Journal of the Pancreas Open Access

Keywords

Amyloid; Diabetes Mellitus, Type 2; Islets of Langerhans; Islets of Langerhans Transplantation; Molecular Chaperones; Protein Conformation

Abbreviations

AGE: advanced glycation endproduct; eNO: endothelial nitric oxide; eNOS: endothelial nitric oxide synthase; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated degradation; IAPP: islet amyloid polypeptide; ISG: insulin secretory granule; RNS, reactive nitrogen species;

Summary

Conformational diseases are conditions that arise from the dysfunctional aggregation of proteins in non-native conformations. Type 2 diabetes mellitus can be defined as a conformational disease because a constituent beta cell protein, islet amyloid polypeptide, undergoes a change in tertiary structure followed by self-association and tissue deposition. Type 2 diabetes mellitus is associated with multiple metabolic derangements that result in the excessive production of reactive oxygen species and oxidative stress. These reactive oxygen species set in motion a host of redox reactions which can result in unstable nitrogen and thiol species that contribute to additional redox stress. The ability of a cell to deal with reactive oxygen species and oxidative stress requires functional chaperones, antioxidant production, protein degradation and a cascade of intracellular events collectively known as the unfolded protein response. It is known that beta cells are particularly susceptible to perturbations in this quality control system and that reactive oxygen species play an important role in the development and/or progression of diabetes mellitus. Oxidative stress and increased insulin production contribute to endoplasmic reticulum stress, protein misfolding, and induction of the unfolded protein response. As the cell’s quality control system becomes overwhelmed, conformational changes occur to islet amyloid polypeptide intermediates, generating stable oligomers with an anti-parallel crossed betapleated sheet structure that eventually accumulate as space-occupying lesions within the islets. By approaching type 2 diabetes mellitus as a conformational disease in which there is a structural transition from physiological protein to pathological protein, it is possible that the relentless nature of disease progression can be understood in relation to other conformational diseases.

Introduction to Conformational Diseases

Conformational diseases occur when an endogenous protein undergoes a change in shape that leads to self-association of these proteins and tissue deposition [1]. In the course of normal protein biosynthesis, misfolding does occur, and intracellular mechanisms have evolved to shuttle and degrade these aberrant proteins/polypeptides [2]. Although conformational changes occur with normal protein processing, a particular protein’s susceptibility to aggregation, and a genetic or environmental predisposition to disease may overwhelm the cell’s quality control mechanisms. In the setting of significant and sustained endoplasmic reticulum (ER) stress, these quality control mechanisms prove insufficient. High concentrations of mutant protein lead to aggregation and slow deposition into tissues over time. This time-requiring sequence of events may partially explain the relatively late clinical presentation of many conformational diseases.

Table 1 is adapted from Carrell and Lomas’s original disease classification [1] and illustrates the wide array of recognized conformational diseases. These diseases arise from secondary or tertiary structural changes within constituent proteins, with subsequent aggregation of those altered proteins. For example, in alpha1-antitrypsin deficiency, a single amino acid substitution results in the destruction of a salt bridge that affects the secondary structure of alpha1-antitrypsin [3]. This perturbation leads to a molecular interaction between the A sheet of one molecule with the reactive center loop of another [4]. This polymerization results in an accumulation of this enzyme in the ER, activation of the unfolded protein response (UPR) (described below) and ultimately, apoptosis. In the prion diseases Kuru and Creutzfeld-Jakob disease, proteins that have primarily helical structure convert to a betapleated sheet configuration [5]. In fact, conformational diseases often feature a protein that aggregates in beta-sheet linkages. Beta-pleated sheets are formed by alternating peptide strands that are linked by hydrogen bonding between their aligned pleated structures. This is a feature of the systemic amyloidoses, neurodegenerative diseases and type 2 diabetes mellitus (T2DM). The diverse clinical presentations of these diseases, as well as the fact that some are almost solely rooted in genetic deficiencies (e.g., Huntington’s disease) whereas others such as T2DM can have a relatively strong environmental component (obesity), may seem to controvert the single grouping of them on the basis of conformational abnormalities. However, one utility of the designation ‘conformational disease’ is that it denotes the mechanisms underlying the sometimes odd and delayed presentation of these diseases. Although fibril formation is a defining feature of these diseases, they are composed of different aggregated proteins, sharing structural properties. Another reason for giving diseases this label is that it suggests common avenues of therapy. It is intriguing to consider that the manipulation of protein structure and/or aggregate assembly can be a platform for the development of novel therapies. For example, processes that result in the tissue deposition of beta-pleated sheets can be inhibited by compounds such as glycosaminoglycan mimetics.

Treating T2DM as a conformational disease does not imply that the disease begins with or can be holistically described as inappropriate protein deposition. Other associated processes, such as the development of insulin resistance, require other models to explain disease pathogenesis. Like Alzheimer’s disease, where brain amyloid represents the culmination of multiple previous events and cannot wholly explain cognitive deficits, so in T2DM, islet amyloid is both cause and consequence of several disease processes. The sole purpose of this review is to interpret how protein abnormalities can be understood in the context of other conformational disease processes and specifically how they could arise in T2DM.

Islet Amyloid: The Conformational Problem of T2DM

The contribution of islet amyloidosis to disease pathogenesis has been vigorously debated [6]. Islet amyloid polypeptide (IAPP) oligomers that precede islet amyloid deposition are likely more toxic to beta cells than islet amyloid itself [7]. Islet amyloid is present at autopsy in as many as 96% of patients with T2DM [8]. With accumulations of toxic, misfolded IAPP oligomers and deposition of crossed beta-pleated sheets, T2DM is similar to other protein conformational diseases. It is interesting to note that the human, feline, and non-human primate forms of the IAPP molecule are known to be amyloidogenic, and these are the only members of the animal kingdom that develop spontaneous T2DM. Lower mammals, on the other hand, do not share this feature of having amyloidogenic IAPP due to proline substitutions at positions 25, 28,and 29, and they do not develop spontaneous T2DM [9, 10]. Most animal models of insulin resistance do not feature islet amyloid except for transgenic mice that express human IAPP [11]. Clinically, it is clear that aggregates of misfolded IAPP are a prominent pathological feature in the development of T2DM (reviewed in [12]). The human IAPP polypeptide is presented in Figure 1.

