FEBS Letters xxx (2013) xxx–xxx

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Review

Mechanisms of islet amyloidosis toxicity in type 2 diabetes

0014-5793/$36.00 � 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.febslet.2013.01.017

Terms and definitions: Amyloid fibrils, protein aggregates having a cross-bstructure and other characeristics, e.g., specific dye-binding; Amyloidosis, anypathological state associated with the formation of extracellular amyloid deposits;Functional amyloid, an amyloid structure found to have a beneficial function inliving systems; Oligomers, clusters of small or large numbers of protein or peptidemolecules without a fibrillar appearance; Protein deposition disease, any patho-logical state with the formation of intracellular or extracellular protein deposits;Protein misfolding, the conversion of a protein into a structure that differs from itsnative state⇑ Corresponding author. Fax: +1 212 263 9497.

E-mail address: Andisheh.Abedini@nyumc.org (A. Abedini).

Please cite this article in press as: Abedini, A. and Schmidt, A.M. Mechanisms of islet amyloidosis toxicity in type 2 diabetes. FEBS Lett. (2013),dx.doi.org/10.1016/j.febslet.2013.01.017

Andisheh Abedini ⇑, Ann Marie SchmidtDiabetes Research Program, Division of Endocrinology, Department of Medicine, New York University Medical Center, 550 First Avenue, Smilow 906, New York, NY 10016,United States

a r t i c l e i n f o

Article history:Received 20 December 2012Revised 10 January 2013Accepted 10 January 2013Available online xxxx

Edited by Wilhelm Just

Keywords:AmylinIslet amyloid polypeptideAmyloidosisType 2 diabetesMetabolic disease

a b s t r a c t

Amyloid formation by the neuropancreatic hormone, islet amyloid polypeptide (IAPP or amylin),one of the most amyloidogenic sequences known, leads to islet amyloidosis in type 2 diabetesand to islet transplant failure. Under normal conditions, IAPP plays a role in the maintenance ofenergy homeostasis by regulating several metabolic parameters, such as satiety, blood glucose lev-els, adiposity and body weight. The mechanisms of IAPP amyloid formation, the nature of IAPP toxicspecies and the cellular pathways that lead to pancreatic b-cell toxicity are not well characterized.Several mechanisms of toxicity, including receptor and non-receptor-mediated events, have beenproposed. Analogs of IAPP have been approved for the treatment of diabetes and are under investi-gation for the treatment of obesity.� 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

Amyloids are partially ordered, fibrillar, protein aggregates thatare rich in b-sheet structure. Amyloid formation has been impli-cated in more than 30 different human disorders including suchdebilitating diseases as Alzheimer’s disease (AD), Parkinson’s dis-ease (PD) and type 2 diabetes (T2D) (Table 1). Amyloid formationis not restricted to in vivo pathological conditions; a large numberof proteins that do not form amyloid in vivo can be induced to doso in vitro under non-physiological conditions [1–4]. Amyloid can,in some cases, be functional and beneficial [4]. In this review, wefocus on amyloid formation by islet amyloid polypeptide (IAPP,amylin), a neuropancreatic hormone that forms pancreatic isletamyloid in T2D and contributes to b-cell dysfunction and death.We first outline the biosynthesis of IAPP and describe its normalphysiological roles. We then discuss IAPP amyloid formation, with

emphasis on potential mechanisms of toxicity, drawing analogy toproteins and physiological consequences documented in otheramyloidosis diseases, which have not yet been characterized for is-let amyloid. We conclude with a brief description of the clinicalapplications of IAPP analogs.

The kinetics of amyloid formation is complex and displays a sig-moidal profile with three observable phases. The initial steps ofaggregation, which lead to formation of an active seed, occur inthe lag phase and represent the rate limiting process. In this phase,monomers oligomerize and convert into species that nucleate anexponential fibril growth phase. Fibrils elongate by addition ofpeptide to their ends. Secondary nucleation that involves the cata-lyst of fibril formation from existing fibrils also occurs. This may in-volve breakage of existing fibrils to increase the concentration offree ends, or the templating of new fibrils on the surface of existingones. Finally, a steady state is reached where soluble peptide is atequilibrium with amyloid fibrils. Off-pathway steps leading toamorphous aggregates also occur. Amyloid formation can be accel-erated by the addition of small amounts of preformed fibrils in aprocess known as ‘‘seeding’’ (Fig. 1A).

Although there is no sequence homology or structural similaritybetween the proteins that form amyloid, all amyloid deposits sharecommon characteristics. Amyloid fibrils are typically unbranched,5–10 nm in width, variable in length, polymorphic, and form across b-sheet structure. This structure is defined by perpendicularorientation of the individual polypeptide chains to the long axis ofthe fibril; with the interchain hydrogen bonds aligned parallel to

http://

Table 1Common pathological amyloidoses and their major protein components.

