Fig 1.
Liver-specific dpp4 silencing does not improve glucose metabolism.
(A) Dose-response curve upon transfection of PC3 cells with siRNA (n = 3). (B) Pilot mouse study to evaluate the siRNA efficacy. Wild-type C57BL/6J mice were injected three-times with dpp4 siRNA (siDpp4, 1 mg/kg), negative control siRNA (luciferase; siRNA-NT) or factor VII siRNA (siFVII) and dpp4 mRNA expression in the liver was analyzed by RT-PCR 7 days after treatment (n = 3; **p<0.005 vs. siRNA-NT (ANOVA, Tukey's multiple comparisons test). (C) Illustration of the mouse study design. Liver samples were taken after study termination (day 13) and dpp4 mRNA expression (D) was analyzed by RT-PCR or protein abundance (E) by Western blot. (F) Representative Western Blot images show dpp4 as well as Gapdh protein levels in liver samples. (G) Circulating dpp4 protein levels as well as (E) circulating dpp4 activity was measured three days after last dosing on day 13. (F) Quantification of active GLP-1 and (G) GIP at day 13. (K) Oral glucose tolerance test was performed after the 3rd injection, immediately before sacrifice. (L) Area under the curve during the oral glucose tolerance test. (M) Insulin levels before (-30 min) and after the oral glucose load (15 min). Data are mean values ± SEM, n = 6–8, **p<0.005 and ***p<0.0001 as indicated (ANOVA, Tukey's multiple comparisons test). FC = fold change.
Fig 2.
siRNA-mediated knock-down of hepatic dpp4 in db/db mice.
(A) Schematic illustration of the study design. (B, C and D) Liver samples were taken after study termination at day 30. (B) dpp4 mRNA expression was analyzed by real-time PCR after indicated treatment. (C) Quantification of dpp4 protein abundance. (D) Representative Western Blot images show dpp4 protein levels after indicated treatment. (E) Circulating dpp4 protein levels as well as (F) circulating dpp4 activity was measured three days after last dosing at day 30. (G) Quantification of active GLP-1 and (H) GIP at day 30. Data are mean values ± SEM, n = 6–8, *p<0.05, **p<0.005 and ***p<0.0001 as indicated (ANOVA, Tukey's multiple comparisons test). FC = fold change.
Fig 3.
: Liver-specific dpp4 silencing does not improve whole-body glucose metabolism.
(A) Body weight and (B) food intake was measured during the study. (C) Fasting blood glucose and (D) fasting insulin was measured after study termination. (E) Oral glucose tolerance test was preformed 7 days after the 5th injection on day 28. (F) Baseline-corrected area under the curve during the oral glucose tolerance test. (G) Insulin concentrations before (-30 min) and after the oral glucose load (15 min).
Fig 4.
Effects of dpp4 silencing on hepatic histology and lipid metabolism in db/db mice.
(A) H&E images of liver depicting lipid accumulation (steatosis) in hepatocytes. All images at 100x magnification; pv = portal vein. (B) Quantitative analysis of the total lipid area, sub-analyzed for the small and large droplet area. (C) Circulating concentrations of triglycerides, cholesterol and adiponectin. Each solid circle represents liver sample from an individual mouse for each group (n = 5–8). Data are mean values ± standard deviation (SD), n = 5–8, * p<0.05.
Fig 5.
Hepatic gene expressions assessed by microfluidic card PCR.
(A) Heatmap view showing a clustering of all assessed specific genes. A blue color indicates downregulation of the respective gene, whereas red square indicated up-regulation versus the mean of all samples. (B) Quantification view on selected single genes averaged for the treatment groups. Data are mean values ± SD, n = 5–8, *p<0.05 comparing obese db/db to lean db/- samples; #p<0.05 comparing within the obese samples to the db/db control group (ANOVA, Tukey's multiple comparisons test).