Proteins must properly fold into threedimensional structures in order to carry out their proper functions within the cell and organism. One model of dysfunctional protein aggregation represented in Figure 2 involves the following intracellular events: 1) misfolding or unfolding of native protein exposes hydrophobic regions; 2) conformational changes result in unstable intermediates that have a propensity to form oligomers; 3) oligomers form pathogenic subunits and crossed beta-pleated sheets; and 4) in the case of T2DM, amyloid fibrils are formed with subsequent stabilization by accessory molecules, such as serum amyloid P, perlecan, and apolipoprotein E [6]. When precision folding goes awry, the misfolded, soluble oligomeric proteins begin to accumulate, become toxic, and promote apoptosis [7, 13]. Misfolded IAPP stabilizes into crossed beta-pleated formations that are deposited within the adjacent surrounding extracellular matrix, resulting in spaceoccupying lesions within the islets of the pancreas. The following discussion outlines cellular stressors in T2DM that contribute to protein misfolding and aggregation. One of the most important stressors leading to these protein conformation abnormalities is redox stress (discussed later in the review).

Once unfolded, IAPP may become refolded in the ER-Golgi complex, accompanied by the support of ATP-dependent chaperone proteins. Kinetic refolding experiments using intermediate proteins associated with known conformational diseases have revealed that there is a higher energy requirement to achieve successful refolding due to the increased exposure of hydrophobic regions in unfolded or partially-folded proteins. Therefore, exposed hydrogen ions may cause a folding pathway to produce a relatively stable intermediate form of protein that is 'kinetically trapped' if the ER cannot overcome this higher energy barrier [14].

When this quality control system is overwhelmed and IAPP is not capable of being correctly refolded, this protein can become a soluble toxic monomer due to the innate amyloidogenic properties of the NFGAILSS region of IAPP in amino acid positions 22-29 [15].

Soluble IAPP oligomers have been shown to be cytotoxic and possibly responsible for beta cell apoptosis in T2DM [7, 16, 17]. Additionally, beta cells with a high turnover (replication) rate have been found to be more susceptible to apoptosis by IAPP oligomers [17]. It is the delicate balance between refolding and degradation, maintained by the quality control system that determines the amount of mutant protein allowed to accumulate. The adaptive and apoptotic mechanisms of the quality control system are so selective that even minor perturbations in protein folding efficiency can cause the rejection of nascent IAPP proteins and, consequently, their accumulation or degradation. Accumulation of mature islet amyloid is responsible for the spaceoccupying lesion with associated secretory and absorptive defects within the islet and is accelerated by free radical polymerization due to reactive oxygen species (ROS). These concepts are summarized in Figure 3.

Researchers have also addressed whether genetic differences in IAPP could predispose to T2DM development. To date, only one missense mutation has been identified in human IAPP. It is the S20G mutation (glycine is substituted for serine at position 20), and this change in the amino acid sequence results in more rapid amyloid formation and early onset T2DM in Japanese, Korean, Chinese and New Zealand Maori populations (1.9- 2.6% of subjects studied thus far) [18, 19, 20, 21, 22, 23, 24, 25]. Although this mutation is restricted to the ethnic groups mentioned, its existence points to the possibility of it being more widespread and that other mutations might contribute to the amyloidogenic properties of IAPP.

Under adverse conditions, and due to its intrinsic conformational instability, the normally soluble IAPP protein quite readily undergoes the structural change to form the crossed beta pleated sheet necessary for aggregation. Due to its propensity to aggregate, IAPP is able to endure free radical polymerization; a process that is further promoted through the cell’s decreased ability to clear misfolded proteins. This is the very beginning of the pre-diabetic condition [26]. Once formed, islet amyloid is quite resistant to the normal proteolytic defenses within the body and is therefore allowed to accumulate and undergo an even more rapid free radical polymerization in an islet milieu of increased reactive oxygen species (ROS) [27, 28]. The question arises, how do inherent beta cell characteristics contribute to the development of abnormally folded proteins which culminate as islet amyloid?

Beta Cell and ER Stress

As is true for other cells performing protein synthesis, beta cells regulate the production of their synthesized protein indirectly via glucose sensors and not directly via the levels of insulin which they produce. These same glucose sensors act on molecular pathways in other endocrine cells that maintain glycolysis and nutrient homeostasis. Thus, transcription in beta cells is not regulated by insulin itself but by translational and post-translational events that are themselves regulated by extracellular glucose levels [29, 30, 31]. Chronic activation of the beta cells’ quality control system favors the induction of more apoptotic pathways of the UPR [2] which is described in greater detail later in this review. The islet beta cell is known to have a highly developed ER, apparently due to an excessive demand for compensatory insulin secretion [32]. An increased demand for insulin secretion may result in beta cell overload ultimately leading to deficient insulin secretion. Beta cell mass (both number and volume of cells) is reduced in the later phases of T2DM as a result of apoptosis, especially in rapidly replicating beta cells [33]. The toxic effects of oligomeric IAPP result in apoptosis of the beta cells, but for a period of time, the more primordial ductal cells of the exocrine pancreas (replicative pool) can replace the damaged, apoptotic beta cells and continue the compensatory hyperinsulinemia causing further beta cell damage. These effects culminate in the development of a defective diffusion barrier within the islet [34]. Table 2 presents a putative model of how accumulations of IAPP-derived protein aggregates may relate to T2DM pathogenesis.

