Figure 7.

IL-1β, SNAP and AICAR alter β-cell calcium homeostasis, and direct activation of AMPK impairs SERCA2 activity. To assess cytosolic Ca²⁺ levels, Calcium 6 fluorescence and fura-2/AM fluorescence ratios were measured as described under Materials and Methods. For Calcium 6 measurements (a–d), INS-1 cells were pretreated with dimethyl sulfoxide (DMSO) (CT), 5 ng/ml IL-1β (IL) combined with or without 0.5 mM  l-NMMA (LN), 300 mM SNAP (SN) or 2 mM AICAR (AC) for 24 h. (e–h) For fura-2/AM assays, INS-1 cells were pretreated with DMSO (CT) or 2 mM AICAR for 3 or 16 h. Quantitative results of the basal cytosolic Ca²⁺ level (F0) and the relative ER Ca²⁺ extrusion described as a normalized ratio according to the formula ΔF/F0 performed in the presence (a and b) or absence (c and d) of 2.5 mM CaCl₂ are shown. (e) Schematic illustrating the calculation of ΔF/F0 ratios from fura-2/AM imaging experiments. The Ca²⁺ clearance rate was used as an estimate of SERCA2 activity and was analyzed using linear regression to calculate the slope of the fura-2/AM ratio following withdrawal of caffeine. (f) Representative trace from untreated INS-1 cells (CT) and INS-1 cells treated with AICAR for 3 or 16 h. (g–h) Quantitative results of the ΔF/F0 ratio and slope from INS-1 cells untreated (CT) or treated with AICAR for 3 or 16 h. Indicated comparisons are significantly different (*P<0.01 and ***P<0.001)