Amyloidosis Disease Amyloidogenic Protein Deposition Type

Alzheimer’s Disease; Inclusion-body myositis; Down’s syndrome; Cerebral b-amyloidangiopathy

Amyloid-b peptides (1–40 and 1–42) Neurodegenerative

Hereditary cerebral haemorrhage with amyloidosis Mutants of Amyloid-b peptides NeurodegenerativeHuntington’s disease (HD) Autosomal dominant mutation of human huntingtin

leading to expanded polyglutamine insertsNeurodegenerative

Parkinson’s Disease and other synucleinopathies a-Synuclein NeurodegenerativeFamilial amyotrophic lateral sclerosis (ALS); also known as motor neuron disease or Lou

Gehrig’s diseaseMutations in Superoxide dismutase (SOD1); TDP-43and FUS/TLS

Neurodegenerative

Serpinopathies Mutations in members of the serine proteaseinhibitor or serpin superfamily of proteins (Serpins)

Neurodegenerativeor Local

Bovine spongiform encephalopathies (BSE or Mad Cow disease); Creutzfeldt-Jakobdisease

Prions in the Scrapie form (PrpSc) Neurodegenerative

Intracytoplasmic neurofibrillary tangles; Tauopathies; Alzheimer’s Disease Tau protein NeurodegenerativeIcelandic hereditary cerebral amyloid angiopathy (CAA); also known as hereditary

cyctatin C amyloid angiopathyMutant of cystatin C Neurodegenerative

Familial British dementia ABri polypeptide (ABriPP) NeurodegenerativeFamilial Danish dementia ADan polypeptide (ADanPP) NeurodegenerativeType 2 diabetes; pancreatic islet amyloidosis Amylin, also known as Islet Amyloid Polypeptide

(IAPP)Local

Aortic medial amyloidosis Medin (a fragment of lactadherin) LocalAtrial amyloidosis Atrial natriuretic factor LocalMedullary carcinoma of the thyroid (MTC) Pro-calcitonin LocalInjection-localized amyloidosis Insulin LocalCritical illness myopathy (CIM) Hyperproteolytic state of myosin ubiquitination LocalLichen amyloidosis Keratins LocalRestrictive amyloid heart; also known as cardiac amyloidosis, amyloid cardiomyophathy

or ApoA-I amyloidosisApolipoprotein A-I (Apo-A1) Local or Systemic

Cataract Crystallin family of proteins LocalPituitary prolactinoma Prolactin LocalPulmonary alveolar proteinosis (PAP) Pulmonary surfactant protein C LocalFamilial amyloid polyneuropathy (FAP); Familial amyloid cardiopathy (FAC); Senile

systemic amyloidosis (SAA)Transthyretin (TTR) Systemic

Familial amyloidosis of Finnish type (FAF) Fragments of gelsolin mutants SystemicAmyloid light chain (AL) amyloidosis; also known as Primary systemic amyloidosis (PSA) Immunoglobulin light chains SystemicAmyloid heavy chain (AH) amyloidosis Immunoglobulin heavy chains SystemicDialysis-related amyloidosis b2-microglobulin (b2m) SystemicCorneal amyloidosis associated with trichiasis Variation of lactoferrin (LF) LocalHereditary lattice corneal dystrophy Mainly C-terminal fragments of kerato-epithelin LocalAA amyloidosis or Secondary amyloidosis (associated with inflammatory disorders such

as tuberculosis, rheumatoid arthritis, bronchiectasis, ulcerative colitis, Crohn’s disease,renal cell carcinoma, ankylosing spondylitis, nephritic syndrome, chronicosteomyelitis, Hodgkin disease, familial Mediterranean fever)

Serum amyloid A (SAA) protein Systemic

Cerebral autosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL)

Mutations in the Notch3 gene Neurodegenerativeor Systemic

ApoA-II amyloidosis Apolipoprotein A-II (ApoA2) SystemicApoA-IV amyloidosis N-terminal fragment of apolipoprotein A-IV (ApoA4) SystemicFibrinogen amyloidosis Variants of fibrinogen a-chain SystemicLysozyme amyloidosis Mutants of lysozyme Systemic

Fig. 1. Amyloid formation by IAPP. (A) Schematic diagram of amyloid formation (solid blue curve). During the lag phase monomers associate to form oligomeric specieswhich then assemble to nucleate an exponential growth phase. Secondary nucleation and off-pathway steps such as formation of amorphous aggregates are omitted forclarity. Amyloid formation can be accelerated by the addition of small amounts of preformed fibrils (dashed red curve). (B) The primary sequence of human IAPP. The peptidehas a free N-terminus, an amidated C-terminus and an intramolecular disulfide bond between residues 2 and 7.

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Please cite this article in press as: Abedini, A. and Schmidt, A.M. Mechanisms of islet amyloidosis toxicity in type 2 diabetes. FEBS Lett. (2013), http://dx.doi.org/10.1016/j.febslet.2013.01.017

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the long axis [4]. Amyloids can form from proteins that fold to acompact tertiary structure in their unaggregated state, or they canoriginate from ‘intrinsically disordered polypeptides’ that fail toadopt compact tertiary structures in their soluble native state [4].Amyloid formation from a folded precursor normally involves globalor local unfolding to an aggregation prone state, while intrinsicallydisordered proteins can aggregate directly from their native ensem-ble. Well studied examples of globular proteins that form amyloidinclude b2-microglobulin, transthyretin (TTR) and mutants of hu-man lysozyme. Stabilization of the native fold can prevent amyloidby this class of proteins; this is the basis of the first clinically ap-proved, rationally designed, small molecule anti-amyloid agent[5]. Ab, a-synuclein, and IAPP are important examples of intrinsi-cally disordered polypeptides that form amyloid in vivo [2,6].