The beta cell ER has unique responses to unfolded or misfolded proteins [32]. The first response is up-regulation of genes encoding antioxidants and ER chaperone proteins, such as BiP/GRP78 and GRP94, to increase protein folding activity and prevent protein aggregation [35, 36, 37]. The second response consists of translational attenuation to reduce the load of new protein synthesis and prevent further accumulation of unfolded proteins. The third is degradation of misfolded proteins in the ER (endoplasmic reticulum-associated degradation, ERAD) [35]. The misfolded proteins are transported from the ER to the cytosol, where most are tagged with ubiquitin-conjugating enzymes for degradation by the 26S proteasome, as well as the lysosome [38]. The fourth is transcriptionally-activated apoptosis, which occurs when the ER is chronically overwhelmed and its function has been severely impaired [35]. These mechanisms will be described in greater detail in a forthcoming section of this review.

Insulin Secretory Granule in a Conformational Disease

Post-translational processing of pro-IAPP in the insulin secretory granule (ISG) yields the soluble, functional IAPP hormone. The same prohormone convertases 1, 2 and 3 process pro-IAPP and pro-insulin, and cosecrete the cleaved forms of both into the circulation [39]. It has been proposed that a balance of these ISG components, including C-peptide, Ca2+ and Zn2+, contributes to maintaining IAPP and insulin in their mature native conformations, thereby hindering aggregate formation [40]. The authors of this study conjectured that if these factors are in an inappropriate concentration, conditions could favor IAPP aggregation. Further, in T2DM, alterations in the proportions of insulin and IAPP in granules could favor fibril formation [41]. Preserving a normal physiologic ratio of proinsulin to insulin in ISGs disfavors fibril formation and beta-pleated sheet formation of IAPP [41]. Several studies have demonstrated, in both human and animal models of T2DM and fasting hyperglycemia, a disproportionate ratio of proinsulin to insulin, relative to the overall increase of both in plasma concentrations [42, 43, 44, 45, 46]. Whether this increase in proinsulin is due to chronic hyperglycemia or impaired glucose tolerance, the resulting disturbance to the proper functioning of the ISG could exacerbate protein misfolding. The abnormal processing of proIAPP with incomplete conversion to IAPP could result in increased IAPP-derived islet amyloid deposition, as proIAPP is also amyloidogenic [40, 47, 48]. Thus, incomplete processing of proinsulin and proIAPP could each present a mode for increased aggregation of misfolded proteins.

The glycosaminoglycan called perlecan is a ubiquitous part of beta cell’s basement membrane, synthesized in the islets [49].

Although not a structural element, perlecan does provide stability to amyloid fibrils by allowing binding of IAPP to the basement membranes surrounding islet capillaries. This pathologically promiscuous binding decreases the secretory response of the ISG as a result of an adsorptive barrier created by thickened basement membranes. This condition may well be the beginning of structural transformations within the islet which provides a located environment with a predilection toward a disproportionate ratio of IAPP to insulin secretion

Chaperones and Conformational Disease

Molecular chaperones are ubiquitous, highly conserved small proteins present in all eukaryotic cells (reviewed in [50]). As noted above, their overall purpose is to minimize aggregation by assisting target proteins, such as IAPP, in proper folding and to covalently transport functional proteins across extracellular space. Under conditions of sustained redox stress, the ability of chaperones to regenerate the redox potential of the cell is compromised due to the increased occupation of chaperones by nascent polypeptides (reviewed in [51]). Consequently, protein misfolding occurs, amplifying the chaperone requirements, and in the case of T2DM, causes islets to be more susceptible to the deleterious effects of redox stress.

In most conformational diseases studied, the soluble, partially-folded intermediates contain an area of exposed hydrophobic regions that are, in the protein’s native state, buried and protected against non-native interactions. These areas of increased hydrophobicity have been implicated in allowing non-native interactions to occur that result in the crossed beta-pleated sheet structure seen in protein aggregates [14] and Figure 2. Thus, it becomes necessary to overcome a higher energy barrier in order for the folding process to continue to completion. Chaperones assure that the correct stoichiometric amounts of folding co-factors are present so that these non-native isoforms can achieve their functional quaternary structure. As very important components of the quality control repertoire, the cell dedicates a substantial amount of metabolic energy to performing chaperone functions.

The Unfolded Protein Response (UPR) and the Balance of Quality Control Mechanisms

An accumulation of misfolded/unfolded polypeptides in the ER of cells presents a challenge to chaperones in the cell [52, 53]. Due to prolonged interactions with these mutant proteins, chaperones are challenged to fulfill their folding duties in a timely manner. The overall function of molecular chaperones is to minimize protein aggregation by ensuring proper protein folding and providing transport to target proteins through covalent cross-linking [54] (see section below). Under challenge, a process known as the UPR recruits existing proteases and ubiquitination enzymes to help deal with this accumulation [55, 56]. As a point of fact, in normal cells, many of the substrates for proteases 'are' misfolded proteins, reflecting the importance of conformation in determining protein selection for degradation.

After existing proteases have been consumed, the UPR will induce survival and apoptotic pathways in response to the particular stress. Survival responses include transcriptional regulation (antioxidant and chaperone production) and translational regulation (protein synthesis inhibition). Apoptotic responses of the UPR include protease synthesis (26S proteasome), ubiquitinconjugating enzymes, and caspases (in the case of chronic stress/severe ER impairment) [57, 58, 59]. The integrity of the quality control system is of paramount importance at this step. Maintaining a balance between folding and degradation determines the amount of mutant protein that can accumulate and potentially cause conformational diseases [1]. An imbalance in the quality control system created by, among other things, abnormal temperatures, high/low glucose concentrations, glycosylation inhibitors, influx of Ca2+ ions, alterations in local pH, or redox stress will cause further accumulation of misfolded/unfolded proteins.

