Maintenance of glucose homeostasis requires' cross-talk' between pancreatic insulin secretion and insulin signaling in the peripheral tissues. Both insulin secretion and glucose uptake are regulated exocytosis processes, mediated by Syntaxin-based SNARE protein complexes. The competitive binding of a Munc 18 protein for the plasma membrane Syntaxin further regulates SNARE complex formation. While it has been established by this investigator and others that genetic perturbation resulting in increased Munc18c or diminished Syntaxin 4 (Syn4) protein will lead to dysregulation of glucose uptake, it is still unclear how important the Munc 18c-Syn4 complex is to insulin secretion. The objective of this application is to delineate the mechanism(s) underlying the regulation of insulin secretion in islet cells and glucose uptake in adipocytes by the Syntaxin 4-Munc 18c complex, and evaluate the combination of these tissue-specific effects to the overall control of whole-body glucose homeostasis in vivo. Preliminary data indicate that increased Munc 18c-Syn4 complex formation is correlated with increased insulin sensitivity, whereas deficiency leads to dysregulated insulin secretion and insulin action. New data also show Munc 18c and Syn4 to be post-translationally modified by phosphorylation and glycosylation in response to stimuli in 3T3L1 adipocytes and MIN6 beta ceils.
The specific aims of this project are: 1) Establish the role of Munc 18c-Syn4 complexes in insulin secretion and glucose homeostasis in vivo; 2) Identify sites of post-translational modification(s) regulating the binding of Munc 18c to Syn4; 3) Define the range of Munc 18c:Syntaxin 4 protein ratio sufficient to support regulated exocytosis. To accomplish these aims, the investigators will quantitate biphasic insulin secretion from Munc18c or Syn4 'knockdown' islet cell models. Sites of glycosylation and phosphorylation will be identified using in vitro labeling of truncated and mutagenized forms of Munc 18c, and the effect on Munc 18c-Syn4 complex formations will also be assessed. In addition, Munc 18c and Syn4 protein levels will be modulated in a novel tetracycline-repressible Munc 18c and Syn4 transgenic mouse model, in addition to Syn4 (-/+) islets transduced with tetracycline-repressible Syn4 adenoviral expression. This work will help towards the identification of the functional and structural regulatory features of the Munc 18c-Syn4 complex in insulin secretion, as well as define the physiologically optimal molecular ratio of Munc18c:Syntaxin 4 in both insulin-secreting and insulin-sensitive tissues. These results are expected to provide new targets for therapeutic intervention and early detection of insulin resistance and diabetes.
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