Amyloidosis diseases can be divided into three broad classes:neurodegenerative, systemic and local amyloidosis [7]. In neurode-generative diseases, amyloids are deposited in the brain and spinalcord. Important examples of this class include AD, PD and Hunting-ton’s disease (HD). In systemic amyloidoses, such as in the case ofamyloid light-chain (AL) amyloidosis, aggregation occurs in multi-ple organs and tissues. A subset of systemic amyloidoses includeslysozyme amyloidosis; senile systemic amyloidosis; familial trans-thyretin-associated amyloidosis, which arises from deposition ofwild-type or one of more than 50 mutated forms of TTR; and dis-eases of chronic inflammation, in which an N-terminal fragmentof the acute phase protein serum amyloid A (SAA) forms amyloiddeposits. In the non-neurological, localized amyloidoses, deposi-tion of amyloid occurs in one target organ, usually proximal tothe production site of the amyloidogenic peptide. Important exam-ples of this third class include amyloid formation by the crystallinsassociated with cataract; atrial amyloid, caused by atrial natri-uretic factor; and islet amyloidosis in T2D. The association be-tween amyloid formation and disease pathogenesis is commonfor all amyloidoses and the cytotoxic properties of amyloidogenicpeptides may be similar [8].

2. IAPP is one of the most amyloidogenic sequences known

2.1. Biosynthesis of IAPP

IAPP is synthesized as an 89 residue pre-prohormone. Removalof the 22 residue signal sequence leads to the 67 residue pro-IAPP,which is further processed in the Golgi and in the insulin secretorygranule to the 37 residue mature hormone [9,10]. Additional posttranslational modifications include formation of an intramoleculardisulfide bridge between residues 2 and 7, and amidation of the C-terminus (Fig. 1B). Mature IAPP is stored in the insulin secretorygranule at a ratio of 1:50 to 1:100 relative to insulin and is co-se-creted with insulin [11].

IAPP is also subject to spontaneous, non-enzymatic, post-trans-lational modifications that may impact its function and its ten-dency to aggregate. For example, the human polypeptidecontains six chemically liable Asn residues which can undergospontaneous deamidation to yield Asp or iso-Asp residues. Thismodification results in the replacement of a neutral amide sidechain with a carboxyl group and will lower the net charge of hu-man IAPP at physiological pH, and presumably decrease its solubil-ity. Deamidation of human IAPP accelerates amyloid formationin vitro, but its role in vivo is not understood [12].

2.2. The primary sequence of IAPP correlates with in vivoamyloidogenicity

IAPP is a member of the calcitonin like family of polypeptidesand has been found in all animals studied, although not all speciesform amyloid [13,14]. Mice and rats do not develop islet amyloid,

Please cite this article in press as: Abedini, A. and Schmidt, A.M. Mechanismsdx.doi.org/10.1016/j.febslet.2013.01.017

but cats, non-human primates and humans do. The human poly-peptide is extremely amyloidogenic in vitro, while rat IAPP isnot, even though the two polypeptides differ at only six positions.Notably, rat IAPP contains three proline residues within the 20–29sequence and these are believed to be responsible for its inabilityto form amyloid. Much attention has been focused on the sequencewithin the 20–29 region and the role it plays in controlling amyloidformation. This portion of the polypeptide chain is considered to beone of the major determinants of the ability of IAPP variants toform amyloid. The characterization of designed variants of IAPPsupport this conjecture; analogs of IAPP which contain prolinesor N-methylated residues at other positions within the 20–29 seg-ment are considerably less amyloidogenic than wild type humanIAPP [15–17]. Proline and N-methyl amino acids are well knownto disrupt b-sheets. There is one known mutation in vivo withinthis region, a Ser20Gly substitution, which enhances amyloid for-mation [18,19]. While there is a strong correlation between the pri-mary sequence of the 20–29 segment and in vitroamyloidogenicity, mutations outside of this region can abolishamyloid formation, indicating that it cannot be the sole factor con-trolling IAPP’s amyloidogenicity [20–22]. For more informationabout the biophysics and the sequence determinants of IAPP amy-loid formation the reader is referred to the accompanying article byCao. et. al. in this issue.

2.3. Physiological function of IAPP

The physiological roles of IAPP are receptor mediated. The IAPPreceptor is formed from a complex of the calcitonin receptor with areceptor activity modifying protein (RAMP) [23,24]. IAPP binds thecalcitonin receptor (CTR) in the absence of RAMPs, but the affinityis low. The affinity of IAPP for the CTR-RAMP complex is higher,with an IC50 reported to be on the order of 8 nM for the CTR–RAMP-1 complex [25]. Six different subtypes of the IAPP receptorare generated by different combinations of the two splice variantsof the calcitonin receptor with different RAMPs, but the distribu-tion of the subtypes is not fully characterized [24,25].