In the setting of hyperinsulinemia, the beta cell likely has significantly increased ER activity and can become stressed in responding to the demands of the peripheral tissues to prevent hyperglycemia [32]. Previously, this process has been referred to as ‘beta cell fatigue’, but a more encompassing explanation can invoke ER stress and an overwhelmed UPR. The basic pathways of the UPR have been exhaustively explored in recent years [60]. The significance of these studies is that they can partly explain how a stressed cell becomes committed to survival or to apoptosis [57]. The UPR is initiated under various conditions of stress which compromise protein folding in the ER. Three major survival pathways are employed as the transcriptional response to this ER stress (Figure 4). Each pathway involves an ER-resident transmembrane protein that senses ER stress or the presence of unfolded proteins: IRE1, ATF4/PERK and ATF6. IRE1 and ATF4/PERK, which both have cytoplasmic serine/threonine kinase domains, are activated by ER stress and undergo homodimerization and phosphorylation [61, 62, 63]. The accumulation of inappropriately folded proteins in the ER lumen results in ATF6 translocation to the ER [55]. ATF6 cleaves its cytosolic domain then translocates to the nucleus to activate transcription of the chaperones GRP78 (BiP) and other co-factors of ER stress target genes [64]. The downstream sequelae of the UPR are transcriptional activation of chaperones, antioxidants, co-factors and regulators involved in ER-associated protein degradation (i.e. ERAD) and inhibition of new protein synthesis. These responses presumably conserve cellular resources in the face of increasing stress. Activation of UPR pathways is also intrinsically important in the initiation of proapoptotic responses (protease and caspase synthesis). Chaperone participation in these responses, as well, provides an additional quality control mechanism. However, the means by which cells commit to apoptosis rather than survival are less well understood.

Redox Stress in the Formation of Islet Amyloid

1. Reactive Oxygen Species (ROS)

Multiple biochemical pathways and mechanisms of action have been implicated in the deleterious effects of chronic hyperglycemia and oxidative stress on the function of the kidney, retina, vascular tissues and, to a lesser extent, pancreatic islets in T2DM [65]. There is currently wide acceptance for the destructive potential of oxidative stress on the islets and arterial vessel walls in patients with T2DM. The damaging effects of ROS and other free radicals on proteins, nucleic acids and fats is key to a better understanding of the formation of amyloid within the islets of patients with T2DM. Excessive redox stress may lead to protein accumulation and aggregation in the ER, with severe consequences for the cell [66].

ROS may impact disulfide bond formation [67] and subsequently influence the development of IAPP misfolding. Disulfide bonds formed in newly synthesized proteins in the ER of cells are important for proper protein folding, protein structure, biological activity, and stability of many secreted and membrane proteins [66, 68, 69]. Protein folding in eukaryotes takes place in the ER with assistance from many redox-sensitive chaperones and oxidoreductases (e.g., protein disulfide isomerase, ERp44, ERp72, ERp57, GRP58, Hsp33) [69]. The effects of excessive ROS on native IAPP within the ER may cause covalent breakage of the disulfide bond at positions Cys2 and Cys7 in this 37 amino acid polypeptide, allowing it to unfold or preventing it from properly folding.

ROS may have an effect on both proteins and lipids within the ER by altering the ER bilipid membrane [70]. Hyperglycemia may have an additive effect by altering the protein content of the ER membrane through formation of advanced glycation endproducts (AGEs) [71]. Oxidative stress, combined with hyperglycemia, has been shown to alter a protein’s susceptibility to glycation (the process of forming irreversible Amadori products from reversible Schiff bases with non-enzymatic rearrangement reactions) [72, 73]. These AGEs, which are prone to crosslinking and aggregation, can modify IAPP through post-translational attachments [74]. Upon proteoglycan binding, there is an observed increase and acceleration in total amyloid fibril formation. Amadori products are currently used as a clinical marker of glucose control because they exist in equilibrium with glucose levels [75]. AGEs, on the other hand, become irreversibly bound to protein. It is this long-term consequence of glycation, leading to the formation of AGEs that can take years to complete and can be detrimental to the patient with diabetes.

These processes (oxidation and glycation) may contribute to a dysfunctional ER membrane, allowing the abnormal leakage of misfolded proteins into the cytosol before they are properly folded into their native 3- dimensional conformation. This same ER membrane leak may also allow the influx of ROS into the ER lumen, disrupting the redoxsensitive milieu within, and allowing for an even greater unfolding and misfolding of proteins to occur.

2. Reactive Nitrogen Species (RNS)

RNS are increased in T2DM [76] and could contribute to protein misfolding. Growing evidence implicates both ROS and RNS (such as the reaction of superoxide anion (O2 -) with nitric oxide (NO) to form peroxynitrite and other RNS) as important molecules in the development of diabetes [77, 78, 79, 80, 81]. In other conformational diseases, such as Alzheimer's disease and Parkinson's disease, abnormal NO production is involved in protein misfolding leading to aggregates and proteasome dysfunction on ubiquinated material [82]. Peroxynitrite is an RNS important in the evolution of diabetes [76]. Peroxynitrite reacts relatively slowly with most biological molecules and as a result becomes a potent selective oxidant. Peroxynitrite specifically modifies tyrosine in proteins/polypeptides to create nitrotyrosine, which leaves an indelible footprint detectable in vivo. Nitrotyrosine and nitrosylated arginine (nitroarginine), known biomarkers of redox stress, are capable of competing with the natural substrate L-arginine for the production of endothelial nitric oxide (eNO) via the endothelial nitric oxide synthase (eNOS) reaction [83]. The presence of RNS in the plasma of diabetic patients suggests a possible involvement of peroxynitrite in the development of diabetic complications [84]. Could RNS-induced protein modifications also increase the propensity for IAPP to become misfolded? Post-translational events, such as nitrosylation, can affect the 3- dimensional configuration of proteins [85]. Nitrosylation of various amino groups could therefore result in prevention of the proper folding of IAPP.

