Figure 6.

V-ATPase is required for insulin secretion and PKA activation in the pancreatic β-cell line Min6. (A) V-ATPase activity is regulated by glucose in Min6 cells. Min6 cells were incubated with low glucose (2.5 mM) medium for 4 h before incubation with KRBH buffer (2.5 mM glucose) for 30 min in the absence or presence of Concanamycin A. Cells were incubated with KRBH containing DAMP, glucose and drug as indicated for 30 min before cells were fixed and DAMP was visualized by immunofluorescence. For control, Min6 cells were grown in high glucose (25 mM) media and incubated with DAMP for 30 min before fixation. (B) V-ATPase inhibition decreases glucose-stimulated insulin secretion. Supernatants of cells in (A) were collected immediately before fixation and assayed for insulin content. A representative result of four independent experiments is shown. Error bars indicating s.d. (σ) and P-value obtained from a t-test are shown. (C, D) V-ATPase contributes to the activation of PKA in Min6 cells. (C) Cells were grown as in (A) and samples were taken for preparation of total lysates 10 min after glucose stimulation and blotted for phospho-S122 CREB1 and phospho-ERK1/2. Equal amounts of extract were blotted for CREB1 and ERK1/2 for control. (D) Cells were incubated with serum-free medium in the presence of the phosphodieasterase inhibitor IBMX and treated with Concanamycin A or drug vehicle for 30 min before determination of cAMP. A representative result of three independent experiments is presented as in (B).