Figure 6.

AMPK activation modulates IL-1β-induced iNOS expression and is required for SNAP-induced downregulation of SERCA2. (a–i) INS-1 cells or isolated rat islets were treated with dimethyl sulfoxide (DMSO) (CT), CC, 5 ng/ml IL-1β (IL) with or without CC (IL-CC) or transduced with an HA-tagged AMPK-DN or DN or control adenovirus (Luci or Lu) before treatment with or without 5 ng/ml IL-1β. Total protein was isolated, and immunoblot was performed using antibodies against iNOS and actin. Quantitative protein levels of iNOS are shown graphically (b, c and e, f). Total mRNA was isolated from INS-1 cells, and reverse-transcribed RNA was subjected to real-time PCR for quantification of iNOS and actin transcript levels (h and i). (g) INS-1 culture media was collected at treatment end, and nitrite concentration measurement was performed. (j–m) Next, INS-1 cells were treated with DMSO (CT), 300 mM of SNAP (SN) combined with or without 10 μM of CC, or AICAR at the indicated doses for 24 h. Total protein was isolated, and immunoblot was performed using antibodies against SERCA2, pAMPKa, IκBα and actin. (k and m) Quantitative protein levels of SERCA2 are shown graphically. Indicated comparisons are significantly different (*P<0.05, **P<0.01 and ***P<0.001)