3. Reactive Thiol Species

Although thiols are traditionally viewed as non-enzymatic antioxidants, reactive thiol species may be yet another consequence of redox stress that promotes conformational disease. In all antioxidant reactions employing thiols, thiyl radicals are simultaneously formed from the reduction of the disulfide bridges and subsequent oxidation of the sulfhydryl groups [86]. Maintaining a balance in the redox state of the cell ensures that thiols can continue their biological action as necessary antioxidants, and, just as importantly, that these thiyl radicals are efficiently reduced to thiols again. An elevated and sustained tension of redox stress in the ER, such as with T2DM, has the potential to disrupt this delicate balance, initiating a process termed 'disulfide reshuffling', in which newly synthesized polypeptides undergo disulfide rearrangement with free thiol groups [86]. These thiol/disulfide exchange reactions promote polymerization of other amyloidogenic proteins such as prion protein PrP found in spongiform encephalopathies [87] and can therefore presumably stabilize aggregates of other proteins, considering the generally reducing environment of the cytoplasm. Hence, not only are IAPP aggregates stabilized by the extensive non-covalent hydrogen bonding of crossed beta-sheet formation, but also this conformational change may provide covalent protection to intermolecular disulfide bonds, hindering any attempt of the UPR at aggregate disassembly. This concept lends support to the widely held belief that amyloid fibrils form via a nucleation-dependent kinetic process [6, 9, 88].

4. Redox Stress in T2DM: Conclusion

The beta cell is poorly equipped to handle redox stress as compared to other cells such as hepatocytes [76, 83], and this very sensitivity has allowed researchers to use the oxidizing agents streptozotocin and alloxan to create diabetic animal models. Not only is the islet inundated with ROS but also the beta cell within is known to be deficient in the classic antioxidants to protect itself from the surrounding redox stress [65, 89]. Additionally, once overt T2DM has developed, the antioxidant reserve is known to be compromised with a systemic deficiency of catalase, superoxide dismutase, and glutathione peroxidase [90, 91, 92, 93].

Redox stress, as manifested by increased ROS, RNS and reactive thiol species, may significantly post-translationally modify IAPP to the extent that protein misfolding is favored. Additionally, these redox stressors can overwhelm the beta cell’s ER folding complex, chaperone-induction signaling mechanism, lysosome-proteasome pathway and attenuate the secretory capacity of this cell [94, 95]. These effects likely result in augmented beta cell apoptosis and the accumulation of islet amyloid.

Conclusion

If T2DM is viewed as a conformational disease, it may be possible to rationally design therapies that specifically focus on the forces which lead to protein misfolding and deposition. This can include decreasing the redox stress associated with increased metabolic demand in obesity or promoting plaque destabilization. New small molecule therapeutics can modify the kinetics of amyloid formation or promote their amyloid resorption. For example, small molecule drugs can be used to stabilize the amyloidogenic protein precursor, or to act on the partially folded intermediates in the folding process, or to actually interact with mature amyloid fibrils to weaken their structural stability. Displacing important cofactors of amyloid deposits such as glycosaminoglycans and serum amyloid P component with these small molecules can favor dissolution of the fibril aggregate [96]. Antibodies can also be used to reduce the ability of an amyloidogenic protein to form partly unfolded species and can be an effective method of preventing its aggregation [97]. Thus, it is possible that in the future, as these therapeutics are developed, it will be possible to slow or prevent the inexorable progression of disease so frequently seen in conformational disease.

Seeing T2DM as another type of conformational disease may facilitate a broader understanding of islet biology beyond the regularly understood parameters of this disease. For example, islet cell stress may lead to a form of T2DM in type 1 diabetic patients bearing islet transplants. It is known that human islets rapidly form amyloid when transplanted into immunodeficient mice [98]. In this relatively successful ‘Edmonton era’ of islet transplantation, the hope for a cure for type 1 diabetes is diminished by a disappointing loss of function in a significant percentage of recipients [99] which can occur only months after transplantation. It is intriguing that a foundational component of modern era transplant success resides in sustaining an optimal islet mass [100]. Could gradual loss of islet cells due to immune attack be compounded by a conformational disease akin to T2DM in limiting the long term success of islet transplants? This and other questions may be addressed if T2DM and non-autoimmune beta cell dysfunction are viewed as a conformational disease. New avenues of therapy that are directed at minimizing forces leading to deleterious accumulations of proteins may offer hope to patients at risk for T2DM.

