Fig 2: eGLP1-ASO-mediated GLP1R internalization in GLP1R-HEK293 cells. Representative confocal images of GLP1R localization in GLP1R-HEK293 cells following incubation with vehicle control (a) or 127I2-eGLP1-34S15-ASO (100 nM) for the time points indicated (b). Nuclei were counterstained with DAPI. (c) GLP1R mean fluorescence intensity was calculated from a region of interest encompassing the cell border. (d) GLP1R-HEK293 cells were incubated with 127I2-eGLP1-34S15-ASO or exenatide for 5–30 min at indicated concentrations and image analysis performed as in (b). For each condition, cells from at least 10 fields of view were analyzed in duplicate wells and data were expressed as mean ± SEM. Scale bar = 10 µm.

Fig 3: Characterizing GLP1R signaling pathways and pharmacology activated by eGLP1-MALAT1-ASO (•) and eGLP1-FOXO1-ASO (▪) compared to eGLP1 peptide (○) and exenatide (ρ).(A) Displacement of 125I-GLP1 in membranes from GLP1R overexpressing HEK293. eGLP1-MALAT1-ASO (•) and eGLP1-FOXO1-ASO (▪) displaced 125I-GLP1 equally well as the eGLP1 peptide (○) and exenatide (ρ). Functional assays measuring G protein signaling by (B) DMR and (C) cAMP accumulation in GLP1R-HEK293 cells showed that eGLP1-MALAT1-ASO (•) and eGLP1-FOXO1-ASO (▪) were equally potent as the eGLP1 peptide (○) and exenatide (△), with only minor impact from conjugation of MALAT1-ASO and FOXO1-ASO to the eGLP1 peptide. (D) β-Arrestin2 recruitment in GLP1R-CHO-K1 and (E) receptor internalization in GLP1R-U2OS using PathHunter assays showed similar potencies for eGLP1, exenatide, and ASO-conjugated eGLP1, but with reduced maximal effect for both eGLP1-MALAT1-ASO (•) and eGLP1-FOXO1-ASO (▪). Data are presented as mean ± SEM using exenatide as reference for 100% effect. (F) Effect of 10 nM exenatide (ρ), eGLP1-Ctrl-ASO (x), FOXO1-ASO (□), eGLP1-FOXO1-ASO (▪), MALAT1-ASO (○), or eGLP1- MALAT1-ASO (•) on glucose GSIS in human reconstituted islet microtissues. Islets were treated with high glucose, and the compounds and insulin content were measured in the culture medium. Data were normalized to secretion at 11 mM glucose and presented as geometric mean ± 95% confidence interval (CI). Statistical analysis by one-way analysis of variance (ANOVA) adjusted for multiple comparisons.

Fig 4: Verification of 127I2-eGLP1-34S15-ASO function. (a) GLP1R internalization assessed in U2OS cells using the PathHunter assay. (b) Malat1 RNA expression in GLP1R-HEK293 cells after incubation with unlabeled or labeled eGLP1-ASO. Data shown as mean ± SEM for 3 independent experiments performed in duplicate.

Fig 5: Schematic structure of a fluorescently labeled eGLP1-conjugated MALAT1 ASO and internalization of fluorescent eGLP1 and eGLP1-MALAT1-ASO.(A) Schematic structure of the Cy3-labeled eGLP1-MALAT1-ASO conjugate. (B) Fluorescence imaging of (a) wild-type and (b and c) GLP1R-HEK293 cells treated with 100 nM BODIPY–labeled (green) eGLP1 peptide for 15 min (b) or 60 min (a and c) at 37°C. (C) Fluorescence imaging of (a) wild-type and (b and c) GLP1R-HEK293 cells treated with 33 nM Cy3–labeled (red) eGLP1-MALAT1-ASO for 15 min at 0°C (b) or for 60 min at 37°C (a and c). Cells were stained, fixed, and imaged on an ImageXpress fluorescence microscope. Nuclei were stained with Hoechst 32585 (blue). Scale bars, 10 μm.