR-NanoLuc, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; results were fitted to 3-parameter dose response curves to obtain Emax and logEC50 for both receptors, with comparisons between these parameters included; n = 5. (B) As for (A) but for GLP-1R vs GIPR endosomal localization dose response curves from NanoBRET assays performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing Rab5-Venus and GLP-1R- or GIPR-NanoLuc, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; n = 5. Data are mean ± SEM, compared by paired t tests or two-way ANOVA with Sidak's test; * P < .05.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Expressing

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: GLP-1R vs GIPR clustering propensities in beta cells. (A) Representative images from RICS analysis of GLP-1R vs GIPR clustering, showing SNAP-Surface 488-labeled GLP-1Rs or GIPRs imaged in the basolateral plane of INS-1 832/3 SNAP-GLP-1R vs SNAP-GIPR cells after 5 minutes treatment in vehicle (Veh) and either 100 nM GLP-1 or GIP. Diffusion coefficients for individual ROIs are indicated on each image, with corresponding intensity traces, as well as 2D and fitted 3D autocorrelation maps for each ROI, are also depicted. (B) Average RICS diffusion coefficients for each receptor and treatment for each cell analyzed from n = 4 experiments. (C) GLP-1R clustering kinetics measured by TR-FRET in INS-1 832/3 SNAP-GLP-1R cells treated with 100 nM agonist as indicated, with vehicle-corrected AUCs included; n = 4. (D) GIPR clustering kinetics measured by TR-FRET in INS-1 832/3 SNAP-GIPR cells treated with 100 nM agonist as indicated, with vehicle-corrected AUCs included; n = 4. Data are mean ± SEM, compared by one-way ANOVA with Sidak's test; **** P < .0001; ns: non-significant.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Labeling, Diffusion-based Assay

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Beta cell GLP-1r vs GIPR degradation propensities. (A) Western blot assessment of SNAP-GLP-1R or GIPR over tubulin levels in INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells with or without stimulation with 100 nM GLP-1 or GIP, respectively, for 6 hours in the presence of the protein synthesis inhibitor cycloheximide; n = 3. (B) Representative Western blot results from (A). Note that the top bands were used to quantify the SNAP-receptor levels, as they correspond to the glycosylated forms of the receptors, known to be biologically active and correctly inserted at the plasma membrane . (C) Percentage of GLP-1R vs GIPR, labeled with the cell-permeable SNAP-tag probe BG-OG, and corresponding representative images from INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells with or without stimulation with 100 nM GLP-1 or GIP, respectively, for the indicated times in the presence of cycloheximide; n = 4. (D) Percentage of co-localization (Mander's coefficient) and representative images of SNAP-GLP-1R vs -GIPR (labeled with SNAP-Surface 649) with Lysotracker Green in INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells stimulated with 100 nM GLP-1 or GIP for 1 hour; n = 5. Data are mean ± SEM, compared by ratio-paired or unpaired t test or two-way ANOVA with Sidak's post hoc test; *** P < .001, **** P < .0001.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Western Blot, Labeling

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Beta cell GLP-1R vs GIPR G protein subtype and β-arrestin 2 recruitment characteristics. (A) Kinetics of Gα s , Gα q , Gα i , and β-arrestin 2 recruitment to the GLP-1R assessed by NanoBiT complementation assay in INS-1 832/3 GLP-1R KO cells transiently expressing GLP-1R-SmBiT and the corresponding mini-G protein subtype or β-arrestin 2. Responses to 100 nM GLP-1 normalized to vehicle and corresponding AUCs are shown; n = 5. (B) Kinetics of Gα s , Gα q , Gα i , and β-arrestin 2 recruitment to the GIPR assessed by NanoBiT complementation assay in INS-1 832/3 GIPR KO cells transiently expressing GIPR-SmBiT and the corresponding mini-G protein subtype or β-arrestin 2. Responses to 100 nM GIP normalized to vehicle and corresponding AUCs are shown; n = 5. (C) NanoBRET assessment of GLP-1R vs GIPR recruitment of Gα s , performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing mini-Gs-Venus and either GLP-1R- or GIPR-NanoLuc, after stimulation with 100 nM GLP-1 or GIP, respectively, with corresponding AUCs also shown; n = 4. Data are mean ± SEM, compared by paired t test; *** P < .05.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Expressing

