The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient β cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin-null mice. The enhanced secretion in mutant β cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a–Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin-null β cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.

The results obtained suggest that the changes of amount of RANKL and OPG may be involved in bone resorption as a response to compression force.

†These authors contributed equally to this work.We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic b cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.

The monomeric small GTPase Rab27a is specifically localized on both secretory granules and lysosome-related organelles. Although natural mutations of the Rab27a gene in human Griscelli syndrome and in ashen mice cause partial albinism and immunodeficiency reflecting the dysfunction of lysosome-related organelles, phenotypes resulting from the defective exocytosis of secretory granules have not been reported. To explore the roles of Rab27a in secretory granules, we analyzed insulin secretion profiles in ashen mice. Ashen mice showed glucose intolerance after a glucose load without signs of insulin resistance in peripheral tissues or insulin deficiency in the pancreas. Insulin secretion from isolated islets was decreased specifically in response to high glucose concentrations but not other nonphysiological secretagogues such as high K + concentrations, forskolin, or phorbol ester. Neither the intracellular Ca 2+ concentration nor the dynamics of fusion pore opening after glucose stimulation were altered. There were, however, marked reductions in the exocytosis from insulin granules predocked on the plasma membrane and in the replenishment of docked granules during glucose stimulation. These results provide the first genetic evidence to our knowledge for the role of Rab27a in the exocytosis of secretory granules and suggest that the Rab27a/effector system mediates glucose-specific signals for the exocytosis of insulin granules in pancreatic β cells.

These findings suggest that low-energy laser irradiation stimulates the velocity of tooth movement via induction of RANK and RANKL.