References

1. Carrell RW, Lomas DA. Conformational disease. Lancet 1997; 350:134-8. [PMID 9228977]
2. Goldberg AL. Protein degradation and protection against misfolded or damaged proteins. Nature 2003; 426:895-9. [PMID 14685250]
3. McCracken AA, Kruse KB, Brown JL. Molecular basis for defective secretion of the Z variant of human alpha-1-proteinase inhibitor: secretion of variants having altered potential for salt bridge formation between amino acids 290 and 342. Mol Cell Biol 1989; 9:1406-14. [PMID 2786139]
4. Lomas DA, Evans DL, Finch JT, Carrell RW. The mechanism of Z alpha 1-antitrypsin accumulation in the liver. Nature 1992; 357:605-7. [PMID 1608473]
5. Riek R, Hornemann S, Wider G, Billeter M, Glockshuber R, Wuthrich K. NMR structure of the mouse prion protein domain PrP(121-321). Nature 1996; 382:180-2. [PMID 8700211]
6. Kahn SE, Andrikopoulos S, Verchere CB. Islet amyloid: a long-recognized but underappreciated pathological feature of type 2 diabetes. Diabetes 1999; 48:241-53. [PMID 10334297]
7. Butler AE, Janson J, Soeller WC, Butler PC. Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formationrather than direct action of amyloid. Diabetes 2003;52:2304-14. [PMID 12941770]
8. Clark A, de Koning EJ, Hattersley AT, Hansen BC, Yajnik CS, Poulton J. Pancreatic pathology in noninsulin dependent diabetes (NIDDM). Diabetes Res ClinPract 1995; 28(Suppl):S39-47. [PMID 8529518]
9. Kapurniotu A. Amyloidogenicity and cytotoxicity of islet amyloid polypeptide. Biopolymers 2001; 60:438-59. [PMID 12209476]
10. Westermark P, Engstrom U, Johnson KH, Westermark GT, Betsholtz C. Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation. ProcNatlAcadSci U S A 1990; 87:5036-40. [PMID 2195544]
11. Soeller WC, Janson J, Hart SE, Parker JC, Carty MD, Stevenson RW, et al. Islet amyloid-associated diabetes in obese A(vy)/a mice expressing human islet amyloid polypeptide. Diabetes 1998; 47:743-50. [PMID 9588445]
12. Hayden MR, Tyagi SC. A' is for amylin and amyloid in type 2 diabetes mellitus. JOP. J Pancreas (Online) 2001; 2:124-39. [PMID 11875249]
13. Butler AE, Jang J, Gurlo T, Carty MD, Soeller WC, Butler PCl. Diabetes due to a progressive defect in beta-cell mass in rats transgenic for human islet amyloid polypeptide (HIP Rat): a new model for type 2 diabetes. Diabetes 2004; 53:1509-16. [PMID 15161755]
14. Chiti F, Taddei N, Baroni F, Capanni C, Stefani M, Ramponi G, Dobson CM. Kinetic partitioning of protein folding and aggregation. Nat StructBiol 2002; 9:137-43. [PMID 11799398]
15. Kapurniotu A, Schmauder A, Tenidis K. Structurebased design and study of non-amyloidogenic, double N- methylated IAPP amyloid core sequences as inhibitors of IAPP amyloid formation and cytotoxicity. J MolBiol 2002; 315:339-50. [PMID 11786016]
16. Janson J, Ashley RH, Harrison D, McIntyre S, Butler PC. The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles. Diabetes 1999; 48:491-98. [PMID 10078548]
17. Ritzel RA, Butler PC. Replication increases betacell vulnerability to human islet amyloid polypeptideinduced apoptosis. Diabetes 2003; 52:1701-08. [PMID 12829636]
18. Hayakawa T, Nagai Y, Ando H, Yamashita H, Takamura T, Abe T, et al. S20G mutation of the amylin gene in Japanese patients with type 2 diabetes. Diabetes Res ClinPract 2000; 49:195-7. [PMID 10963832]
19. Lee SC, Hashim Y, Li JK, Ko GT, Critchley JA, Cockram CS, Chan JC. The islet amyloid polypeptide (amylin) gene S20G mutation in Chinese subjects:evidence for associations with type 2 diabetes andcholesterol levels. ClinEndocrinol (Oxf) 2001; 54:541-6. [PMID 11318791]
20. Ma Z, Westermark GT, Sakagashira S, Sanke T, Gustavsson A, Sakamoto H, et al. Enhanced in vitro production of amyloid-like fibrils from mutant (S20G) islet amyloid polypeptide. Amyloid 2001; 8:242-9. [PMID 11791616]
21. Poa NR, Cooper GJ, Edgar PF. Amylin gene promoter mutations predispose to Type 2 diabetes in New Zealand Maori. Diabetologia 2003; 46:574-8. [PMID 12679865]
22. Sakagashira S, Sanke T, Hanabusa T, Shimomura H, Ohagi S, Kumagaye KY, et al. Missense mutation of amylin gene (S20G) in Japanese NIDDM patients. Diabetes 1996; 45:1279-81. [PMID 8772735]
23. Seino S, Study Group of Comprehensive Analysis of Genetic Factors in Diabetes Mellitus. S20G mutation of the amylin gene is associated with Type II diabetes in Japanese. Study Group of Comprehensive Analysis of Genetic Factors in Diabetes Mellitus. Diabetologia 2001; 44:906-9. [PMID 11508277]
24. Shimomura H, Sanke T, Hanabusa T, et al. Nonsense mutation of islet-1 gene (Q310X) found in a type 2 diabetic patient with a strong family history. Diabetes 2000; 49:1597-1600. [PMID 10969846]
25. Xiang K, Zheng T, Lu H. The impact of the missense mutation-ser20gly in islet amyloid polypeptide gene on NIDDM in Chinese. Zhonghua Yi XueZaZhi 1998; 78:817-20. [PMID 11038774]
26. Hayden MR. Islet amyloid, metabolic syndrome, and the natural progressive history of type 2 diabetes mellitus. JOP. J Pancreas (Online) 2002; 3:126-38. [PMID 12221327]
27. Badman MK, Pryce RA, Charge SB, Morris JF, Clark A. Fibrillar islet amyloid polypeptide (amylin) is internalised by macrophages but resists proteolytic degradation. Cell Tissue Res 1998; 291:285-94. [PMID 9426315]
28. deKoning EJ, van den Brand JJ, Mott VL, Charge SB, Hansen BC, Bodkin NL, et al. Macrophages and pancreatic islet amyloidosis. Amyloid 1998; 5:247-54. [PMID 10036582]
29. Grupe A, Hultgren B, Ryan A, Ma YH, Bauer M, Stewart TA. Transgenic knockouts reveal a critical requirement for pancreatic beta cell glucokinase in maintaining glucose homeostasis. Cell 1995; 83:69-78. [PMID 7553875]
