Figure 3.

Glucose regulates MafA abundance and insulin expression through AMPK. A and B, MIN6 cells (A) or isolated mouse islets (B) grown in high-glucose (Hi, 20 mm) or low-glucose (Lo, 2 mm) medium were treated with the AMPK inhibitor dorsomorphin (Dorso, 10 μm) for 16 h, and whole-cell extracts were analyzed by immunoblotting using the indicated antibodies. C, the insulin promoter reporter plasmid pGL4/h-ins-p (0.5 μg) and a Renilla luciferase expression plasmid, pEF-Rluc (0.5 μg), were transfected into MIN6 cells, and the cells were incubated in medium containing high (20 mm) or low (2 mm) glucose with or without dorsomorphin (10 μm) for 16 h. Data are expressed relative to the activity in cells that received the reporter plasmid at high glucose concentration without dorsomorphin. Data are mean ± S.E. of five independent experiments. *, p < 0.05 (Student's t test). D, MIN6 cells were grown in high-glucose (20 mm) or low-glucose (2 mm) medium and treated with dorsomorphin (Dorso, 10 μm) for 16 h. Total RNA was then isolated and subjected to quantitative RT-PCR analysis. Data are mean ± S.E. of three independent experiments. *, p < 0.05 (Student's t test). E, the DN form of AMPKα1 was overexpressed in MIN6 cells grown under low-glucose conditions, and the cell extracts were analyzed by immunoblotting. The data in A, B, and E are representative of two independent biological experiments. The intensity of the bands was quantified using ImageJ software, and the relative amounts (averages of two independent experiments) are indicated below the bands.