Figure 5.

BNLN regulates glucose-stimulated insulin secretion

(A) Glucose stimulated insulin secretion in INS-1 cells overexpressed with FLAG-GFP-BNLN in low glucose (2.5 mmol/L) or high glucose (17 mmol/L) for 1 h each (n = 3–4 experimental replicates). The data were normalized to insulin content. Representative western blots of BNLN expression are shown on the top of panel. (B) GSIS in INS-1 cells overexpressed with the indicated constructs for 48 h (n = 3–4 experimental replicates). The data were normalized to low glucose. (C) GSIS measured in isolated islets from mice fed a regular diet (RD) (N = 5–10 age-matched male mice; 12 weeks) followed transduction of adeno-GFP (CT) or adeno-BNLN constructs (48 h). The data were normalized to insulin content. (D) GSIS measured in isolated islets from mice transduced with Ad-shLacZ and Ad-shTunar (n = 3–4 biological replicates). The data were normalized to insulin content. (E) Levels of mRNAs encoding for Tunar in islets isolated from mice in (D). The data were normalized to Hprt expression in the islets (n = 3–4 biological replicates). (F) GSIS measured in isolated islets from mice fed a HFD (N = 3–6 age-matched male mice biological replicates; 12 weeks on HFD) followed transduction of adeno-GFP (CT) or adeno-BNLN constructs (48 h). The data were normalized to insulin content. (G) Levels of mRNAs encoding for BNLN in islets isolated from mice fed a RD or a HFD (12 weeks). The data were normalized to Hprt expression in the islets (n = 3–4 biological replicates). (H–K) Representative GSIS measured in islets from human donors followed transduction of adeno-GFP (CT) or adeno-BNLN constructs (48 h; N = 5–7 human donors). The data were normalized to insulin content. In (F), GSIS was performed on a male non-diabetic human donor of 69 years of age, 27.2 BMI; in (G), GSIS was performed on a male non-diabetic human donor of 18 years of age, 19 BMI. (I) Insulin content measured in islets from human donors followed transduction of adeno-GFP (CT) or adeno-BNLN constructs (48 h). Data are represented as a difference in intracellular insulin content after performing GSIS between CT and BNLN human islets. Data are shown as means ± SEM. ∗p value equal to 0.05, ∗∗p value equal to 0.01, ∗∗∗∗p value less than 0.0001 when compared to treatment of low glucose in cells/islets with same type of construct in (A), (C), (D), and (G), to the CT vector group in (B) and (E), to the RD group in (F); #p value less than 0.05, ##p value less than 0.01, ###p value less than 0.0001 when compared to the CT group in cells/islets incubated with the same concentrations of glucose in (C), (D), and (G). Statistical significance was determined by Student’s t test in (E), (F), (J), and (K), and ANOVA followed by Tukey’s multiple comparisons test in (A), (B), (C), (D), and (G), p < 0.05.