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Aims/hypothesis: Actin and focal adhesion (FA) remodelling are essential for glucose-stimulated insulin secretion (GSIS). Non-muscle myosin II (NM II) isoforms have been implicated in such remodelling in other cell types, and myosin light chain kinase (MLCK) and Rho-associated coiled-coil-containing kinase (ROCK) are upstream regulators of NM II, which is known to be involved in GSIS. The aim of this work was to elucidate the implication and regulation of NM IIA and IIB in beta cell actin and FA remodelling, granule trafficking and GSIS. Methods: Inhibitors of MLCK, ROCK and NM II were used to study NM II activity, and knockdown of NM IIA and IIB to determine isoform specificity, using sorted primary rat beta cells. Insulin was measured by radioimmunoassay. Protein phosphorylation and subcellular distribution were determined by western blot and confocal immunofluorescence. Dynamic changes were monitored by live cell imaging and total internal reflection fluorescence microscopy using MIN6B1 cells. Results: NM II and MLCK inhibition decreased GSIS, associated with shortening of peripheral actin stress fibres, and reduced numbers of FAs and insulin granules in close proximity to the basal membrane. By contrast, ROCK inhibition increased GSIS and caused disassembly of glucose-induced central actin stress fibres, resulting in large FAs without any effect on FA number. Only glucose-induced NM IIA reorganisation was blunted by MLCK inhibition. NM IIA knockdown decreased GSIS, levels of FA proteins and glucose-induced extracellular signal-regulated kinase 1/2 phosphorylation. Conclusions/interpretation: Our data indicate that MLCK-NM IIA may modulate translocation of secretory granules, resulting in enhanced insulin secretion through actin and FA remodelling, and regulation of FA protein levels

Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes.

Aims/hypothesis: Actin and focal adhesion (FA) remodelling are essential for glucose-stimulated insulin secretion (GSIS). Non-muscle myosin II (NM II) isoforms have been implicated in such remodelling in other cell types, and myosin light chain kinase (MLCK) and Rho-associated coiled-coil-containing kinase (ROCK) are upstream regulators of NM II, which is known to be involved in GSIS. The aim of this work was to elucidate the implication and regulation of NM IIA and IIB in beta cell actin and FA remodelling, granule trafficking and GSIS. Methods: Inhibitors of MLCK, ROCK and NM II were used to study NM II activity, and knockdown of NM IIA and IIB to determine isoform specificity, using sorted primary rat beta cells. Insulin was measured by radioimmunoassay. Protein phosphorylation and subcellular distribution were determined by western blot and confocal immunofluorescence. Dynamic changes were monitored by live cell imaging and total internal reflection fluorescence microscopy using MIN6B1 cells. Results: NM II and MLCK inhibition decreased GSIS, associated with shortening of peripheral actin stress fibres, and reduced numbers of FAs and insulin granules in close proximity to the basal membrane. By contrast, ROCK inhibition increased GSIS and caused disassembly of glucose-induced central actin stress fibres, resulting in large FAs without any effect on FA number. Only glucose-induced NM IIA reorganisation was blunted by MLCK inhibition. NM IIA knockdown decreased GSIS, levels of FA proteins and glucose-induced extracellular signal-regulated kinase 1/2 phosphorylation. Conclusions/interpretation: Our data indicate that MLCK-NM IIA may modulate translocation of secretory granules, resulting in enhanced insulin secretion through actin and FA remodelling, and regulation of FA protein levels