In Vivo Misfolding of Proinsulin Below the Threshold of Frank Diabetes

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical Center, Ann Arbor, Michigan, USA.
Diabetes (Impact Factor: 8.1). 06/2011; 60(8):2092-101. DOI: 10.2337/db10-1671
Source: PubMed


Endoplasmic 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.
In 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.
At 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.
The 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.

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    • "However, upregulation of UPR and ERAD seems to have a protective effect [34]. In vivo expression of the same proinsulin mutant driven by the weak Ins1 promoter induced both ER stress and pancreatic compensation [35]. Altogether these data demonstrate a clear link between misfolding of proinsulin and ER stress induction. "
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    ABSTRACT: Pancreatic β cell failure leads to diabetes development. During disease progression, β cells adapt their secretory capacity to compensate the elevated glycaemia and the peripheral insulin resistance. This compensatory mechanism involves a fine-tuned regulation to modulate the endoplasmic reticulum (ER) capacity and quality control to prevent unfolded proinsulin accumulation, a major protein synthetized within the β cell. These signalling pathways are collectively termed unfolded protein response (UPR). The UPR machinery is required to preserve ER homeostasis and β cell integrity. Moreover, UPR actors play a key role by regulating ER folding capacity, increasing the degradation of misfolded proteins, and limiting the mRNA translation rate. Recent genetic and biochemical studies on mouse models and human UPR sensor mutations demonstrate a clear requirement of the UPR machinery to prevent β cell failure and increase β cell mass and adaptation throughout the progression of diabetes. In this review we will highlight the specific role of UPR actors in β cell compensation and failure during diabetes.
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    • "These findings have stimulated renewed interest in the biosynthesis of insulin [21] [22] [23] and the structural basis of disulfide pairing [24] [25] [26] [27] [28] [29]. The foundational importance of this problem and its clinical relevance have motivated construction of novel molecular reagents and animal models [30] [31] [32] [33]. Such studies have led in parallel to an enhanced understanding of structure–function relationships [13] and cellular mechanisms of proinsulin folding and insulin biosynthesis [1]. "
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    ABSTRACT: Dominant mutations in the human insulin gene can lead to pancreatic β-cell dysfunction and diabetes mellitus due to toxic folding of a mutant proinsulin. Analogous to a classical mouse model (the Akita mouse), this monogenic syndrome highlights the susceptibility of human β-cells to endoreticular stress due to protein misfolding and aberrant aggregation. The clinical mutations directly or indirectly perturb native disulfide pairing. Whereas the majority of mutations introduce or remove a cysteine (leading in either case to an unpaired residue), non-cysteine-related mutations identify key determinants of folding efficiency. Studies of such mutations suggest that the evolution of insulin has been constrained not only by its structure and function, but also by the susceptibility of its single-chain precursor to impaired foldability.
    Preview · Article · May 2013 · FEBS letters
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    • "Interestingly, subpopulations of cells have been reported for the Akita mouse, with some cells showing less ER‐accumulation of proinsulin than others1. It has been suggested that these less‐affected β‐cells represent younger cells that have yet to accumulate significant amounts of misfolded mutant proinsulin in the ER1. "
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    ABSTRACT: Aims/Introduction The human insulin gene/preproinsulin protein mutation C43G disrupts disulfide bond formation and causes diabetes in humans. Previous in vitro studies showed that these mutant proteins are retained in the endoplasmic reticulum (ER), are not secreted and are associated with decreased secretion of wild‐type insulin. The current study extends this work to an in vivo zebrafish model. We hypothesized that C43G‐green fluorescent protein (GFP) would be retained in the ER, disrupt β‐cell function and lead to impaired glucose homeostasis. Materials and Methods Islets from adult transgenic zebrafish expressing GFP‐tagged human proinsulin mutant C43G (C43G‐GFP) or wild‐type human proinsulin (Cpep‐GFP) were analyzed histologically across a range of ages. Blood glucose concentration was determined under fasting conditions and in response to glucose injection. Insulin secretion was assessed by measuring circulating GFP and endogenous C‐peptide levels after glucose injection. Results The majority of β‐cells expressing C43G proinsulin showed excessive accumulation of C43G‐GFP in the ER. Western blotting showed that C43G‐GFP was present only as proinsulin, indicating defective processing. GFP was poorly secreted in C43G mutants compared with controls. Despite these defects, blood glucose homeostasis was normal. Mutant fish maintained β‐cell mass well into maturity and secreted endogenous C‐peptide. Conclusions In this model, the C43G proinsulin mutation does not impair glucose homeostasis or cause significant loss of β‐cell mass. This model might be useful for identifying potential therapeutic targets for proper trafficking of intracellular insulin or for maintenance of β‐cell mass in early‐stage diabetic patients.
    Full-text · Article · Mar 2013
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