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: GLP-1R vs GIPR endosomal vs plasma membrane activity in beta cells. (A) GLP-1R vs GIPR plasma membrane activity dose response curves from bystander NanoBiT signaling assays performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing Nb37-SmBiT, LgBiT-CAAX and SNAP-GLP-1R or -GIPR, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; 30-minute AUC for each agonist concentration tested were fitted to 3-parameter dose response curves to obtain plasma membrane Emax and logEC50 for both receptors, with comparisons between these parameters included; n = 4. (B) As for (A) but for GLP-1R vs GIPR endosomal activity in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing Nb37-SmBiT, Endofin-LgBiT and SNAP-GLP-1R or -GIPR, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; n = 4. Data are mean ± SEM, compared by paired t tests; * P < .05, *** P < .001.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Activity Assay, Expressing, Concentration Assay

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Functional analysis of GLP-1R vs GIPR signaling in beta cells. (A) cAMP dose response curves to GLP-1 vs GIP assessed in INS-1 832/3 cells by HTRF assay; results were fitted to 3-parameter dose response curves to obtain plasma membrane Emax and logEC50 for both receptors, depicted here combined as log(Emax/EC50) for each receptor; n = 5. (B) cAMP FRET responses to 100 nM GLP-1 or GIP from isolated and 4-hydroxytamoxifen-treated Pdx1-Cre ERT /CAMPER mouse islets; including agonist AUCs calculated for each receptor; n = 4. (C) INS-1 832/3 calcium responses (using the calcium indicator Cal520-AM) to 100 nM GLP-1 or GIP, including agonist AUCs calculated for 0-1 minute and 1-5 minutes responses for each receptor; n = 4. (D) Calcium responses to 100 nM GLP-1 vs GIP from purified WT mouse islets loaded with Cal520-AM, including agonist AUCs for each receptor; n = 4. (E) Insulin secretion responses to 100 nM GLP-1 vs GIP, expressed as fold increases to 11 mM glucose (G11) secretion levels from INS-1 832/3 WT ( n = 3), GLP-1R KO ( n = 5) and GIPR KO ( n = 4) cells. Data are mean ± SEM, compared by paired t tests; * P < .05; ns, nonsignificant.

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Functional Assay, HTRF Assay, Isolation, Purification

Journal: Endocrinology

Article Title: Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells

doi: 10.1210/endocr/bqad028

Figure Lengend Snippet: Summary of trafficking, coupling, and cellular outputs of GLP-1R vs GIPR in response to cognate agonists

Article Snippet: Here the SmBiT was cloned in frame at the C-terminus of the GLP-1R and the GIPR by substitution of the Tango sequence on FLAG-tagged GLP-1R-Tango or GIPR-Tango (a gift from Prof. Bryan Roth, University of North Carolina, USA; Addgene plasmids #66291 and #66294), respectively.

Techniques: Expressing

Journal: Endocrinology

Article Title: GLP-1 Receptor Expression Within the Human Heart

doi: 10.1210/en.2018-00004

Figure Lengend Snippet: Antibodies Used

Article Snippet: Immunoprecipitations were performed using 1 mg/mL of cell or tissue protein extract and 2 μg/mL of rabbit polyclonal anti-human GLP-1R antibody [Novus 19400002; Research Resource Identifier (RRID) AB_10004637 ; Bio-Techne, Oakville, ON, Canada] overnight at 4°C.