30. Schuit FC, Huypens P, Heimberg H, Pipeleers DG. Glucose sensing in pancreatic beta-cells: a model for the study of other glucose-regulated cells in gut, pancreas, and hypothalamus. Diabetes 2001; 50:1-11. [PMID 11147773]
31. Wu L, Nicholson W, Knobel SM, Steffner RJ, May JM, Piston DW, Powers AC. Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines. J BiolChem 2004; 279:12126-34. [PMID 14688272]
32. Oyadomari S, Koizumi A, Takeda K, Gotoh T, Akira S, Araki E, Mori M. Targeted disruption of the Chop gene delays endoplasmic reticulum stressmediated diabetes. J Clin Invest 2002; 109:525-32. [PMID 11854325]
33. Butler PC. Apoptosis and the beta-cell in type 1 and type 2 diabetes. Horm Res 2004; 62(Suppl 3):66.
34. Jones LC, Clark A. beta-cell neogenesis in type 2 diabetes. Diabetes 2001; 50:S186-7. [PMID 11272189]
35. Oyadomari S, Araki E, Mori M. Endoplasmic reticulum stress-mediated apoptosis in pancreatic betacells. Apoptosis 2002; 7:335-45. [PMID 12101393]
36. Araki E, Oyadomari S, Mori M. Endoplasmic reticulum stress and diabetes mellitus. Intern Med 2003; 42:7-14. [PMID 12583611]
37. Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 2004; 11:381-9. [PMID 14685163]
38. White MF. IRS proteins and the common path to diabetes. Am J PhysiolEndocrinolMetab 2002; 283:E413-22. [PMID 12169433]
39. Marzban L, Trigo-Gonzalez G, Zhu X, Rhodes CJ, Halban PA, Steiner DF, Verchere CB. Role of beta-cell prohormoneconvertase (PC)1/3 in processing of proislet amyloid polypeptide. Diabetes 2004; 53:141-8. [PMID 14693708]
40. Westermark P, Li ZC, Westermark GT, Leckstrom A, Steiner DF. Effects of beta cell granule components on human islet amyloid polypeptide fibril formation. FEBS Lett 1996; 379:203-6. [PMID 8603689]
41. Jaikaran ET, Nilsson MR, Clark A. Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation. Biochem J 2004; 377:709-16. [PMID 14565847]
42. Temple RC, Carrington CA, Luzio SD, Owens DR, Schneider AE, Sobey WJ, Hales CN. Insulin deficiency in non-insulin-dependent diabetes. Lancet 1989; 1:293-5. [PMID 2563455]
43. Ward WK, LaCava EC, Paquette TL, Beard JC, Wallum BJ, Porte D Jr. Disproportionate elevation of immunoreactiveproinsulin in type 2 (non-insulindependent) diabetes mellitus and in experimental insulin resistance. Diabetologia 1987; 30:698-702. [PMID 3322910]
44. Deacon CF, Schleser-Mohr S, Ballmann M, Willms B, Conlon JM, Creutzfeldt W. Preferential release of proinsulin relative to insulin in non-insulindependentdiabetes mellitus. ActaEndocrinol (Copenh)1988; 119:549-54. [PMID 3059740]
45. Yoshioka N, Kuzuya T, Matsuda A, Taniguchi M, Iwamoto Y. Serum proinsulin levels at fasting and after oral glucose load in patients with type 2 (non-insulindependent) diabetes mellitus. Diabetologia 1988; 31:355-60. [PMID 3046976]
46. Leahy JL, Halban PA, Weir GC. Relative hypersecretion of proinsulin in rat model of NIDDM. Diabetes 1991; 40:985-9. [PMID 1860563]
47. Krampert M, Bernhagen J, Schmucker J, Horn A, Schmauder A, Brunner H, et al. Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP). ChemBiol 2000; 7:855-71. [PMID 11094339]
48. Hull RL, Andrikopoulos S, Verchere CB, Vidal J, Wang F, Cnop M, et al. Increased dietary fat promotes islet amyloid formation and beta-cell secretory dysfunction in a transgenic mouse model of islet amyloid. Diabetes 2003; 52:372-9. [PMID 12540610]
49. Marzban L, Park K, Verchere CB. Islet amyloid polypeptide and type 2 diabetes. ExpGerontol 2003; 38:347-51. [PMID 12670620]
50. Fink AL. Chaperone-mediated protein folding. Physiol Rev 1999; 79:425-49. [PMID 10221986]
51. Papp E, Nardai G, Soti C, Csermely P. Molecular chaperones, stress proteins and redox homeostasis. Biofactors 2003; 17:249-57. [PMID 12897446]
52. Jaikaran ET, Clark A. Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology. BiochimBiophysActa 2001; 1537:179-203. [PMID 11731221]
53. Stefani M, Dobson CM. Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 2003; 81:678-99. [PMID 12942175]
54. Ben-Zvi AP, Goloubinoff P. Review: mechanisms of disaggregation and refolding of stable protein aggregates by molecular chaperones. J StructBiol 2001; 135:84-93. [PMID 11580258]
55. Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R, et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell 2000; 6:1355-64. [PMID 11163209]
56. Haynes CM, Caldwell S, Cooper AA. An HRD/DER-independent ER quality control mechanism involves Rsp5p-dependent ubiquitination and ERGolgi transport. J Cell Biol 2002; 158:91-101. [PMID 12105183]
57. Rutkowski DT, Kaufman RJ. A trip to the ER:coping with stress. Trends Cell Biol 2004; 14:20-28.[PMID 14729177]
58. VanSlyke JK, Musil LS. Dislocation and degradation from the ER are regulated by cytosolic stress. J Cell Biol 2002; 157:381-94. [PMID 11980915]
59. Szegezdi E, Fitzgerald U, Samali A. Caspase-12 and ER-stress-mediated apoptosis: the story so far. Ann N Y AcadSci 2003; 1010:186-94. [PMID 15033718]
60. Harding HP, Calfon M, Urano F, Novoa I, Ron D. Transcriptional and translational control in the Mammalian unfolded protein response. Annu Rev Cell DevBiol 2002; 18:575-99. [PMID 12142265]
61. Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic-reticulumresident kinase. Nature 1999; 397:271-74. [PMID 9930704]
62. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol 2000; 2:326-32. [PMID 10854322]
63. Liu CY, Schroder M, Kaufman RJ. Ligandindependent dimerization activates the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum. J BiolChem 2000; 275:24881- 85. [PMID 10835430]
64. vanHuizen R, Martindale JL, Gorospe M, Holbrook NJ. P58IPK, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eIF2alpha signaling. J BiolChem 2003; 278:15558- 64. [PMID 12601012]
65. Robertson RP. Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J BiolChem 2004; 279:42351-54. [PMID 15258147]
