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Aggregation and Lack of Secretion of Most Newly Synthesized Proinsulin in Non-ß-Cell Lines

Department of Pharmacology, Yale School of Medicine and Yale School of Nursing, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Priscilla S. Dannies, Yale University School of Medicine, Department of Pharmacology, 333 Cedar Street, New Haven, Connecticut 06520-8066. E-mail: priscilla.dannies{at}yale.edu.

Myoblasts transfected with HB10D insulin secrete more hormone than those transfected with wild-type insulin, as published previously, indicating that production of wild-type insulin is not efficient in these cells. The ability of non-ß-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH₄C₁ cells stably transfected with proinsulin, two thirds of ³⁵S-proinsulin was degraded within 3 h of synthesis, whereas ³⁵S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of ³⁵S-proinsulin was degraded within 3 h after synthesis, whereas ³⁵S-GH was stable. In transiently transfected fibroblast COS cells, ³⁵S-proinsulin was stable for longer, but less than 10% was secreted 8 h after synthesis. Proinsulin formed a concentrated patch detected by immunofluorescence in transfected cells that did not colocalize with calreticulin or BiP, markers for the endoplasmic reticulum, but did colocalize with membrin, a marker for the cis-medial Golgi complex. Proinsulin formed a Lubrol-insoluble aggregate within 30 min after synthesis in non-ß-cells but not in INS-1E cells, a ß-cell line that normally produces insulin. More than 45% of ³⁵S-HB10D proinsulin was secreted from COS cells 3 h after synthesis, and this mutant formed less Lubrol-insoluble aggregate in the cells than did wild-type hormone. These results indicate that proinsulin production from these non-ß-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of ß-cells may prevent aggregation of proinsulin to allow efficient production.