Advanced Glycation End Products Impair Glucose-Stimulated Insulin Secretion of a Pancreatic β-Cell Line INS-1-3 by Disturbance of Microtubule Cytoskeleton via p38/MAPK Activation

Figure 4

AGEs activate p38/MAPK signaling pathway that disrupts microtubule dynamics. (a) Immunoblots of INS-1-3 cells cultured without (CON) or with 200 μg/mL AGEs for 48 hours (AGEs-48 h) or 72 hours (AGEs-72 h). The levels of phosphorylated and total p38/MAPK were immunoblotted by phosphospecific and general anti-p38 antibodies, respectively. β-actin was used as loading control. (b) Immunostaining images of α-tubulin (green) in INS-1-3 cells. INS-1-3 cells were cultured without (CON) or with 200 μg/mL AGEs in the absence or presence of 50 μM SB202190 (SB) for 48 hours. The boxed areas are shown at higher magnification in the right panels to illustrate the details of the microtubules. Scale bar: 25 μm. (c) Quantification of free tubulin (FT) and polymerized tubulin (MT) fractions of INS-1-3 cells by immunoblots. One of the representative results is shown. Statistical analysis was performed based on three immunoblotting results. The polymerization status of tubulin is presented as the percentage of polymerized tubulin divided by the total tubulin content. ; AGE group versus CON, SB, or SB + AGEs group. (d) Relative insulin secretion from INS-1-3 cells in response to 2.5 mM or 25 mM glucose, normalized to the total insulin content, measured by ELISA. Data were derived from three independent experiments and shown as . versus control.

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