Fig. 3.

Disruption of lipid rafts abolishes intracellular calcium signaling induced by insulin. A: confocal images of HepG2 cells loaded with Fluo-4/AM (6 µM) and stimulated with insulin (300 nM). A and D: control (A) and metyl-β-cyclodextrin (MβCD)-treated cells (D) were analyzed. Images were pseudocolored according to the scale shown at bottom. Scale bar = 10 μm. B and E: observe that MβCD treatment nearly abolished the amplitude of Ca²⁺ signaling. Graphical representation of the fluorescence increase in the nucleus (blue traces) and cytosol (red traces) of a control (B) and an MβCD-treated cell (E), stimulated with insulin, pointed with an arrow. C and F: summary of insulin stimulation studies. G and J: confocal images of hepatocytes loaded with Fluo-4/AM (6 µM) and stimulated with insulin (300 nM). Control (G) and MβCD-treated hepatocytes (J) were analyzed. H and K: Graphical representation of the fluorescence increase in the nucleus (blue traces) and cytosol (red traces) of a control (H) and an MCBD-treated cell (K) stimulated with insulin. I and L: summary of insulin stimulation studies (HepG2: control nucleus = 221.3 ± 12%, control cytosol = 164 ± 5%; n = at least 50 cells for each condition; hepatocytes: control nucleus = 142.3 ± 2%, control cytosol = 58 ± 2,2%; n = at least 50 cells for each condition. Values are means ± SD of the peak Fluo-4 fluorescence acquired during the observation period, expressed as %baseline. *P < 0.01, difference between groups was statistically significant; ns, not significant. Data were analyzed by one-way ANOVA followed by Bonferroni posttests.