Techniques: Sequencing

Journal: Endocrinology

Article Title: GLP-1 Receptor Expression Within the Human Heart

doi: 10.1210/en.2018-00004

Figure Lengend Snippet: GLP1R mRNA transcript levels in the human heart are comparable to those in human pancreas and islets. (A) GLP1R mRNA levels were measured via qPCR analysis in multiple human tissues and in transfected BHK cells that express low levels of the human GLP-1R. For data represented without standard error bars, each single RNA sample was analyzed in duplicate; for isolates depicted with error bars, peripheral blood lymphocyte samples were analyzed in duplicate from two different sources, and at least three different samples were analyzed in duplicate by qPCR for RNAs from islet, bone marrow, left atria (LA), right atria (RA), left ventricle (LV), and right ventricle (RV). (B) qPCR analysis of GLP1R and tissue- or cell-type–specific gene expression in the indicated samples as confirmation of RNA/cDNA integrity. For (A) and (B), data are expressed as cycle threshold (Ct) values because none of the housekeeping genes examined (ACTB, GPI, PSMB4, CHMP2A, and EMC7) exhibited consistent expression levels in all tissues examined. Values are mean ± standard error (where appropriate); n = 1 to 3 samples per tissue. LA samples are from patients P01371, P01430, and P01504. RA samples are from patients P01262, P01371, and P01377. LV samples are from patients P01262, P01430, and P01371. RV samples are from patients P01262, P01371, and P01504 (see Supplemental Table 1 and Figure 4). Islet samples are from donors R177, R199, and R200 (see Supplemental Table 3). CA EC, coronary artery endothelial cells; CA SMC, coronary artery smooth muscle cells; PBL, peripheral blood lymphocytes; Card FB, cardiac fibroblasts.

Article Snippet: Immunoprecipitations were performed using 1 mg/mL of cell or tissue protein extract and 2 μg/mL of rabbit polyclonal anti-human GLP-1R antibody [Novus 19400002; Research Resource Identifier (RRID) AB_10004637 ; Bio-Techne, Oakville, ON, Canada] overnight at 4°C.

Techniques: Transfection, Expressing

Journal: Endocrinology

Article Title: GLP-1 Receptor Expression Within the Human Heart

doi: 10.1210/en.2018-00004

Figure Lengend Snippet: (A) Western blot analysis of whole cell or human islet tissue extracts using GLP-1R antibodies against the human GLP-1R, Mab 3F52, or polyclonal Novus. Molecular mass standards (kDa) are indicated in the center. Blots on the left contain whole cell extracts from BHK pcDNA3 (lane 1), BHK clone #12A (lane 2), BHK clone #13 (lane 3), BHK clone #27 (lane 4), and CFPAC-1 cells (lane 5). Blots on the right contain whole cell extracts from BHK clone #12A (lane 1) and whole tissue extracts from human islets #167 (lane 2), #177 (lane 3), #186 (lane 4), and 199 (lane 5). The blot on the far right was immunoblotted with Akt and Erk1/2 antibodies as loading controls. (B) Western blot analysis of whole cell or human cardiac tissue extracts analyzed using the indicated GLP-1R antibody. Molecular mass standards (kDa) are indicated in the center. Immunoblotting with Akt and Erk1/2 antibodies (bottom panels) was used as loading controls. In (A) and (B), the brackets indicate predicted migration positions of immunoreactive GLP-1R protein.

Article Snippet: Immunoprecipitations were performed using 1 mg/mL of cell or tissue protein extract and 2 μg/mL of rabbit polyclonal anti-human GLP-1R antibody [Novus 19400002; Research Resource Identifier (RRID) AB_10004637 ; Bio-Techne, Oakville, ON, Canada] overnight at 4°C.

Techniques: Western Blot, Migration

Journal: Endocrinology

Article Title: GLP-1 Receptor Expression Within the Human Heart

doi: 10.1210/en.2018-00004

Figure Lengend Snippet: (A) Whole cell/tissue extracts after immunoprecipitation (IP) and Western blotting with the indicated GLP-1R antibodies. Lanes 1 and 2 are whole cell extracts, and lanes 3 through 8 correspond to immunoprecipitated samples. Lane 1, BHK pcDNA3; lane 2, BHK clone #12A; lane 3, BHK clone #12A; lane 4, BHK clone #13; lane 5, human islet #167; lane 6, human islet #177; lane 7, human islet #186; and lane 8, human islet #199. Molecular mass standards (kDa) are indicated on the left. (B) Whole tissue extracts from human heart chamber samples after IP and Western blotting with the indicated GLP-1R antibodies. Molecular mass standards (kDa) are indicated on the far left and right of the blots. In (A) and (B), the brackets indicate predicted migration positions of immunoreactive GLP-1R protein.

