Fig 1: GLP1R-dependent uptake of ASO and knockdown of gene expression in mice treated with eGLP1-ASO conjugates in vivo.(A) Representative pancreatic sections stained for ASO by IHC and MALAT1 RNA by ISH from mice treated for 2 weeks with three subcutaneous injections of (a) saline, (b) MALAT1-ASO (1 μmol/kg), or (c) eGLP1-MALAT1-ASO (1 μmol/kg). (B) ASO uptake by IHC and MALAT1 RNA levels by ISH in pancreatic sections from mice treated for 2 weeks with three intravenous injections of (a) saline or (b) eGLP1-MALAT1-ASO (1 μmol/kg). (C) MALAT1 gene expression by ISH in (a) wild-type (WT) and (b) GLP1R knockout (KO) mice 72 hours after a single subcutaneous dose of saline or eGLP1-MALAT1-ASO (1 μmol/kg). (D) Pancreatic section from wild-type mice stained using fluorescence in situ probes for MALAT1 (purple, arrows), insulin (green), and glucagon (red). Pancreatic sections were collected 72 hours after one subcutaneous administration of (a) saline or (b) MALAT1-ASO and (c) eGLP1-control-ASO or (d) eGLP1-MALAT1-ASO, and all compounds were dosed at 1 μmol/kg. Scale bars, 200 μm. Islets are circled in blue in (A) to (C).
Fig 2: Representative confocal images of GLP1R and LAMP1 following 30 min incubation with vehicle control (a), 100 nM exenatide (b), or 100 nM 127I2-eGLP1-34S15-ASO (c). Nuclei were counterstained with DAPI. Nuclei were counterstained with DAPI. Pearson’s correlation co-efficient for GLP1R colocalization with LAMP1 was calculated from cytoplasmic ROIs (d). All analyses are presented as mean ± SEM for 3 independent experiments. For each condition, cells from at least 10 fields of view were analyzed from at least 2 technical replicates per experiment. 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: Effects of ASO and eGLP1-ASO conjugates on gene expression and protein levels in vitro in cell lines and primary mouse islet cells.(A) MALAT1 RNA measured in GLP1R-HEK293 cells treated overnight with increasing concentrations of either MALAT1-ASO (○) or eGLP1-MALAT1-ASO (•), and data were normalized to MALAT1 expression in untreated cells (Ctrl). Data are plotted as mean ± SD of six replicates. (B) MALAT1 and FOXO1 transcripts measured in primary mouse islets treated overnight with 1 μM MALAT1-ASO and 1 μM FOXO1-ASO (open bars), 1 μM eGLP1-MALAT1-ASO, and 1 μM eGLP1-FOXO1-ASO (closed bars), as indicated in the graph. Data were normalized to corresponding expression in untreated islets (Ctrl). Representative data from one of three independent experiments. (C) MALAT1 RNA measured in human islets treated with 1 μM MALAT1-ASO (○) and 1 μM eGLP1-MALAT1-ASO (•) and compared with expression in untreated islets (x). (D) FOXO1 mRNA measured in mouse islets treated for 96 hours with 1 μM eGLP1-Ctrl-ASO (x), 1 μM FOXO1-ASO (□), or 1 μM eGLP1-FOXO1-ASO (▪). Data were normalized to FOXO1 expression measured in untreated islets (Ctrl) and plotted as dot plots for individual animals and geometrical mean ± 95% CI. (E) FOXO1 protein levels in the islets by Western blot for the untreated and islets treated with 1 μM eGLP1-FOXO1-ASO for 96 hours. (F) Relative protein levels were quantified by normalizing the band intensity to α-tubulin, and only one biological replicate per treatment was evaluated (therefore having no statistical analysis).
Fig 5: 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.
Supplier Page from DiscoverX for PathHunter® eXpress GLP1R Activated GPCR Internalization Assay