GCGR ASO treatment increases islet GLP-1 and insulin content. (A) Islets were isolated as described in Methods from 12-week-old male db/db mice (n = 5–6 per treatment group), which had been treated twice per week (every 3.5 days) by subcutaneous injection with saline (black bar) or GCGR ASO 180475 (white bar) at 25 mg/kg for a total of 9 doses. Five replicates of 10 islets from each animal were extracted with acid ethanol overnight at 4–C, and GLP-1 was assayed by ELISA. Results are expressed as mean ± SEM. P < 0.05. (B) Islet insulin content was assayed by RIA using samples prepared as described in (A). Results are expressed as mean ± SEM. P < 0.05. (C) Real-time quantitative RT-PCR was used to profile gene expression from islets of 10-week-old male db/db mice (n = 9 per treatment group), which had been treated twice per week (every 3.5 days) by subcutaneous injection with saline (black bar) or GCGR ASO 180475 (white bar) at 25 mg/kg for a total of 9 doses. Islets were isolated as described in Methods, and 200 islets from 3 individuals were pooled to give one sample for RNA extraction. Islet GCGR, preproglucagon (proGCG), Brain-4 (Brn4), and insulin-1 (Ins1) levels showed significant differences when compared to saline-treated animals (P < 0.05 using Student’s t test). Differences in the mRNA levels of insulin-2 (Ins2), glucose transporter-2 (Glut2) and glucokinase (GCK) were not observed. Mouse 36B4 ribosomal phosphoprotein mRNA was measured and used to normalize RNA. Data are the mean values ± SEM of 3 samples per treatment group.