Regulation of pancreatic β-cell insulin secretion by actin cytoskeleton remodelling: role of gelsolin and cooperation with the MAPK signalling pathway

We have previously isolated two MIN6 beta-cell sublines, B1, highly responsive to glucose-stimulated insulin secretion, and C3, markedly refractory (Lilla, V., Webb, G., Rickenbach, K., Maturana, A., Steiner, D. F., Halban, P. A. and Irminger, J. C. (2003) Endocrinology 144, 1368-1379). We now demonstrate that C3 cells have substantially increased amounts of F-actin stress fibres whereas B1 cells have shorter cortical F-actin. Consistent with these data, B1 cells display glucose-dependent actin remodelling whereas, in C3 cells, F-actin is refractory to this secretagogue. Furthermore, F-actin depolymerisation with latrunculin B restores glucose-stimulated insulin secretion in C3 cells. In parallel, glucose-stimulated ERK1/2 activation is greater in B1 than in C3 cells, and is potentiated in both sublines following F-actin depolymerisation. Glucose-activated phosphoERK1/2 accumulates at actin filament tips adjacent to the plasma membrane, indicating that these are the main sites of action for this kinase during insulin secretion. In addition, B1 cell expression of the calcium-dependent F-actin severing protein gelsolin is >100-fold higher than that of C3 cells. Knock-down of gelsolin reduced glucose-stimulated insulin secretion, whereas gelsolin over-expression potentiated secretion from B1 cells. Gelsolin localised along depolymerised actin fibres after glucose stimulation. Taken together, these data demonstrate that F-actin reorganization prior to insulin secretion requires gelsolin and plays a role in the glucose-dependent MAPK signal transduction that regulates beta-cell insulin secretion

Regulation of insulin secretion by phosphatidylinositol-4,5-bisphosphate.

The role of PIP(2) in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP(2) with PH-PLC-GFP or PIP5KIgamma RNAi did not impact on glucose-stimulated secretion although susceptibility to apoptosis was increased. Over-expression of PIP5KIgamma improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F-actin, PIP(2), transferrin receptor, caveolin 1, Arf6 and the insulin granule membrane protein phogrin but not insulin. Expression of constitutively active Arf6 Q67L also resulted in vacuole formation and inhibition of secretion, which was reversed by PH-PLC-GFP co-expression. PIP(2) co-localized with gelsolin and F-actin, and gelsolin co-expression partially reversed the secretory defect of PIP5KIgamma-over-expressing cells. RhoA/ROCK inhibition increased actin depolymerization and secretion, which was prevented by over-expressing PIP5KIgamma, while blocking PIP(2) reduced constitutively active RhoA V14-induced F-actin polymerization. In conclusion, although PIP(2) plays a pro-survival role in MIN6B1 cells, excessive PIP(2) production because of PIP5KIgamma over-expression inhibits secretion because of both a defective Arf6/PIP5KIgamma-dependent endocytic recycling of secretory membrane and secretory membrane components such as phogrin and the RhoA/ROCK/PIP5KIgamma-dependent perturbation of F-actin cytoskeleton remodelling