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-beta-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH(4)C(1) cells stably transfected with proinsulin, two thirds of (35)S-proinsulin was degraded within 3 h of synthesis, whereas (35)S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of (35)S-proinsulin was degraded within 3 h after synthesis, whereas (35)S-GH was stable. In transiently transfected fibroblast COS cells, (35)S-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-beta-cells but not in INS-1E cells, a beta-cell line that normally produces insulin. More than 45% of (35)S-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-beta-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of beta-cells may prevent aggregation of proinsulin to allow efficient production.

Misfolded proinsulin in the endoplasmic reticulum during development of beta cell failure in diabetes

“…55 Presumably, beta cells are more optimized than other cells to promote proinsulin folding and solubility, as recombinant proinsulin expression in heterologous cell types is thought to fare worse than endogenous proinsulin expressed in beta cells. 56 Nevertheless, as described throughout this review, beta cells remain quite susceptible to proinsulin misfolding. In the last decade, it has been suggested that a portion of proinsulin in beta cells may be recovered in non-native oligomers or aggregate, the formation of which is susceptible to disturbances in beta cell energy production, calcium changes, reductive stress, and inflammatory cytokine exposure.…”

Proinsulin misfolding and endoplasmic reticulum stress during the development and progression of diabetes☆

“…However, under condition of physiological pH within the ER, the folding environment seems to be less favorable for proinsulin folding. A study examining stability, secretion and aggregation of wild-type proinsulin in several non-beta cell lines found that none of them could efficiently fold proinsulin (Zhu et al, 2004). In fact, accumulating evidence suggests that, even in normal beta cells, up to 20% of newly synthesized wild-type proinsulin fails to achieve its native folding structure (Liu et al, 2010a; Liu et al, 2005; Wang et al, 2011; Wang and Osei, 2011), suggesting that during biosynthesis, a certain amount of misfolded proinsulin is expected as a by-product.…”

Diabetes mellitus due to the toxic misfolding of proinsulin variants

“…Defining such an “ER proteome” of β‐cell‐specific foldases represents a major goal of current studies. Altered ER proteomes in non‐neurosecretory cell lines is likely to underlie their impaired ability to support the efficient folding and secretion of proinsulin [102].…”