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The delivery of newly-formed secretory content to the granule inventory occurs through direct fusion of recently formed granules and mature granules. The introduction of knockout mice allowed us to investigate the characteristics of the delivery process and to determine the core protein machinery required for granule growth. The SNARE machinery mediates membrane fusion and is essential for the granule lifecycle. In the current work, we use VAMP8 knockout mice to show that the SNARE machinery plays a critical role in the process of granule homotypic fusion. Consistent with this, the mutated mouse pancreatic acinar secretory granules are significantly smaller compared to the control group, demonstrating few granule profiles that might be the result of homotypic fusion. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Previous studies have demonstrated that the VAMP8 protein plays a complex role in the control of granule secretion, transport vesicle trafficking, phagocytosis, and endocytosis. The present study was aimed to investigate the role of VAMP8 in mediating GLUT4 trafficking and therefore insulin action in mice. RESEARCH DESIGN AND METHODS--Physiological parameters were measured using Oxymax indirect calorimetry system in 12-week-old VAMP8 null mice. Dynamic analysis of glucose homeostasis was assessed using euglycemic-hyperinsulinemic clamp coupled with tracer radioactively labeled 2-deoxyglucose. Insulin stimulated GLUT4 protein expressions on muscle cell surface were examined by immunofluorescence microscopy. RESULTS--VAMP8 null mice display reduced adiposity with increased energy expenditure despite normal food intake and reduced spontaneous locomotor activity. In parallel, the VAMP8 null mice also had fasting hypoglycemia (84 ± 11 vs. 115 ± 4) and enhanced glucose tolerance with increased insulin sensitivity due to increases in both basal and insulin-stimulated glucose uptake in skeletal muscle (0.19 ± 0.04 vs. 0.09 ± 0.01 mmol/kg/ min during basal, 0.6 ± 0.04 vs. 0.31 ± 0.06 mmol/kg/min during clamp in red-gastrocnemius muscle, P < 0.05). Consistent with a role for VAMP8 in the endocytosis of the insulin-responsive GLUT4, sarcolemma GLUT4 protein levels were increased in both the basal and insulin-stimulated states without any significant change in the total amount of GLUT4 protein or related facilitative glucose transporters present in skeletal muscle, GLUT1, GLUT3, and GLUT11. CONCLUSIONS--These data demonstrate that, in the absence of VAMP8, the relative subcellular distribution of GLUT4 is altered, resulting in increased sarcolemma levels that can account for increased glucose clearance and insulin sensitivity. Diabetes 60:30-38, 2011 [ABSTRACT FROM AUTHOR]

Abstract: A spontaneous lymphoma was detected in mice, which was caused by a recessive autosomal mutation. The genetic basis was revealed to be a 5-bp deletion at the splicing donor site of the first intron of the FasL gene, resulting in aberrant transcripts coding for non-functional proteins. This mutation of the FasL gene caused development of lymphoma in all four mouse genetic backgrounds tested and the lymphoma was characterized by an expansion of leucocytes that were TCR+CD3+B220+CD19−CD4−CD8−. Accordingly, severe splenomegaly developed in the mutant mice. Interestingly, thymic hyperplasia was observed in mutant mice at later stages. These results underscore the functional importance of the splicing donor site in the function of the FasL gene and provide an independent evidence for a role of FasL in normal development of lymophocytes. The mutant mice offer another genetically defined mouse model for further studies of the role and mechanism of action of FasL. [Copyright &y& Elsevier]

Despite our general understanding that members of the SNARE superfamily participate in diverse intracellular docking/fusion events, the physiological role of the majority of SNAREs in the intact organism remains elusive. In this study, through targeted gene knockout in mice, we establish that VAMP8/endobrevin is a major player in regulated exocytosis of the exocrine pancreas. VAMP8 is enriched on the membrane of zymogen granules and exists in a complex with syntaxin 4 and SNAP-23. VAMP8-/- mice developed normally but showed severe defects in the pancreas. VAMP8 null acinar cells contained three times more zymogen granules than control acinar cells. Furthermore, secretagogue-stimulated secretion was abolished in pancreatic fragments derived from VAMP8-/- mice. In addition, VAMP8-/- mice were partially resistant to supramaximal caerulein-induced pancreatitis. These results suggest a major physiological role of VAMP8 in regulated exocytosis of pancreatic acinar cells by serving as a v-SNARE of zymogen granules. [Copyright &y& Elsevier]