Circulating concentrations of IAPP have been reported to be be-tween 3–5 picomolar in rats, rising to 15–20 picomolar with in-creased blood glucose levels [26]. However these values areunlikely to be relevant to amyloid formation since IAPP is storedat a much higher level in the insulin secretory granule, between500 micromolar to several millimolar. This implies that the localconcentration of IAPP after release from the granule will be tempo-rarily much higher than the circulating concentration.

The normal physiological roles of adaptive IAPP are not com-pletely understood in humans, but studies in rodent models showthat IAPP is involved in the suppression of satiety and adiposity, aswell as the regulation of glucose homeostasis via inhibition of glu-cose-stimulated insulin secretion (GSIS), gastric emptying, sup-pression of glucagon release, vasodilatation, and the excretion ofcalcium, potassium and sodium [26–35]. IAPP’s anorectic effect ap-pears to be mediated mainly at the area postrema (AP) of the CNS.Several recent reviews provide a critical and in depth examinationof the physiological roles of IAPP [26–29,34,35].

3. IAPP impacts T1D, T2D and islet cell transplantation

Amyloid accumulates in the pancreatic Islets of Langerhans inthe majority of individuals with T2D. Pancreatic amyloid depositswere first described more than 100 years ago, but the protein com-ponent was not identified until much later when, in 1987 twogroups independently isolated a 37 residue polypeptide fromex vivo samples of pancreatic amyloid [36–38]. Interest in isletamyloid has undergone a resurgence in the last ten years withthe realization that b-cell dysfunction and the loss of b-cell mass

of islet amyloidosis toxicity in type 2 diabetes. FEBS Lett. (2013), http://

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are key features of T2D [39]. The decline in b-cell mass and func-tion is attributed to several factors, including islet inflammation,glucolipotoxicity, accumulation of cholesterol and islet amyloidformation [40–43].

Amyloid deposition is also an important factor in the failure ofislet cell transplants, and correlates with graft failure [44–46]. IAPPamyloid forms rapidly upon transplantation of human islets intonude mice and occurs before the recurrence of hyperglycaemia;this is correlated with the loss of b-cells [44,45]. Islet amyloidhas been detected in transplanted human islets in a patient thatsuffered islet graft failure [46]. Conversely, prevention of amyloidformation by transplantation of porcine islets prolongs islet graftsurvival [47].

In T2D, formation of islet amyloid by IAPP is a significant prob-lem and the clinical goal is to inhibit amyloidosis-induced toxicity.The situation is different in type 1 diabetes (T1D); here the issue isa lack of production of adaptive IAPP. IAPP is produced and re-leased with insulin by pancreatic b-cells, thus it is absent in T1D,as is insulin. The absence of physiologic concentrations of IAPP inT1D and late stage T2D may have deleterious effects, motivatingthe development of non-toxic bioactive analogs of human IAPPfor the clinical goal of hormone replacement therapy, as is donewith insulin. One analog has been approved as an adjunct for insu-lin therapy for the treatment of diabetes and its administration isreported to improve glycemic control. In contrast, there are no clin-ically approved inhibitors of pathologic IAPP amyloid formationindicated for T2D [48]. The nature of toxic species produced duringIAPP amyloidosis is not known, making it difficult to design thera-peutics that target the pathological form of the polypeptide. Cur-rently, drug design aimed at preventing amyloid formation is anactive area of research.

3.1. Islet amyloid includes other components which may influence thekinetics of amyloid formation and fibril stability

In vivo, amyloid formation takes place in a heterogeneous envi-ronment with the potential for interactions with molecules of theextracellular matrix and with membranes, as well as with otherfactors. Like other amyloid deposits, islet amyloid contains serumamyloid P component (SAP), apolipoprotein E (apoE), and the hep-aran sulfate proteoglycan (HSPG) perlecan [49–52]. There is a welldocumented correlation between the e4 allele of the apolipopro-tein E gene (apoe4) and AD [53]. The e4 allele plays an importantrole in AD and has more widespread effects than any other geneticfactor that has been implicated in sporadic, late-onset AD. Studieswith apoE knockout mice have shown that this is not the case forT2D [51]. Interactions between SAP and IAPP also do not appearto play a role in amyloid deposition, but interactions with the gly-cosaminoglycan (GAG) component of HSPGs might. GAG chains ofHSPGs have been shown to significantly accelerate amyloid forma-tion by IAPP and partially processed forms of pro-IAPP in vitro [54].

One model for IAPP amyloid formation in vivo assigns an impor-tant role to the interaction of incorrectly processed proIAPP withperlecan [55]. Perlecan is a component of the extracellular matrixand is associated with islet amyloid. A fraction of the IAPP that issecreted in T2D is incompletely processed and includes the N-ter-minal flanking peptide. Impaired processing leads to increasedamyloid [56,57], and the processing intermediate, denoted hereas IAPP-Npro, is found in islet amyloid. IAPP-Npro is less amyloido-genic in solution than mature IAPP, but it interacts more effectivelywith GAGs. Interactions with model GAGs significantly enhancethe rate of in vitro amyloid formation by IAPP-Npro and the result-ing fibrils are able to seed amyloid formation by fully processedIAPP [58]. Hence release of increased amounts of incorrectly pro-cessed IAPP-Npro which bind to GAGs could generate a high localconcentration of the polypeptide and initiate amyloid formation.

