Diabetes affects more than 25 million Americans, or 8% of the total population, with a growth of nearly 2 million new cases in the year 2010. This disease contributes to hundreds of thousands of deaths annually, as well as hundreds of billions of dollars in medical costs. Type II diabetes is the most common form of the disease and is caused by the failure of the body to maintain proper blood-glucose levels due to either an inability to produce insulin or insulin-resistance of muscle and adipose (fat) cells, which are responsible for the postprandial absorption of glucose from the bloodstream. Healthy muscle and adipose cells maintain correct glucose homeostasis by trafficking the Glucose transporter 4 (Glut4) to the plasma membrane in response to insulin stimulation. A basal level of Glut4 is maintained at the cell surface via endosomal recycling pathways, but the large response to insulin signaling is mediated by pools of the transport protein sequestered in Glut4 storage vesicles (GSVs). In the absence of stimulation, GSVs lie beneath the cell surface, but in response to insulin, they fuse with the plasma membrane, releasing their Glut4 cargo into the lipid bilayer. Therefore, characterizing the mechanism by which the fusion process occurs and identifying its modes of regulation will provide a better understanding of type II diabetes and could lead to therapeutic targets. The proteins that are responsible for generating the requisite force to fuse the bilayers belong to the SNARE family;in the case of have primarily been examined as overexpression or knockdown products in whole cell systems, thus their exact influences at the single molecular level have not yet been pinpointed. On the other hand, the neuronal fusion mechanism that regulates synaptic transmission has been well-characterized in cellular, structural, and biophysical studies. Interestingly, several of the proteins implicated in GSV SNARE regulation are related to those involved in the neuronal fusion process. It is our goal to determine whether the regulation of the SNARE-mediated fusion process is conserved across these different cell types, or whether the different requirements depending on the environment give rise to distinct modes of regulation. To achieve this, we will perform fusion assays using total internal reflection fluorescence microscopy (TIRFM) on supported bilayers in microfluidic flow cells, an experiment that was developed in our lab. This technique provides the versatility of a bulk liposome fusion assay with the spatial and temporal resolution of cellular fluorescence microscopy, allowing us to abstract the specific function of each protein separate from the complexities of a living cell, as well as measure the biophysical parameters of vesicle docking and fusion. Overall, these experiments will provide a deeper understanding of the exact mode of regulation of GSV fusion in response to insulin stimulation, a process that is vital for glucose homeostasis, and whose disruption leads to type II diabetes. GSV fusion, the active SNAREs are syntaxin 4, SNAP-23, and VAMP2. Additional proteins are known to regulate fusion by directly interacting with the SNAREs, including Munc18c and Doc2b, but
Diabetes affects more than 25 million Americans, or 8% of the total population, and contributes to hundreds of thousands of deaths annually. Type II diabetes is the most common form of the disease and is caused by the failure of the body to maintain proper blood-glucose levels due to insulin-resistance of muscle and adipose (fat) cells. Glucose homeostasis is controlled by trafficking of the glucose transporter protein to the cell membrane in response to insulin stimulation;therefore, characterizing the mechanism by which this process occurs and identifying its modes of regulation will provide a better understanding of this disease and could lead to therapeutic targets.
