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Dysfunctional mitochondria are observed in β-cells of diabetic patients, which are eventually removed by autophagy. Vesicle-associated membrane protein (VAMP)7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic β-cells. However, its molecular mechanism is largely unknown. In this study, we investigated the molecular mechanism of VAMP7-dependent autophagosome formation using VAMP7-deficient β-cells and β-cell–derived Min6 cells. VAMP7 localized in autophagy-related (Atg)9a–resident vesicles of recycling endosomes (REs), which contributed to autophagosome formation, and it interacted with Hrb, Syntaxin16, and SNAP-47. Hrb recruited VAMP7 and Atg9a from the plasma membrane to REs. Syntaxin16 and SNAP-47 mediated autophagosome formation at a step later than the proper localization of VAMP7 to Atg9a-resident vesicles. Knockdown of Hrb, Syntaxin16, and SNAP-47 resulted in defective autophagosome formation, accumulation of dysfunctional mitochondria, and impairment of glucose-stimulated insulin secretion. Our data indicate that VAMP7 and Atg9a are initially recruited to REs to organize VAMP7 and Atg9a-resident vesicles in an Hrb-dependent manner. Additionally, VAMP7 forms a SNARE complex with Syntaxin16 and SNAP-47, which may cause fusions of Atg9a-resident vesicles during autophagosome formation. Thus, VAMP7 participates in autophagosome formation by supporting Atg9a functions that contribute to maintenance of mitochondrial quality.

Pancreatic beta-cells are susceptible to reactive oxygen species (ROS), which are known to be generated by high or low glucose (LG), hypoxic, or cytokine-producing conditions. When we cultured mouse beta-cell-derived MIN6 cells in a LG condition, we detected a significant generation of ROS, including hydrogen peroxide, which was comparable to the ROS production in hypoxic or cytokine-treated conditions. ROS accumulation induced by the LG culture led to cell death, which was prevented by the ROS scavengers N-acetylcysteine and manganese(III)tetrakis(4-benzoic acid) porphyrin. We next investigated the mechanism of stress-activated protein kinases (SAPKs), c-jun N-terminal kinase (JNK) and p38, in ROS-induced MIN6 cell death. Activation of p38 occurred immediately after the LG culture, whereas JNK activation increased slowly 8 h later. Adenoviral p38 expression decreased MIN6 cell death, whereas the JNK expression increased it. Consistently, blocking p38 activation by inhibitors increased beta-cell death, whereas JNK inhibitors decreased it. We then examined the role of MAPK phosphatases (MKPs) specific for stress-activated protein kinases in beta-cell death. We found that MKP-1 presented an increase in its oxidized product after the LG culture. ROS scavengers prevented the appearance of this oxidized product and JNK activation. Thus, ROS-induced MKP inactivation causes sustained activation of JNK, which contributes to beta-cell death. Adenoviral overexpression of MKP-1 and MKP-7 prevented the phosphorylation of JNK at 36 h after the LG culture, and decreased MIN6 beta-cell death. We suggest that beta-cell death is regulated by interactions between JNK and its specific MKPs.

We recently identified a novel set of Rab and its effector, Rab27a/granuphilin, that possibly regulates the exocytosis of insulin-containing, dense core granules in pancreatic beta-cells. In the present study we characterized another isoform, Rab27b, to further investigate the function of the Rab27 subfamily. Among the tissues examined, Rab27b is most abundantly expressed in pituitary tissue, where Rab27a is preferentially expressed. It is also significantly expressed in brain and spleen, where Rab27a is minimally expressed. Rab27a and Rab27b are differentially expressed in pituitary cell types that secrete different peptide hormones. Rab27b associates with secretory granules and granuphilin in the pituitary endocrine cell line AtT20. Furthermore, overexpression of its inactive mutant, Rab27b N133I, significantly inhibits basal and forskolin-induced ACTH secretion in AtT20 cells. These findings indicate that Rab27b is involved in pituitary hormone secretion, which further supports the idea that members of the Rab27 subfamily regulate the exocytosis of dense core granules containing peptide hormones in endocrine cells.