Please cite this article in press as: Abedini, A. and Schmidt, A.M. Mechanismsdx.doi.org/10.1016/j.febslet.2013.01.017

These deposits might recruit mature IAPP and additional IAPP-Npro. Are interactions with GAGs important in vivo? The answeris not known, but over-expression of heparanase inhibits amyloidformation in a transgenic mouse that over-expresses IAPP, andthe inhibition of GAG synthesis in cultured islets has been demon-strated to reduce amyloid deposition [59,60].

Binding of HSPGs to amyloid deposits could influence their sta-bility and their clearance. Salts accelerate IAPP amyloid formationand also stabilize IAPP fibrils by reducing electrostatic repulsion[61,62]. The polyanionic GAG chains of HSPGs could be even moreeffective than simple salts since the spacing of the negative chargesin GAGs can match the spacing of the positively charged sites inamyloid fibrils [54]. The effect of GAGS on IAPP amyloid stabilityand on the clearance of islet amyloid is a largely unexplored area.

3.2. The initiation site of islet amyloid formation is controversial

Determining if islet amyloid originates intracellularly or extra-cellularly is important because it will directly impact therapeuticapproaches. The location of the initial site of islet amyloid deposi-tion in vivo is currently controversial [27,63,64]. Amyloid depositsfound in T2D appear to be extracellular and initial histologicalstudies with rodent models argue in favor of an extracellular ori-gin. Transgenic rodent models that over-express human IAPP areconsistent with an intracellular origin [63]. In contrast, work witha cultured islet model is consistent with an extracellular origin ofislet amyloid [64]. That study showed that the secretion of IAPPis an important factor in islet amyloid formation and b-cell toxicity.The effect of reagents that increased IAPP secretion, but did not in-crease the amount of IAPP produced, were used together with re-agents that inhibited IAPP secretion, but maintained the level ofproduction of IAPP. Increasing secretion increased amyloid forma-tion and toxicity, while inhibiting secretion reduced amyloid for-mation and toxicity [64]. The conflicting results may be relatedto the level of IAPP produced and to the techniques used in thedetection of amyloid [27,63–65].

4. Mechanisms of toxicity in other amyloidosis-potentiallessons for islet amyloidosis

A range of mechanisms has been proposed for the general toxiceffects of amyloidosis, however, the exact mechanisms of celldeath are still not completely clear. In some cases, amyloid fibrildeposits physically disrupt tissue architecture and lead to organdysfunction, however, in most cases, activation of multiple over-lapping cellular mechanisms and downstream signaling pathwayshave been proposed to lead to disease pathogenesis. These includereceptor-mediated interactions and non-receptor mediatedphenomena.

Non-receptor based mechanisms have primarily focused onmembrane disruption and permeabilization by soluble oligomersand amyloid fibrils. Membrane disruption leads to an increase inintracellular Ca2+ and has been shown to activate several patho-genic pathways, including production of reactive oxygen species(ROS) [66], altered signaling pathways [67,68] and mitochondrialdysfunction [69]. Membrane permeabilization by amyloid oligo-mers may also induce an oxidative stress response in cells[66,70]. Permeabilization of mitochondrial membranes by amyloidoligomers and the accumulation of Ca2+ in the matrix of mitochon-dria lead to an increase in ROS production, cytochrome C releaseand apoptosis [71,72]. ER stress has also been proposed to contrib-ute to cytotoxicity [73].

Receptor-mediated mechanisms of toxicity include FAS (alsoknown as APO-1, APT or CD95), p75NTR (p75 neurotrophin recep-tor) and RAGE (receptor for advanced glycation end products). Inparticular, FAS and p75NTR have been investigated in IAPP and

of islet amyloidosis toxicity in type 2 diabetes. FEBS Lett. (2013), http://

Fig. 2. Physiological and pathophysiological effects of IAPP. According to this scheme, amyloidosis by adaptive IAPP leads to the production of cytotoxic species. The toxicspecies of IAPP may form either intracellularly or extracellularly, leading to a series of parallel, overlapping and potentially additive or synergistic pathogenic pathways.

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Ab toxicity [74,75], while RAGE has been shown to engage amyloi-dogenic species of Ab1–40 and Ab1–42, serum amylin A (SAA) andprion-derived peptide, among others [76]. RAGE was originallynamed for its ability to bind advanced glycation end products(AGEs), but is now recognized as a multi-ligand pattern recognitionreceptor with several classes of ligands including amyloid formingpolypeptides and proteins. RAGE not only elicits signaling path-ways that lead to inflammation and apoptosis, but is also involvedin internalization of bound amyloidogenic ligands. RAGE-Ab inter-actions have received prominence [76–78]; however, the role ofRAGE in islet amyloidosis has not been investigated.

