Journal: Molecular Metabolism

Article Title: Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A

doi: 10.1016/j.molmet.2015.01.010

Figure Lengend Snippet: PKA activity correlates with loss of GLP-1R from the cell surface . MIN6 cells were transfected with a GFP-tagged GLP-1R (GLP-1R-GFP) expressed under the control of the constitutive CMV promoter. (A) GLP-1R-GFP transfected MIN6 cells were cultured at low glucose (3 mM; LG) or high glucose (25 mM; HG) for 4 h in the presence or the absence of the PKA inhibitor, H89 (HG + H89). GFP was visualized in green, nuclei stained with dapi (blue) and β-catenin immune-stained to mark the plasma membrane (red). (B) GLP-1R-GFP transfected MIN6 cells were infected with a recombinant adenovirus expressing a constitutively active PKA catalytic subunit (caPKA). Cells were cultured at low glucose (LG), and the localization of the GLP-1R-GFP was determined by fluorescence microscopy. Cells infected with the caPKA adenovirus were identified using an antibody against the FLAG-tag of the caPKA (red). (C) GLP-1R-GFP transfected MIN6 cells were cultured for 4 h at high glucose (HG) with: forskolin (Fsk), to raise cAMP levels through the activation of adenylyl cyclases; H89, to inhibit PKA; or expression of a dominantly negative PKA regulatory subunit (dnPKA). GLP-1-GFP was visualized in green, nuclei by staining with dapi (blue), and the FLAG-tag epitope on the dnPKA in red.

Article Snippet: Culture media were supplemented with exendin-4 at 10 nM (American Peptide Co. cat. # 46-3-12A), forskolin at 2 μM (Sigma–Aldrich, cat. # F6886), and H89 at 20 μM (Cell Signaling Technologies, cat # 9844).

Techniques: Activity Assay, Transfection, Control, Cell Culture, Staining, Clinical Proteomics, Membrane, Infection, Recombinant, Expressing, Fluorescence, Microscopy, FLAG-tag, Activation Assay

Journal: Molecular Metabolism

Article Title: Chronic hyperglycemia downregulates GLP-1 receptor signaling in pancreatic β-cells via protein kinase A

doi: 10.1016/j.molmet.2015.01.010

Figure Lengend Snippet: Chronic exposure of β-cells to the GLP-1R agonist exendin-4 . (A) MIN6 cells were chronically cultured (20 h) at low glucose in the absence (LG) or the presence (LG + Ex4) of the GLP-1R agonist exendin-4, fixed and incubated with fluorescein-tagged exendin-4 to bind cell surface (but not internal) GLP-1R. (B) MIN6 cells transfected with GLP-1R-GFP were cultured for 4 h at high glucose with exendin-4 in the absence (HG + Ex) or the presence of H89 (HG + Ex + H89), and GLP-1R-GFP localization was determined by fluorescence microscopy (green). Cells were stained for β-catenin to mark membranes (red) and dapi to identify nuclei (blue). (C) To determine the in vivo effects of chronic exendin-4 exposure, male mice were administered exendin-4 at 4–6 h intervals for 24 h or were administered saline at each time-point (controls). Mice were then given a 5 μg/kg body exendin-4 dose and an hour later a 3 g/kg intraperitoneal glucose bolus. Plasma insulin levels were measured for 15 min following the glucose challenge (C) and blood glucose levels for 120 min (D). Data were analyzed by 2-way ANOVA with Bonferroni post hoc tests. *, P < 0.05; ***, P < 0.001. n = 6–8 mice for both C and D).

Article Snippet: Culture media were supplemented with exendin-4 at 10 nM (American Peptide Co. cat. # 46-3-12A), forskolin at 2 μM (Sigma–Aldrich, cat. # F6886), and H89 at 20 μM (Cell Signaling Technologies, cat # 9844).

Techniques: Cell Culture, Incubation, Transfection, Fluorescence, Microscopy, Staining, In Vivo, Saline, Clinical Proteomics