Article Snippet: Immunoprecipitations were performed using 1 mg/mL of cell or tissue protein extract and 2 μg/mL of rabbit polyclonal anti-human GLP-1R antibody [Novus 19400002; Research Resource Identifier (RRID) AB_10004637 ; Bio-Techne, Oakville, ON, Canada] overnight at 4°C.

Techniques: Immunoprecipitation, Western Blot, Migration

Journal: Endocrinology

Article Title: GLP-1 Receptor Expression Within the Human Heart

doi: 10.1210/en.2018-00004

Figure Lengend Snippet: GLP-1R immunohistochemistry with Mab 3F52 does not detect GLP-1R–immunoreactive cells in human ventricular tissue. Immunostaining of hyperplastic human Brunner glands with isotype control antibody (A) and GLP-1R Mab 3F52 (B). The Novus 19400002 antibody did not reliably detect GLP-1R–immunopositive cells in sections containing human Brunner glands (data not shown). Immunostaining of human pancreas with isotype control antibody (C) and GLP-1R Mab 3F52 (D). (E–P) GLP-1R Mab 3F52 immunostaining of human cardiac tissues from individual subjects [(E) 193856; (F) 61704; (G) 70150; (H) 70847; (I) 81858; (J) 164011; (K) 116603; (L) 166469; (M) 179268; (N) 171263; (O) 182651; (P), 52411; see Supplemental Table 2].

Article Snippet: Immunoprecipitations were performed using 1 mg/mL of cell or tissue protein extract and 2 μg/mL of rabbit polyclonal anti-human GLP-1R antibody [Novus 19400002; Research Resource Identifier (RRID) AB_10004637 ; Bio-Techne, Oakville, ON, Canada] overnight at 4°C.

Techniques: Immunohistochemistry, Immunostaining

Journal: Molecular Medicine

Article Title: Exenatide regulates Th17/Treg balance via PI3K/Akt/FoxO1 pathway in db/db mice

doi: 10.1186/s10020-022-00574-6

Figure Lengend Snippet: Effects of exenatide on Th17/Treg proliferation in vitro. A The expression of GLP-1R in PBMCs from C57BL/6 J mice by western blot. Representative image and gray value analysis of GLP-1R from Control and palmitate (PA) groups. Data are presented as mean ± SEM of three independent experiments. ACTIN was used as a loading control. Naïve CD4 + T cells collected from C57BL/6 J mice were cultured under Th17/Treg-inducing conditions with palmitate (PA), exenatide plus palmitate (Exe + PA) and exenatide, palmitate plus FxoO1 inhibitor AS1842856 (Exe + PA + AS). B Representative flow cytometric plots of Th17 cells from different groups. C Statistical analysis of the percentage of Th17 cells in different groups. D Representative flow cytometric plots of Treg cells with CD4-FITC, CD25-PE and Foxp3 staining. E Statistical analysis of the percentage of Treg cells. Data are presented as mean ± SEM of three independent experiments. ** P < 0.01, * P < 0.05 vs control group; # P < 0.05 vs PA group; & P < 0.05 vs Exe + PA group; ns P > 0.05

Article Snippet: After blocking with 5% non-fat milk in Tris-buffered saline Tween-20 (TBST), the membranes were incubated with primary antibodies against IL-17 (#AO688, Abclonal Technology, China), Foxp3 (#GB11093, Servicebio, China), phosphoinositide 3-kinase (PI3K) (#bsm-33219m, Bioss, China), phospho (p)-PI3K (Tyr317, #bs-5570R, Bioss, China), protein kinase B (Akt) (#GB11689, Servicebio, China), p-Akt (Ser473, #AF0908, Affinity, USA), FoxO1 (#GB11286, Servicebio, China), p-FoxO1 (Thr24, #9464T, Cell Signaling Technology, USA) or GLP-1R (#CSB-PA009514YA01HU, Cusabio, China) at 4°C overnight.

Techniques: In Vitro, Expressing, Western Blot, Control, Cell Culture, Staining