Figure 1.

Nrf2 is necessary for glucose-stimulated β-cell proliferation ex vivo. A: Illustration of the Nrf2 protein structure emphasizing the deletion of exon 5, which expresses the Neh1 domain, in Nrf2^lox/lox mice. Nrf2 antibody (Nrf2 and Nrf2-p) epitopes are indicated. B: Islets from C57BL/6 mice were isolated, dispersed, and incubated in 5.5 or 20 mmol/L glucose for 5 min, followed by immunostaining with insulin and Nrf2-p antibodies. C and D: Dispersed Nrf2^lox/lox mouse islets were transduced with LacZ or Cre adenovirus and cultured in the presence of 20 mmol/L glucose for 48 h, followed by immunostaining using insulin and Nrf2 (C) or insulin and Nrf2-p antibodies (D). E: Dispersed Nrf2^lox/lox islets were incubated with 20 mmol/L glucose for 48 h, followed by RNA isolation. F and G: mRNA expression of Nrf2 and known Nrf2 target genes was measured. Dispersed Nrf2^lox/lox mouse islets were transduced with LacZ or Cre adenovirus and incubated with 50 nmol/L brusatol (Bru) or vehicle (100% ethanol) in the presence of 5.5, 11, or 20 mmol/L glucose for 48 h, followed by immunostaining using insulin and Ki67 antibodies. Percentage of Ki67-positive and insulin-positive cells was then calculated. H: Mouse islets were incubated with 50 nmol/L Bru or vehicle in the presence of 20 mmol/L glucose for 48 h, followed by immunostaining with insulin and Nrf2 antibodies. I and J: Dispersed Nrf2^lox/lox mouse islets were transduced with adenoviruses expressing either LacZ or Cre and cultured in the presence of 20 mmol/L glucose for 48 h, followed by TUNEL assay. The percentage of insulin- and TUNEL-positive cells was calculated. K: Nrf2^lox/lox islets transduced with LacZ or Cre adenovirus were incubated in 20 mmol/L glucose for 24 h, followed by RNA extraction. Expression of cell-cycle regulator genes was measured by RT-PCR. Data are mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.