Upregulation of autophagy has been recognized as a commonprotective response to the accumulation of toxic amyloidogenicaggregates in degenerative diseases [79–82]. However, autophago-cytosis and lysosomal degradation of misfolded amyloidogenicpolypeptides and proteins is not entirely successful as accumula-tion of amyloidogenic aggregates also leads to autophagy-medi-ated lysosomal dysfunction and cell death [80,81].

Chronic inflammation may be an important contributing factorto amyloidosis protein toxicity as it is frequently observed in localand systemic amyloidosis diseases [83–85]. In neurodegenerativediseases, such as AD, PD and HD, activation of microglia [86] andpro-inflammatory processes (e.g. production of cytokines, chemo-kines, nitric oxide (NO), ROS, and arachidonic acid metabolites)can lead to oxidative stress, mitochondrial dysfunction and neuraltoxicity [87–90]; however, the exact mechanisms of cell death arecontroversial. Ab-induced activation of NF-kappaB, a potent imme-diate-early transcriptional regulator of numerous proinflammatorygenes, has been shown to be stimulated by ROS formation in pri-mary neurons derived from AD patients [91]. Activation of theinflammasome protein complex by misfolded protein aggregateshas also been implicated in the pathogenesis of several amyloidosisdiseases such as AD and ALS, providing a common mechanism ofIL-1b cytokine production in these diseases [92].

Amyloid oligomers, which precede mature fibrils, have beenimplicated, albeit indirectly, in the pathogenesis of several dis-eases, including AD, AL and TTR amyloidosis in vitro and in vivo;and have been shown to trigger oxidative stress and activation of

Please cite this article in press as: Abedini, A. and Schmidt, A.M. Mechanismsdx.doi.org/10.1016/j.febslet.2013.01.017

apoptotic pathways [93–96]. The identity and nature of the specifictoxic species has not been addressed for islet amyloidosis.

Disease-specific events and pathways characterize amyloidoses.Mutations that lead to the destabilization, unfolding and/or mis-folding of proteins, are commonly associated with inherited dis-eases. Mutations in proteins can also enhance theamyloidogenicity of polypeptides and proteins resulting in acceler-ation or increase in the formation of toxic amyloidogenic species.Other disease-specific mutations can occur in genes that do not en-code proteins that accumulate as amyloids, but rather impact theproduction of amyloidogenic proteins. Examples include the pre-senilins in AD and parkin in PD. These mutations can have the ef-fect of increasing the concentration of aggregation prone proteinsand polypeptides. Presenilin mutations alter the proteolytic pro-cessing of APP, leading to an increase in the more aggregationprone Ab-42 isoform [97]. Covalent modification of proteins, suchas oxidation, glycation and racemization can also promote unfold-ing or mis-folding and may contribute to amyloid formation [95].

These common pathological mechanisms in amyloidogenic dis-eases may stem from common properties that are shared amongtoxic amyloidogenic species [8,95,96]. Ongoing efforts to elucidatethe mechanisms of cytotoxicity and tissue damage by other amy-loid proteins may ultimately redirect therapeutic efforts in isletamyloidosis.

5. Mechanisms of IAPP-induced b-cell toxicity in isletamyloidosis

A subset of the mechanisms described above has been investi-gated in the context of islet amyloidosis. Cell membrane permeabi-lization or disruption by IAPP aggregates has been suggested to bea mechanism of toxicity. Other mechanisms of IAPP toxicity in-clude localized islet inflammation, defects in autophagy, ER stress,as well as receptor-mediated mechanisms involving FAS and theactivation of downstream signaling pathways such as cJUN N-ter-minal kinase (JNK). The available data suggest that IAPP exertsits toxic effects on b-cells by multiple mechanisms. Several of theseoverlap and share common signaling pathways (Fig. 2).

of islet amyloidosis toxicity in type 2 diabetes. FEBS Lett. (2013), http://

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Amyloid formation by IAPP has been shown to induce apoptosisin cell culture and in isolated human islets [65,98–101]. The path-ways that lead to IAPP induced b-cell apoptosis are not yet fullyelucidated, although there is a growing body of literature and pro-gress is being made [102,103].

It has been proposed that IAPP might exert non-specific cyto-toxicity by permeabilizing cell surface membranes [104,105].Numerous studies have demonstrated clustering of IAPP amyloidfibrils on or near membranes in vivo, and exogenous IAPP disruptscell membranes in vitro. The ability of IAPP to induce ion leakagedepends on the lipid composition of the membrane and the ratioof lipid to peptide examined. An important caveat may be thatmost of the in vitro membrane model systems employed in bio-physical studies of membrane leakage utilize much higher frac-tions of anionic lipids, and very different types of lipids thanthose found innately in b-cells [106]. These membrane mimeticslack cholesterol and gangliosides, which have been shown to playa role in mediating IAPP clearance and membrane interactions[107]. Studies have shown that IAPP variants that are not toxic tob-cells in vivo have the ability to disrupt some of these in vitromodel membrane systems. Permeabilization and loss of membraneintegrity by IAPP may indeed be one mechanism of toxicity, partic-ularly at high peptide concentrations, however, caution should betaken when extrapolating from studies that employ non-physio-logical model membranes and peptide concentrations to the morecomplicated situation in vivo. More multifarious model mem-branes are now being employed and are expected to provide newmechanistic insights under more physiologically relevant condi-tions [106].

