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ß-Cell Lines Derived from Transgenic Cpe^fat/Cpe^fat Mice Are Defective in Carboxypeptidase E and Proinsulin Processing¹

Department of Molecular Pharmacology, Albert Einstein College of Medicine (O.V., L.D.F.), Bronx, New York 10461; the Department of Biochemistry and Molecular Biology (H.F., D.F.S.) and Howard Hughes Medical Institute (D.F.S.), University of Chicago, Chicago, Illinois 60637; and The Jackson Laboratory (S.H.L., E.H.L.), Bar Harbor, Maine 04609

Address all correspondence and requests for reprints to: Lloyd D. Fricker, Ph.D., Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461.

A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene in Cpe^fat/Cpe^fat mice affects proinsulin processing. Cell lines derived from the pancreatic ß-cells of Cpe^fat/Cpe^fat mice were generated by crossing C57BLKS/J-Cpe^fat/+ mice with NOD mice expressing the simian virus 40 large T oncogene under the control of the rat insulin II promoter. Two cell lines, designated NIT-2 and NIT-3, were cultured from adenomatous islets obtained from F2 littermates and were compared with the NIT-1 cell line previously developed from mice with wild-type CPE. Electron microscopy of the cultured NIT-2 and -3 cells showed increased numbers of enlarged and electron-lucent granules compared with NIT-1 cells. Pro-CPE, but not the mature form of CPE, is present in NIT-2 and -3 cells, and neither pro-CPE nor CPE are secreted into the medium. Immunocytochemistry shows the pro-CPE to be localized to an endoplasmic reticulum-like structure in NIT-3 cells. Proinsulin is less extensively processed in NIT-2 and -3 cells than in NIT-1 cells, indicating that the Cpe^fat mutation affects both the endopeptidase and carboxypeptidase reactions. The secretion of insulin/proinsulin from NIT-2 and -3 cells is significantly elevated by secretagogues, indicating that CPE is not required for sorting proinsulin into the regulated pathway.

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