Intracellular Transport of Proinsulin in Pancreatic β-Cells

Abstract

In pancreatic islets, formation of beta-secretory granule cores involves early proinsulin homohexamerization and subsequent insulin condensation. We examined proinsulin conformational maturation by monitoring accessibility of protein disulfide bonds. Proinsulin disulfides are intact immediately upon synthesis, but are > or = 90% sensitive to in vivo reduction with 2 mM dithiothreitol; wash out of dithiothreitol leads to reoxidation, proinsulin transport, and conversion to insulin. With t1/2 approximately 10 min, newly synthesized proinsulin becomes resistant to disulfide reduction, correlating with endoplasmic reticulum (ER) export. However, inhibition of ER export with brefeldin A blocks acquisition of resistance to reduction, and once proinsulin arrives in the Golgi, it resists reduction despite brefeldin treatment. Moreover, in vivo, resistance of proinsulin disulfides is overcome after increasing [dithiothreitol] > 10-fold, or in vitro, in islets lysed in a zinc-free, but not a zinc-containing, medium. Employing 30 mM dithiothreitol in vivo, a further decrease in disulfide accessibility is observed following proinsulin conversion to insulin. Incubation of islets with chloroquine or zinc enhances and diminishes accessibility of insulin disulfides, respectively. We hypothesize that two major conformational changes culminating in granule core formation, proinsulin hexamerization and insulin condensation, are sensitive to zinc and occur upon ER exit and arrival in immature secretory granules, respectively.

Role of the Connecting Peptide in Insulin Biosynthesis

“…Specifically, although the possible number and combination of bioengineered amino acids comprising such a linker is endless and conclusions cannot be drawn about sequences that have never been tested, from our previous and current studies using a simple set of constructs we suggest that insulin disulfide bonds form early during protein folding in the ER (22) with subpopulations of disulfide isomers that are not readily interconverted thereafter (Fig. 8 and Ref.…”

“…Nevertheless, we found surprising the recent result that certain B-chain point mutations, including ones known to provide thermodynamic stability to insulin that has been folded in vitro (such a substitution of insulin HisB10 by Asp), induce insulin disulfide mispairing within the secretory pathway in vivo (12). In this study, we have primarily used analysis by nonreducing SDS-PAGE to assess insulin disulfide maturation, which is generally recognized to occur within the endoplasmic reticulum (22), focusing primarily on the length of the linker peptide contained within SCIs. In an initial series of constructs, we created "artificial C-peptides" from five to nine residues in which the linker sequence begins and ends with a Met residue, flanking a stretch of glycine residues.…”

Proinsulin Is Refractory to Protein Fibrillation

“…3 Proinsulin assembles to form soluble Zn 2ϩ -coordinated hexamers shortly after export from ER to the Golgi apparatus (2,3). Endoproteolytic digestion and conversion to insulin occurs in immature secretory granules followed by morphological condensation (3,4). Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM) (4).…”

Zinc as a paracrine effector in pancreatic islet cell death.

“…Islet -cells contain even more Zn 2 + than do neurons. Zn 2 + in -cells, involved in the formation of insoluble insulin hexamer in secretory granules (11,12), is also co-secreted with insulin after stimulation with secretagogues (13). Zn 2 + released in certain conditions may have an impact on pancreatic -cells if significant local concentration is achieved.…”