Other proposed mechanisms of IAPP-induced b-cell death in-clude defects in autophagy, local inflammation, mitochondrial dys-function and receptor-mediated mechanisms linked to oxidativestress, cytokine production and activation of signaling cascadesleading to apoptosis [73,102,103,108–117]. These toxic mecha-nisms have been shown to be activated by either intracellular orextracellular aggregates. The pro-apoptotic JNK pathway mediatesb-cell apoptosis in cultured cells and in islets exposed to high con-centrations of IAPP, and has recently been shown to become upreg-ulated in response to endogenous IAPP amyloid formation [103]. Inb-cells, JNK becomes activated by a range of events leading to cel-lular stress, including ER stress, ROS formation and oxidativestress, increases in glucose concentration and the production ofpro-inflammatory cytokines. Similar downstream signaling path-ways have also been found to become activated in both the intrin-sic (Bim) and extrinsic (Fas, Fadd) pathways. Interaction of eitherendogenous or exogenous IAPP aggregates with FAS, also knownas the death receptor, leads to caspase 3 activation, while deletionof Fas protects b-cells from IAPP toxicity [74]. These studies aresupported by in vivo experiments that demonstrate that inhibitionof caspase 3 protects b-cells from IAPP-induced b-cell apoptosis[118]. The downstream signaling pathways that regulate b-celldeath in response to IAPP amyloidosis are not fully characterized,and more work is needed to understand how many of these path-ways intertwine and overlap, and whether these intracellular con-sequences are triggered by interactions with cell surface receptorsand/or membrane disruption.

Defects in autophagy have been shown to play a role in amyloi-dosis diseases and have been proposed to be a factor in IAPP toxic-ity. Chaperone-mediated autophagy and macroautophagy ensuresynthesis of cellular components, recycle damaged or dysfunc-tional organelles, and clear ubiquitinated proteins. Studies haveshown that activation of autophagy protects b-cells from IAPP-in-duced apoptosis, while inhibition of autophagy-lysosomal diges-tion promotes IAPP toxicity [110]. Over expression of IAPP in b-cells, before the development of hyperglycemia, has been shownto impair autophagy [110,115]. Thus, impairment in autophagy

Please cite this article in press as: Abedini, A. and Schmidt, A.M. Mechanismsdx.doi.org/10.1016/j.febslet.2013.01.017

could lead to the build up of toxic aggregates and promote b-celldeath.

Local islet inflammation induced by toxic forms of IAPP mayplay a role in b-cell dysfunction and death via activation of pro-inflammatory responses [112,114]. IAPP has been shown to acti-vate inflammasomes, which are multi-protein caspase activatingcomplexes that have been implicated in metabolic disease. Inflam-masome activation triggers signaling cascades leading to the pro-duction of pro-inflammatory cytokines such as Interleukin-1b (IL-1b) [112]. IL-1b has been reported to be a mediator of IAPP-inducedb-cell toxicity, however the exact source(s) of IL-1b productionassociated with islet amyloidosis in vivo is still an open question.

Defects in endoplasmic reticulum associated protein degrada-tion (ERAD), unfolded protein response (UPR) and ER stress havebeen reported to induce b-cell death by IAPP aggregates producedboth intracellularly and extracellularly [108,109,116,117]. In thecase where toxicity arises from intracellular aggregate formation,ProIAPP and not mature IAPP may be the deleterious species, asproIAPP miss-processing has been shown to occur in diabetesand post-translational modification is completed in the Golgi andinsulin secretory granules [56,57]. The exact role of ER stress inIAPP-induced b-cell dysfunction in vivo is currently not wellunderstood. Studies using transgenic animal models that signifi-cantly overexpress IAPP support a role for ER stress, while no ERstress was detected in cultured islets expressing lower levels ofIAPP [111].

One of the major unresolved issues in the field of IAPP biology isthe difficulty in differentiating between the functional and toxicforms of the polypeptide, making it difficult to determine whetherthe outcome of a particular experiment is relevant to the physio-logical or pathophysiological situation. This problem is com-pounded by the large variation in the methods used to prepareIAPP by different workers. Small variations in peptide concentra-tion, residual buffers and co-solvents can potentially alter peptidesecondary structure, affecting stability, aggregation kinetics andpotentially activation of off-target cellular stress responses. Cau-tion should be applied when interpreting studies that use unchar-acterized IAPP for in vitro or ex vivo investigations. Structural andbiochemical characterization of amyloidogenic polypeptides andproteins is traditionally carried out by specialized spectroscopictechniques primarily utilized by biophysicists and chemists. Thecurrent gap between the disciplines of biology and biophysicalchemistry hinder research efforts, not only in the field of islet amy-loidosis, but in the study of amyloidosis diseases in general.

