Mutant INS-Gene Induced Diabetes of Youth: Proinsulin Cysteine Residues Impose Dominant-Negative Inhibition on Wild-Type Proinsulin Transport
Figure 3
Effect of mutant proinsulins on insulin storage derived from co-expressed nonmutant proinsulin in pancreatic beta cells (and effect of mutant proinsulins on proinsulin export in 293T cells).
A. INS832/13 cells, which co-store human insulin in secretory granules, were transiently transfected with pCMS-GFP to co-express mouse preproinsulins (as indicated) and cytosolic GFP. Transfected cells were recovered after 48 h by fluorescence-activated cell sorting and were extracted with acid-ethanol. A human insulin-specific radioimmunoassay was used to measure hormone storage in mature secretory granules, normalized to cell number. When mouse proinsulin-WT was expressed, the amount of human insulin stored served as a positive control (ie, set to 100%). Results shown are the mean ± s.d. in at least three independent experiments. *p<0.05 compared with mouse proinsulin-WT. B. INS-1 cells were co-transfected with wild-type human preproinsulin and wild-type or mutant mouse preproinsulin. Transfected cells were incubated for 28 h with fresh medium before collection for measurement of secreted human insulin using human insulin specific radioimmunoassay. C. 293T cells were co-transfected to express 1) human proinsulin-WT and co-express 2) mouse proinsulin-WT or missense mutants in which the described mutation is within the B-chain, C-peptide, A-chain, or proinsulin-DelCys at a plasmid ratio of 1 : 2. Beginning at 24 h post-transfection, cells were incubated for 16 h with high-glucose DMEM plus 10% FBS. Media were collected and a human proinsulin-specific radioimmunoassay was used to measure secretion of co-expressed human proinsulin-WT. When mouse proinsulin was replaced by empty vector, the amount of human proinsulin-WT secretion served as a positive control (ie, set to 100%). Medium collected from 293T cells expressing only mouse proinsulin-WT served as a negative control for the specificity of the human proinsulin radioimmunoassay (while independent measurements not shown proved ample secretion of rodent proinsulin-WT in these samples). Results are expressed as mean ± s.d. from at least three independent experiments. *p<0.05 compared with mouse proinsulin-WT.