Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted

Abstract

Mutations in the preproinsulin protein that affect processing of preproinsulin to proinsulin or lead to misfolding of proinsulin are associated with diabetes. We examined the subcellular localization and secretion of 13 neonatal diabetes-associated human proinsulin proteins (A24D, G32R, G32S, L35P, C43G, G47V, F48C, G84R, R89C, G90C, C96Y, S101C and Y108C) in rat INS-1 insulinoma cells. These mutant proinsulin proteins accumulate in the endoplasmic reticulum (ER) and are poorly secreted except for G84R and in contrast to wild-type and hyperproinsulinemia-associated mutant proteins (H34D and R89H) which were sorted to secretory granules and efficiently secreted. We also examined the effect of C96Y mutant proinsulin on the synthesis and secretion of wild-type insulin and observed a dominant-negative effect of the mutant proinsulin on the synthesis and secretion of wild-type insulin due to induction of the unfolded protein response and resulting attenuation of overall translation.

Fig. 1

Expression of human proinsulin in INS-1 cells. Cells were transfected with vector (pcDNA3.1) or WT and mutant human proinsulin cDNAs. Cell lysates were prepared 24 h post-transfection and 15 µg of total protein separated by 15% SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with an anti-human proinsulin C-peptide antibody. The lane labeled “Proinsulin” is the recombinant human proinsulin standard.

Fig. 2

Subcellular localization of wild-type and mutant proinsulin. A. INS-1 cells. B. AtT20 cells. The localization of human C-peptide (INS-1) or human insulin (AtT20) immunoreactive protein is shown in green, ER is shown in red and the Golgi marker in cyan. Nuclei were stained with DAPI (blue). Data shown are representative of three independent experiments. Scale bar: 5 µm.

Fig. 2

Subcellular localization of wild-type and mutant proinsulin. A. INS-1 cells. B. AtT20 cells. The localization of human C-peptide (INS-1) or human insulin (AtT20) immunoreactive protein is shown in green, ER is shown in red and the Golgi marker in cyan. Nuclei were stained with DAPI (blue). Data shown are representative of three independent experiments. Scale bar: 5 µm.

Fig. 3

Dominant-negative effect of C96Y proinsulin on wild-type proinsulin biosynthesis. INS-1 cells were transfected with 1 µg of WT insulin cDNA and increasing amounts of C96Y insulin cDNA (0, 0.5, 1.0 and 1.5 µg). Human insulin mRNA and C-peptide levels and rat total and phospho-eIF2a levels were determined 24 h post-transfection by real-time RT-PCR, immunoassay and Western blotting (15 µg of cell lysate), respectively. The results are the mean ± SEM of three independent experiments. A. Relative levels of human mRNA level (normalized to rat TBP mRNA). B. Total human C-peptide levels in cell extracts and media. The C-peptide levels are significantly decreased (P < 0.01) in cells transfected with C96Y insulin cDNA compared to cells transfected with 1 µg of WT insulin cDNA. C. Induction of phospho-eIF2α in cells transfected with pcDNA3.1 (2 µg), WT (2 µg) or C96Y (1 µg) and WT (1 µg) cDNAs. D. Fold induction of phospho-eIF2α.