Non-muscle myosin IIA is involved in focal adhesion and actin remodelling controlling glucose-stimulated insulin secretion.

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.

Oleate-mediated activation of phospholipase D and mammalian target of rapamycin (mTOR) regulates proliferation and rapamycin sensitivity of hepatocarcinoma cells.

AIMS/HYPOTHESIS: A high-fat diet and obesity are associated with increased risk of liver cancer. Because increased delivery of NEFA to the liver occurs in these conditions, we investigated the involvement of the unsaturated fatty acid oleate in hepatocarcinoma cell proliferation using human-derived hepatocarcinoma cell lines as model systems. METHODS: Western blotting, FACS analysis and [(3)H]thymidine incorporation were used to study the signalling pathways and the proliferation of cells cultured for up to 72 h with or without a concentration of oleate considered to be relevant to human pathophysiology (50 µmol/l) or a concentration considered elevated (1 mmol/l). RESULTS: In HepG2 cells, proliferation was increased in the presence of 50 µmol/l oleate, but was decreased at 1 mmol/l. This differential effect was correlated with the activation of the mammalian target of rapamycin complex 1 (mTORC1) and with increased translation of cell cycle regulators. Oleate-mediated mTORC1 activation required phospholipase D activation, which produces phosphatidic acid and is known to render mTORC1 rapamycin resistant. Remarkably, rapamycin resistance was found to affect specifically the mTORC1/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) branch of the mTORC1 pathway in the presence of 50 µmol/l oleate. Furthermore, inhibition of phosphatidic acid production abolished oleate-induced increases in mTORC1 activity and cyclin A production. Importantly, the same differential effects of oleate on mTORC1 activation, cell cycle regulators and rapamycin resistance were found in SK-Hep1 cells. CONCLUSIONS/INTERPRETATION: Oleate stimulates mTORC1 activation and rapamycin resistance. We propose that rapamycin-derived mTOR inhibitors are likely to be of limited therapeutic use to restrain hepatic tumour growth, particularly in the context of associated obesity.