A study on the Intracellular dynamics of insulin granules using a phogrin-GFP fused protein

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1.We constructed an expression vector in which phogrin is bound to the C-terminus of GFP. Using the vector, we successfully expressed. GFP-phogxin in MIN6 cells.2.We got several new findings concerning regulation mechanisms of the insulin granule traffic.(1)Glucose accelerated the insulin granule traffic in MIN6 cells. The calmodulin-inhibitor W-7 inhibited the glucose-induced acceleration of the granule traffic, and myosin light chain kinase inhibited the glucose-induced acceleration of the granule traffic.(2)The blockers to Ca^<2+> channels (nifedipine and nitrendipine) did not inhibit the glucose-induced acceleration of the granule traffic.(3)The K_<ATP>-opener (diazoxide) did not inhibit the glucose-induced acceleration of the granule traffic.(4)The glucose stimulation increased phosphatized myosin light chain in MIN6 cells.3.We investigated the mechanism of B cell loss in transgenic mice with elevated levels of B cell calmodulin.(1)The transgenic mice experienced a sudden rise in blood glucose levels, which was associated with developed of severe hypoinsulinemia and loss of B cells from the islets.(2)Ultrastructural analysis revealed that compromised granule formation and apoptotic changes in the transgenic B cells preceded the onset of hyperglycemia.(3)Intraperitoneal injection of tolbutamide, an antidiabetic sulfonylurea, decreased blood glucose levels but increased the number of apoptotic B cells.(4)Injection of the mice with N^ω-nitro-L-arginine methyl ester, which inhibits nitric oxide synthase activity, prevented hyperglycemia and lessened the changes in number and size of B cells.4.We found that caveolin-1 was expressed in MIN6 cells, and colocalized with γ-tubulin in microtubule-organizing center. The signal of caveolin-1 decreased after glucose stimulation.