It has previously been shown that misfolded mutant Akita proinsulin in the endoplasmic reticulum engages directly in protein complexes either with nonmutant proinsulin or with "hProCpepGFP" (human proinsulin bearing emerald-GFP within the C-peptide), impairing the trafficking of these "bystander" proinsulin molecules (Liu, M., Hodish, I., Rhodes, C. J., and Arvan, P. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 15841-15846). Herein, we generated transgenic mice, which, in addition to expressing endogenous proinsulin, exhibit ␤-cellspecific expression of hProCpepGFP via the Ins1 promoter. In these mice, hProCpepGFP protein levels are physiologically regulated, and hProCpepGFP is packaged and processed to CpepGFP that is co-stored in ␤-secretory granules. Visualization of CpepGFP fluorescence provides a quantifiable measure of pancreatic islet insulin content that can be followed in live animals in states of health and disease. We examined loss of pancreatic insulin in hProCpepGFP transgenic mice mated to Akita mice that develop neonatal diabetes because of the expression of misfolded proinsulin. Loss of bystander insulin in Akita animals is detected initially as a block in CpepGFP/insulin production with intracellular accumulation of the precursor, followed ultimately by loss of pancreatic ␤-cells. The data support that misfolded proinsulin perturbs bystander proinsulin in the endoplasmic reticulum, leading to ␤-cell failure.During the progression of diabetes mellitus, the endocrine pancreas encounters difficulty in meeting insulin requirements (1); ␤-cell dysfunction is recognized as a major contributor to the disease (2-5). One element of ␤-cell dysfunction is ER 3 stress (6 -11) with ER accumulation of misfolded protein (12), especially proinsulin (13, 14). ␤-Cells ordinarily maintain a high level of proinsulin production with finite additional capacity before the biosynthetic apparatus is taxed to the point of ER stress (15). Chronically increased secretory demand, either in animal models or in humans, results in morphological depletion of ␤-secretory granules with a compensatory increase in apparent secretory pathway activity, including distention of the ER (16 -18). These conditions may favor additional proinsulin misfolding (19).The causality between misfolded proinsulin and ␤-cell failure is unequivocally established in congenital diabetes caused by preproinsulin coding sequence mutations, in which diabetes is inherited in an autosomal dominant manner (20 -24). Insulin haploinsufficiency cannot account for the diabetes (25), yet despite three normal proinsulin alleles, both Akita and Munich mice each develop overt diabetes by expressing from a single allele a mutant proinsulin with replacement of one Cys residue that disrupts one of the three proinsulin disulfide bonds (26,27). In addition to being retained in the ER, it has been suggested that misfolded proinsulin may impair normal insulin production via physical interactions between mutant and wild-type proinsulin gene products (26). Indeed, we have direct...

OBJECTIVEEndoplasmic reticulum (ER) stress has been described in pancreatic β-cells after onset of diabetes—a situation in which failing β-cells have exhausted available compensatory mechanisms. Herein we have compared two mouse models expressing equally small amounts of transgenic proinsulin in pancreatic β-cells.RESEARCH DESIGN AND METHODSIn hProCpepGFP mice, human proinsulin (tagged with green fluorescent protein [GFP] within the connecting [C]-peptide) is folded in the ER, exported, converted to human insulin, and secreted. In hProC(A7)Y-CpepGFP mice, misfolding of transgenic mutant proinsulin causes its retention in the ER. Analysis of neonatal pancreas in both transgenic animals shows each β-cell stained positively for endogenous insulin and transgenic protein.RESULTSAt this transgene expression level, most male hProC(A7)Y-CpepGFP mice do not develop frank diabetes, yet the misfolded proinsulin perturbs insulin production from endogenous proinsulin and activates ER stress response. In nondiabetic adult hProC(A7)Y-CpepGFP males, all β-cells continue to abundantly express transgene mRNA. Remarkably, however, a subset of β-cells in each islet becomes largely devoid of endogenous insulin, with some of these cells accumulating large quantities of misfolded mutant proinsulin, whereas another subset of β-cells has much less accumulated misfolded mutant proinsulin, with some of these cells containing abundant endogenous insulin.CONCLUSIONSThe results indicate a source of pancreatic compensation before the development of diabetes caused by proinsulin misfolding with ER stress, i.e., the existence of an important subset of β-cells with relatively limited accumulation of misfolded proinsulin protein and maintenance of endogenous insulin production. Generation and maintenance of such a subset of β-cells may have implications in the avoidance of type 2 diabetes.