6. Therapeutic applications of IAPP for the treatment ofdiabetes and obesity

IAPP is deficient in individuals suffering from T1D and advancedT2D. Co-administration of IAPP with insulin helps to normalizefluctuating glucose levels to a greater degree than is possible withinsulin alone [48,119]. However, the extreme amyloidogenicity ofhuman IAPP prevents its direct use as an adjunct to insulin therapy.Consequently, a non-amyloidogenic analog of human IAPP, de-noted as Pramlintide, which contains proline substitutions at thesame positions found in rat IAPP, was developed and has been ap-proved by the FDA for use in the treatment of diabetes [48].

IAPP and IAPP analogs, such as Pramlintide, are also being ex-plored for the treatment of obesity. Particularly exciting is the po-tential of combining leptin and IAPP [120–122]. Leptin is anadipokine that plays a major role in maintaining energy homeosta-sis. The protein acts on the CNS to suppress appetite and altersmetabolism in peripheral tissue. Treatment of leptin deficient(ob/ob) mice with leptin has been shown to reduce food intakeand lead to weight loss. Individuals that lack functional leptin suf-fer from extreme obesity, which can be reversed by treatment with

of islet amyloidosis toxicity in type 2 diabetes. FEBS Lett. (2013), http://

A. Abedini, A.M. Schmidt / FEBS Letters xxx (2013) xxx–xxx 7

leptin [120]. Unfortunately, administration of exogenous leptindoes not lead to weight loss or reduction of food intake for obeseindividuals who generate normal leptin, and the restoration of lep-tin responsiveness in obese individuals is difficult. This has led tothe concept of leptin resistant obesity, where obese individualsare insensitive to high circulating concentrations of leptin. Themolecular mechanism of leptin signaling has been extensivelystudied, although the origins of leptin resistance are not fully char-acterized. Binding of the protein to its receptor stimulates Janus ki-nase 2 (JAK2) and activates several pathways, including theactivator of transcription and signal transducer pathway (STAT3,STAT5) which are important for the effects of leptin on bodyweight [121–123]. Leptin-induced signaling is negatively con-trolled by feedback systems that involve protein tyrosine phospha-tase1B and suppression of cytokine signaling-3. The negativefeedback loop serves to prevent prolonged leptin receptor activa-tion. Leptin-induced signaling has been recently reviewed [123].

Weight-lowering effects mediated by IAPP have been docu-mented in obese rats and humans. IAPP and IAPP analogs havebeen shown to reduce food intake without producing signs of con-ditioned taste aversion or visceral illness [120,124]. Animal studieswith food-matched controls led to the notion that IAPP-inducedweight loss occurs via mechanisms similar to those found with en-hanced leptin sensitivity [121,124]. Leptin and IAPP have been pro-posed to affect energy homeostasis synergistically [120]. Thissuggests that co-administration of leptin and IAPP might be bene-ficial. Administration of leptin to leptin resistant, diet-induced ob-ese rats showed that leptin by itself is ineffective at inducingweight loss. Treatment with IAPP alone led to modest loss in bodyweight. However, the sustained use of the two polypeptides led tosignificant effects [120,122]. A recent report suggests that some ofthe effects of IAPP and leptin might be additive rather than cooper-ative [125]. In that study, administration of IAPP and leptin wasshown to activate extracellular-regulated kinase (ERK), STAT3,AMP-activated protein kinase (AMPK) and the AKT signaling path-ways in an additive, but not synergistic fashion, and the effectswere abolished by ER stress. The co-administration of IAPP and lep-tin is still potentially an attractive therapeutic strategy in the ab-sence of strong synergy since even additive effects could bebeneficial. Other gastric satiety signals have also been shown tohave beneficial effects when administered with leptin, and this isan active area of research.

7. Perspectives, puzzles and future directions

Increasing evidence supports an adaptive role for IAPP in theregulation of adiposity and energy homeostasis. Under normal cir-cumstances, this neuropancreatic hormone is co-produced, co-stored and co-secreted with insulin from b-cell secretory granulesinto the circulation. In T1D and late stage T2D, lack of production ofadaptive IAPP may have deleterious consequences, while in earlyT2D, overproduction and misprocessing of IAPP, along with otherfactors, lead to islet amyloidosis and b-cell dysfunction and celldeath. Progress has been made in understanding the pathologicaleffects of IAPP amyloid formation in vivo, however many unan-swered questions still remain. These include the mechanisms of is-let amyloid formation and cytotoxicity in vivo and in vitro; theinitial site of amyloid deposition in vivo; the role of membranesand HPSGs in vivo, the properties of the toxic species; and mecha-nisms of clearance. Major hurdles that hinder progress include alack of physiologically relevant model systems for biophysicalstudies, and the lack of detailed biophysical characterization ofIAPP prior to testing in biological assays. The latter is essential inorder to differentiate between adaptive and toxic forms of IAPP.Bridging the gap between the disciplines will be important foraccurate interpretation of ex vivo biological and pharmacological

Please cite this article in press as: Abedini, A. and Schmidt, A.M. Mechanismsdx.doi.org/10.1016/j.febslet.2013.01.017

studies of IAPP using cultured cells and islets, as well as for identi-fication of therapeutic targets and the design of pharmacologicalagents for the treatment of islet amyloidosis.

Acknowledgement

This work was supported by a grant from the United States Na-tional Institutes of Health to A.A. (F32 DK089734-02).

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