Studies On Mechanisms Of Exocytosis Regulation In Rat Pancreatic β Cells

Insulin plays a pivotal role in the balance of blood sugar. The deficiency of insulin secretion from pancreatic βcells will result in non-insulin dependent diabetes mellitus. In the pancreatic βcell, the insulin secretion is regulated by calcium-dependent regulative exocytosis of large dense-cored vesicles. Thus, the study on the molecular mechanism of insulin secretion and its regulation are important for theories, and have guidance meaning for the clinic treatment of diabetes. In this study, by using capacitance measurement, optic image(confocal, TIRFM, FRET), microfluorometric measurement of calcium, flash photolysis of caged Ca²⁺, fluorescence protein and the techniques of molecular biology, we studied the kinetics of exocytosis; the regulation mechanism of protein kinase A and C; the molecular mechanism of Stx1A and Munc18a in regulation of insulin secetion from single rat pancreatic βcells. (1) We found exocytosis of vesicles, induced by a sequence of depolarization stimulus, which coupling to the voltage dependent calcium channel in rat pancreatic βcell, was a calcium dependent process. By using the calcium slope measurement, we found that the so-called calcium-independent secretion induced by LTX was still a calcium dependent process. The fact is that LTX lower the calcium threshold of secretion, and therefore results in the exocytosis at rest calcium level in βcell. By employing capacitance measurement and flash photolysis of caged Ca²⁺, we found there were three types of exocytosis after UV flash stimulus in individual cell: exocytosis, exo-endocytosis and excess retrieval. The exocytosis can be divided into three kinetic components which can be well analysed by using triple exponential and linearity fitting. (2) Glucose and other secretagogues are thought to activate a variety of protein kinases. This study was designed to unravel the sites of action of protein kinase A (PKA) and protein kinase C (PKC) in modulating insulin secretion. By using high time resolution measurements of membrane capacitance and flash photolysis of caged Ca²⁺, we characterize three kinetically different pools of vesicles in rat pancreatic β-cells, namely, a highly calciumsensitive pool (HCSP), a readily releasable pool (RRP), and a reserve pool. The size of the HCSP is ²0 fF under resting conditions, but is dramatically increased by application of either phorbol esters or forskolin. Phorbol esters and forskolin also increase the size of RRP to a lesser extent. The augmenting effect of phorbol esters or forskolin is blocked by various PKC or PKA inhibitors, indicating the involvement of these kinases. The effects of PKC and PKA on the size of the HCSP are not additive, suggesting a convergent mechanism. Using a protocol where membrane depolarization is combined with photorelease of Ca2+, we find that the HCSP is a distinct population of vesicles from those colocalized with Ca2+ channels. We propose that PKA and PKC promote insulin secretion by increasing the number of vesicles that are highly sensitive to Ca2+. (3) Syntaxin 1A (Stx1A) and Munc18a play essential roles in vesicular trafficking and exocytosis. The molecular mechanism and the functional roles of their interaction remain to be explored. In the present study, we have studied the intracellular localization and interaction of Stx1A and Munc18a in insulin secreting INS-1 cells as well as CHO cells by confocal and evanescent field fluorescence imaging. We found that Munc18a colocalized with clusters of Stx1A and its open-form mutant, but not the mutant lacking Habc domain, at the plasma membrane in live INS-1 cells, suggesting the interaction of Munc18a with the Habc domain of Stx1A is necessary for the translocation of Munc18a to the plasma membrane. In CHO cells, where no endogenous Stx1A is reported, Stx1A failed to localize to the plasma membrane even in the presence of coexpressed Munc18a, suggesting Munc18a is not required for the transportation of Stx1A to the plasma membrane. Moreover, in vivo FRET measurement demonstrated the interaction between Munc18a and Stx1A, whereas no significant FRET signal was observed for Munc18a and the open-form Stx1A mutant despite their colocalization. By overexpression of Munc18a in primary cultured pancreatic beta cells, we further showed that Munc18a negatively regulated insulin secretion by inhibiting the recruitment of granules to the readily releasable pool. Overexpression of neither the wild type Stx1A nor the open-form Stx1A-Habc affects exocytosis, suggesting the availability of Stx1A in its open form is not a rate limiting step in insulin secretion. Overexpression of open-form Stx1A-DM alone or Stx1A-DM andMunc18a under the ratio of 1:1 promotes the secretion from SRP and sustained component, excluding RRP, suggesting the pen-form Stx1A participate in the formation of SNARE complex, as well as the interaction of Munc18a with the Habc domain of Stx1A.