In Vivo Misfolding of Proinsulin Below the Threshold of Frank Diabetes

Abstract

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.

Insulin demand regulates β cell number via the unfolded protein response

“…Inducing UPR by transgenic overexpression of mutant proinsulin or the islet amyloid polypeptide (49,50), or by deletion of ER stress cell-death mediators CHOP or DP5 (30,51), was noted to increase islet size or β cell proliferation. In adult mice, reducing PERK expression increased β cell proliferation (52,53).…”

ATF5 regulates β-cell survival during stress

“…Insulin-secreting β cells are notoriously susceptible to stress, including endoplasmic reticulum (ER), cytokine, and oxidative stress (1)(2)(3)(4). Thus, understanding apoptotic cell-fate decisions during stress could provide new targets that could be exploited for the prevention or amelioration of diabetes.…”

Dominant protein interactions that influence the pathogenesis of conformational diseases

“…However, in the presence of the mutant h P r o C ( A 7 ) Y-C p e p G F P (which itself is entrapped in the ER; ref. 22), more cells began to appear with endogenous mPro in an ER-like pattern (colocalizing with calnexin; Figure 6B, top 2 rows). There is published evidence that endogenous proinsulin synthesis/content is heterogeneous in the population of islet β cells (24,25).…”

“…For this, we examined mice expressing the Akita-like mutant hProC(A7)Y-CpepGFP transgene (22), with deletion of either one allele or homozygous knockout of endogenous Ins2 to progressively decrease WT proinsulin expression. We then used mPro-specific immunof luorescence to examine the intracellular distribution of the remaining endogenous (primarily Ins1) gene product.…”

“…Mice expressing hProC(A7)Y-CpepGFP transgene driven by the Ins1 promoter, bearing heterozygous/homozygous disruption of endogenous Ins2, were as previously described (22). A full description of rdw-Tg3xMyc transgenic mice is forthcoming; briefly, a transgene consisting of the bovine Tgn promoter (60) immediately upstream of the full-length mouse Tgn ORF encoding rdw-Tg plus a tripleMyc epitope tag (i.e., rdw-Tg3xMyc) (10) was expressed in C57BL/6 mice.…”