Aggregation and lack of secretion of most newly synthesized proinsulin in non-β-cell lines

Abstract

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-β-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-β-cells but not in INS-1E cells, a β-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-β-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of β-cells may prevent aggregation of proinsulin to allow efficient production.