Mast cell (MC)-induced inflammatory responses involve massive exocytosis of pre-stored granular mediators and secretion of numerous cytokines/chemokines. SNARE proteins are crucial for intracellular membrane fusion during secretion in MCs. Here we have analyzed the role of vesicular associated membrane protein-8 (VAMP-8), a member of the v-SNARE family of fusion proteins in MC secretory events. We show that VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. Using VAMP-8-deficient in bone marrow derived MCs (BMMCs), we observed that release of preformed mediators such as beta-hexosaminidase and histamine is inhibited (~ 50 %) compared to WT BMMCs. Similarly, we found that VAMP-8-deficient mice have reduced plasma histamine levels in passive systemic anaphylaxis experiments. By contrast, VAMP-8-deficient BMMCs secreted equal amounts of TNF, IL-4, IL-6 and MIP-1alpha. Upon stimulation, VAMP-8 engages in SNARE complexes with SNAP-23 and syntaxin-4, known to play a role in preformed mediator release. Furthermore, we showed that while externalisation of SGs is blocked in VAMP-8-deficient MCs, we can detect unprocessed TNF accumulating at the surface colocalizing with a VAMP-3-positive compartmentbut not with VAMP-8. Our findings demonstrate that VAMP-8 segregates MC preformed mediator exocytosis from cytokine trafficking pathways. [ABSTRACT FROM AUTHOR]

Abstract: During an inflammation and upon encountering pathogens, immature dendritic cells (DC) undergo a maturation process to become highly efficient in presenting antigens. This transition from immature to mature state is accompanied by various physiological, functional and morphological changes including reduction of caspase activity and inhibition of phagocytosis in the mature DC. Caspases are cysteine proteases which play essential roles in apoptosis, necrosis and inflammation. Here, we demonstrate that VAMP-8, (a SNARE protein of the early/late endosomes) which has been shown previously to inhibit phagocytosis in DC, is a substrate of caspases. Furthermore, we identified two putative conserved caspase recognition/cleavage sites on the VAMP-8 protein. Consistent with the up-regulation of VAMP-8 expression upon treatment with caspase inhibitor (CI), immature DC treated with CI exhibits lower phagocytosis activity. Thus, our results highlight the role of caspases in regulating VAMP-8 expression and subsequently phagocytosis during maturation of DC. [Copyright &y& Elsevier]

In rodents and humans, alcohol exposure has been shown to predispose the pancreas to cholinergic or viral induction of pancreatitis. We previously developed a rodent model in which exposure to an ethanol (EtOH) diet, followed by carbachol (Cch) stimulation, redirects exocytosis from the apical to the basolateral plasma membrane of acinar cells, resulting in ectopic zymogen enzyme activation and pancreatitis. This redirection of exocytosis involves a soluble NSF attachment receptor (SNARE) complex consisting of syntaxin-4 and synapse-associated protein of 23 kDa (SNAP-23). Here, we investigated the role of the zymogen granule (ZG) SNARE vesicle-associated membrane protein 8 (VAMP8) in mediating basolateral exocytosis. In WT mice, in vitro EtOH exposure or EtOH diet reduced Cch-stimulated amylase release by redirecting apical exocytosis to the basolateral membrane, leading to alcoholic pancreatitis. Further reduction of zymogen secretion, caused by blockade of both apical and basolateral exocytosis and resulting in a more mild induction of alcoholic pancreatitis, was observed in Vamp8(-/-) mice in response to these treatments. In addition, although ZGs accumulated in Vamp8(-/-) acinar cells, ZG-ZG fusions were reduced compared with those in WT acinar cells, as visualized by electron microscopy. This reduction in ZG fusion may account for reduced efficiency of apical exocytosis in Vamp8(-/-) acini. These findings indicate that VAMP8 is the ZG-SNARE that mediates basolateral exocytosis in alcoholic pancreatitis and that VAMP8 is critical for ZG-ZG homotypic fusion. [ABSTRACT FROM AUTHOR]

Phagocytosis is a specialized mechanism utilized by mammalian cells, particularly the cells of the immune system, such as the dendritic cells (DC) and macrophages to protect the host against infection. The process involves a complex cascade of pathways, from the ligation of surface receptors of phagocytes with components of the microorganism's surface, formation of phagosomes and subsequently phagolysosomes, to the eventual presentation of foreign antigens. VAMP-8/endobrevin has been shown previously to function in the endocytic pathways. Our results showed that VAMP-8 co-localized with LAMP-2 and a significant amount of VAMP-8 was recruited to the phagosomes during bacterial ingestion. However, overexpression of VAMP-8 significantly inhibited phagocytosis in DC. We also found that the phagocytic activity of VAMP-8-/- DC was significantly higher than wild-type VAMP-8+/+ DC, thus further confirming that VAMP-8 negatively regulates phagocytosis in immature DC. This work was supported by grants from the Microbiology Vaccine Initiative (R182-005-067-731 to S.H.W) and Academic Research Council, Singapore Ministry of Education (R182-000-099-112 to S.H.W). [ABSTRACT FROM AUTHOR]