66. Kopito RR, Ron D. Conformational disease. Nat Cell Biol 2000; 2:E207-9. [PMID 11056553]
67. Cumming RC, Andon NL, Haynes PA, Park M, Fischer WH, Schubert D. Protein disulfide bond formation in the cytoplasm during oxidative stress. J BiolChem 2004; 279:21749-58. [PMID 15031298]
68. Anelli T, Alessio M, Mezghrani A, Simmen T, Talamo F, Bachi A, Sitia R. ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family. EMBO J 2002; 21:835-44. [PMID 11847130]
69. Fassio A, Sitia R. Formation, isomerisation and reduction of disulphide bonds during protein quality control in the endoplasmic reticulum. Histochem Cell Biol 2002; 117:151-7. [PMID 11935291]
70. Camhi SL, Lee P, Choi AM. The oxidative stress response. New Horiz 1995; 3:170-82. [PMID 7583159]
71. Yue DK, McLennan S, Turtle JR. Non-enzymatic glycosylation of tissue protein in diabetes in the rat. Diabetologia 1983; 24:377-81. [PMID 6873515]
72. Schnider SL, Kohn RR. Glucosylation of human collagen in aging and diabetes mellitus. J Clin Invest 1980; 66:1179-81. [PMID 7430347]
73. Brownlee M, Vlassara H, Cerami A. Nonenzymatic glycosylation and the pathogenesis of diabetic complications. Ann Intern Med 1984; 101:527-37. [PMID 6383165]
74. Ma Z, Westermark P, Westermark GT. Amyloid in human islets of Langerhans: immunologic evidence that islet amyloid polypeptide is modified in amyloidogenesis. Pancreas 2000; 21:212-18. [PMID 10975716]
75. Koenig RJ, Blobstein SH, Cerami A. Structure of carbohydrate of hemoglobin AIc. J BiolChem 1977; 252:2992-7. [PMID 853040]
76. Zou MH, Cohen R, Ullrich V. Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus. Endothelium 2004; 11:89-97. [PMID 15370068]
77. Adeghate E, Parvez SH. Nitric oxide and neuronal and pancreatic beta cell death. Toxicology 2000; 153:143-56. [PMID 11090953]
78. Turko IV, Marcondes S, Murad F. Diabetesassociated nitration of tyrosine and inactivation of succinyl-CoA:3-oxoacid CoA-transferase. Am J Physiol Heart CircPhysiol 2001; 281:H2289-94. [PMID 11709394]
79. Hayden MR, Tyagi SC. Islet redox stress: the manifold toxicities of insulin resistance, metabolic syndrome and amylin derived islet amyloid in type 2 diabetes mellitus. JOP. J Pancreas (Online) 2002; 3:86- 108. [PMID 12110767]
80. Adeghate E. Molecular and cellular basis of the aetiology and management of diabetic cardiomyopathy: a short review. Mol Cell Biochem 2004; 261:187-91. [PMID 15362503]
81. Cowell RM, Russell JW. Nitrosative injury and antioxidant therapy in the management of diabetic neuropathy. J Investig Med 2004; 52:33-44. [PMID 14989368]
82. Emerit J, Edeas M, Bricaire F. Neurodegenerative diseases and oxidative stress. Biomed Pharmacother 2004; 58:39-46. [PMID 14739060]
83. Zou MH, Shi C, Cohen RA. Oxidation of the zincthiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest 2002; 109:817-26. [PMID 11901190]
84. Ceriello A, Mercuri F, Quagliaro L, Assaloni R, Motz E, Tonutti L, Taboga C. Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia 2001; 44:834-38. [PMID 11508267]
85. Hess DT, Matsumoto A, Nudelman R, Stamler JS. S-nitrosylation: spectrum and specificity. Nat Cell Biol 2001; 3:E46-49. [PMID 11175760]
86. Wedemeyer WJ, Welker E, Narayan M, Scheraga HA. Disulfide bonds and protein folding. Biochemistry 2000; 39:4207-16. [PMID 10841785]
87. Welker E, Wedemeyer WJ, Scheraga HA. A role for intermolecular disulfide bonds in prion diseases? ProcNatlAcadSci U S A 2001; 98:4334-36. [PMID 11274354]
88. Kapurniotu A, Bernhagen J, Greenfield N, Al- Abed Y, Teichberg S, Frank RW, et al. Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide. Eur J Biochem 1998; 251:208- 16. [PMID 9492286]
89. Tiedge M, Lortz S, Drinkgern J, Lenzen S. Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 1997; 46:1733-42. [PMID 9356019]
90. Chari SN, Nath N, Rathi AB. Glutathione and its redox system in diabetic polymorphonuclear leukocytes. Am J Med Sci 1984; 287:14-15. [PMID 6731472]
91. Arai K, Iizuka S, Tada Y, Oikawa K, Taniguchi N. Increase in the glucosylated form of erythrocyte Cu- Zn-superoxide dismutase in diabetes and close association of the nonenzymaticglucosylation with the enzyme activity. BiochimBiophysActa 1987; 924:292-6. [PMID 3567220]
92. Jennings PE, Chirico S, Jones AF, Lunec J, Barnett AH. Vitamin C metabolites and microangiopathy in diabetes mellitus. Diabetes Res 1987; 6:151-4. [PMID 3436115]
93. Sinclair AJ, Lunec J, Girling AJ, Barnett AH. Modulators of free radical activity in diabetes mellitus: role of ascorbic acid. EXS 1992; 62:342-52. [PMID 1450596]
94. Sies H. Oxidative stress: from basic research to clinical application. Am J Med 1991; 91:31S-8S. [PMID 1928209]
95. Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D, Nicotera T. Oxidative damage to DNA in diabetes mellitus. Lancet 1996; 347:444-5. [PMID 8618487]
96. De Lorenzi E, Giorgetti S, Grossi S, Merlini G, Caccialanza G, Bellotti V. Pharmaceutical strategies against amyloidosis: old and new drugs in targeting a 'protein misfolding disease'. Curr Med Chem 2004; 11:1065-84. [PMID 15078166]
97. Solomon B. Anti-aggregating antibodies, a new approach towards treatment of conformational diseases. Curr Med Chem 2002; 9:1737-49. [PMID 12369884]
98. Westermark P, Eizirik DL, Pipeleers DG, Hellerstrom C, Andersson A. Rapid deposition of amyloid in human islets transplanted into nude mice. Diabetologia 1995; 38:543-9. [PMID 7489836]
99. Ryan EA, Lakey JR, Paty BW, Imes S, Korbutt GS, Kneteman NM, et al. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 2002; 51:2148- 57. [PMID 12086945]
100. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343:230-8. [PMID 10911004]