Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its Nterminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation

Kinetics of Rab27a-dependent actions on vesicle docking and priming in pancreatic Β-cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65888/1/jphysiol.2008.158477.pd

Proinsulin Atypical Maturation and Disposal Induces Extensive Defects in Mouse Ins2+/Akita β-Cells

Because of its low relative folding rate and plentiful manufacture in β-cells, proinsulin maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) through the integration of maturation and disposal processes. PIHO is susceptible to genetic and environmental influences, and its disorder has been critically linked to defects in β-cells in diabetes. To explore this hypothesis, we performed polymerase chain reaction (PCR), metabolic-labeling, immunoblotting, and histological studies to clarify what defects result from primary disorder of PIHO in model Ins2+/Akita β-cells. We used T antigen-transformed Ins2+/Akita and control Ins2+/+ β-cells established from Akita and wild-type littermate mice. In Ins2+/Akita β-cells, we found no apparent defect at the transcriptional and translational levels to contribute to reduced cellular content of insulin and its precursor and secreted insulin. Glucose response remained normal in proinsulin biosynthesis but was impaired for insulin secretion. The size and number of mature insulin granules were reduced, but the size/number of endoplasmic reticulum, Golgi, mitochondrion, and lysosome organelles and vacuoles were expanded/increased. Moreover, cell death increased, and severe oxidative stress, which manifested as increased reactive oxygen species, thioredoxin-interacting protein, and protein tyrosine nitration, occurred in Ins2+/Akita β-cells and/or islets. These data show the first clear evidence that primary PIHO imbalance induces severe oxidative stress and impairs glucose-stimulated insulin release and β-cell survival as well as producing other toxic consequences. The defects disclosed/clarified in model Ins2+/Akita β-cells further support a role of the genetic and stress-susceptible PIHO disorder in β-cell failure and diabetes

A new general glucose homeostatic model using a proportional-integral-derivative controller

The glucose-insulin system is a challenging process to model due to the feedback mechanisms present, hence the implementation of a model-based approach to the system is an on-going and challenging research area. A new approach is proposed here which provides an effective way of characterising glycaemic regulation. The resulting model is built on the premise that there are three phases of insulin secretion, similar to those seen in a proportional-integral-derivative (PID) type controller used in engineering control problems. The model relates these three phases to a biological understanding of the system, as well as the logical premise that the homeostatic mechanisms will maintain very tight control of the system. It includes states for insulin, glucose, insulin action and a state to simulate an integral function of glucose. Structural identifiability analysis was performed on the model to determine whether a unique set of parameter values could be identified from the available observations, which should permit meaningful conclusions to be drawn from parameter estimation. Although two parameters - glucose production rate and the proportional control coefficient - were found to be unidentifiable, the former is not a concern as this is known to be impossible to measure without a tracer experiment, and the latter can be easily estimated from other means. Subsequent parameter estimation using Intravenous Glucose Tolerance Test (IVGTT) and hyperglycaemic clamp data was performed and subsequent model simulations have shown good agreement with respect to these real data

Direct imaging shows that insulin granule exocytosis occurs by complete vesicle fusion

Confocal imaging of GFP-tagged secretory granules combined with the use of impermeant extracellular dyes permits direct observation of insulin packaged in secretory granules, trafficking of these granules to the plasma membrane, exocytotic fusion of granules with the plasma membrane, and eventually the retrieval of membranes by endocytosis. Most such studies have been done in tumor cell lines, using either confocal methods or total internal reflectance microscopy. Here we compared these methods by using GFP–syncollin or PC3–GFP plus rhodamine dextrans to study insulin granule dynamics in insulinoma cells, normal mouse islets, and primary pancreatic beta cells. We found that most apparently docked granules did not fuse with the plasma membrane after stimulation. Granules that did fuse typically fused completely, but a few dextran-filled granules lingered at the membrane. Direct recycling of granules occurred only rarely. Similar results were obtained with both confocal and total internal reflection microscopy, although each technique had advantages for particular aspects of the granule life cycle. We conclude that insulin exocytosis involves a prolonged interaction of secretory granules with the plasma membrane, and that the majority of exocytotic events occur by full, not partial, fusion