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Physiological and pathophysiological role of islet amyloid

polypeptide (IAPP, amylin)




Gunilla T. Westermark

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
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IAPP has a number of effects which may be of physiological relevance. Islet amyloid,

which earlier was regarded as a non-important degenerative product, most likely

plays a central role in the loss of beta cells in type 2 diabetes and probably in

transplanted human islets. Taken together the results from human and animal studies

show that amyloid develops before beta-cell deficiency and the occurrence of

oligomers and amyloid intracellular induce beta cell death. Prevention of islet amyloid

most likely will save beta-cells and extend hormone secretion.
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Islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) was originally isolated as the major peptide

constituent of the amyloid from an insulinoma [1], and subsequently isolated from

amyloid deposits present in the islet of Langerhans from patients with type 2 diabetes

[2; 3]. The 37 residue polypeptide proved to have an earlier unknown sequence, but

showed an almost 50% identity to the known calcitonin gene related peptide [4].

Other nomenclatures for IAPP are amylin [5], Diabetes Associated Peptide [6] and

IAP Insulinoma Amyloid Peptide [1]. IAPP is phylogenetically well preserved and

found in all mammals where it has been looked for [7; 8; 9; 10], and also in an avian

[11] and fish [12].

    During embryogenesis in mice, IAPP was detected in the primordia at E12 and the

immunoreactivity was restricted to the simultaneously occurring insulin expressing

cells [13]. In human, IAPP immune-reactive cells were demonstrated from week 13 of

gestation and here its expression was preceded by insulin that was present already

at 9 weeks of gestation. In fetal and neonatal pancreas there were a higher number

of insulin positive cells than IAPP positive cells, but this difference did not remain in

the adult pancreas where all beta cells co-express insulin and IAPP [13; 14].

An additional expression pattern in developing mice was described by Wilson et al.

[15] where IAPP and proglucagon/glucagon reactivity co-localized in the primordia at

E.10.5 in cells also expressing PC1/3, a convertase not present in the alpha cells of

the mature pancreas. Instead, PC1/3 is expressed in beta cells and together with

proglucagon in intestinal L-cells. Cells positive for IAPP and glucagon did not express

pdx-1, an activator of the IAPP gene [16] but they expressed brain-4 (Brn-4). Brn-4,

originally described in brain, is also a regulator of glucagon expression in alpha-cells

[17].
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In human, IAPP is almost exclusively produced by the beta-cell where it is stored [18]

and released together with insulin [19] and only minor synthesis occurs in entero-

chromaffine cells in the intestinal tract [20]. IAPP is synthesized as an 89 residues

long prepropeptide [21; 22] from which an 18 residue signal peptide is removed in the

endoplasmatic reticulum. Posttranslational cleavages of proIAPP occur at di-basic

residues and comprise the removal of N- and C-terminal flanking peptides. This

proIAPP processing is initiated in the late transgolgi where cleavage by the

proprotein convertase PC1/3 removes 16 residues at the carboxy terminus [23; 24]

followed by PC2 cleavage in the secretory granules that leads to the removal of an

11 residue peptide at the amino terminus [25]. The residues Lys-Arg that remain at

the C-terminus after PC1/3 cleavage are removed by carboxy peptidase E (CPE)

[26]. To receive full biological activity, IAPP must be cyclized by a disulfide bond

between the cystein residues at position 2 and 7 of the mature IAPP and be C-

terminally amidated [27]. An additional processing site is present at residues 79-80

(Lys-Arg) of the C-terminal flanking peptide, but no extended IAPP peptide has been

described (Figure 1).

    Proinsulin is processed to insulin by the same convertases at the same location

[28], and IAPP and insulin are stored in the same secretory granules [29; 30; 31] . In

the mature granule IAPP and C-peptide occupy the halo region while Zn2+ insulin is

present in the dense core region [32]. The insulin to IAPP ratio varies but is often

reported to be 10 to 1 [32; 33; 34]. However, heterogeneity among beta-cells occurs

[35]. Reported non-stimulated plasma levels of IAPP in man range between 2-20 pM

IAPP [36; 37]. IAPP is cleared by the kidneys [38] and insulin is cleared by the liver

and kidneys [39; 40]. The clearance of IAPP is almost 4 times slower than that

determined for insulin but comparable to that for C-peptide [41].Taking this in
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account, a comparison of IAPP and C-peptide plasma levels might be more accurate

and reflect the actual ratio.

    The IAPP to insulin ratio remains constant under normal circumstances and is not

affected by type of stimuli [42]. However, assays used for IAPP quantification will not

discriminate between the active hormone and the partially or non-processed

hormone.



Regulation of the IAPP gene

The human IAPP gene is a single copy gene situated on the short arm of

chromosome 12 and consists of three exons separated by a 0.3 kb and 5 kb intron,

respectively. Exon1 encodes most of the 5’ untranslated region of the transcribed

RNA while exon 2 encodes the signal peptide and 5 residues of the N-terminal

flanking peptide and exon 3 encodes the remaining residues 6-89 of the preproIAPP

molecule [21; 43; 44; 45; 46]. Transcription of the IAPP gene is controlled by a

promoter situated within the sequence spanning from -2798 to + 450, relative to the

transcriptional start codon [47]. IAPP and insulin genes contain similar promoter

elements [48] and the transcription factor PDX1 regulates the effects of glucose on

both genes [49; 50; 51; 52]. Glucose stimulated beta-cells respond with a parallel

expression pattern of IAPP and insulin [53; 54]. The islet hormones interplay in the

regulation of glucose homeostasis [55], and insulin and glucagon stimulate IAPP

gene expression [16], in contrast to somatostatin that has no effect [56]



Receptor for IAPP

IAPP belongs to the calcitonin family of peptides also including calcitonin [57],

calcitonin gene-related peptides (CGRP) [58], adrenomedullin [59] and intermedin
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[60]. For long, there was a futile search for a specific IAPP receptor, and it was not

until the discovery of the receptor activity modifying proteins (RAMPs) the problem

was solved. RAMPs constitute a family of three different single transmembrane

proteins [61] that by combining with the G protein coupled calcitonin receptor (CTR)

or the calcitonin receptor-like receptor (CLR) [62] determine the ligand specificity and

also increase the receptor repertoire [63]. Paring of RAMP3 with a CT receptor forms

an IAPP specific receptor [64].



IAPP in other species

A more disperse distribution of IAPP is seen in other species. In rat and mouse, IAPP

immunoreactivity co-localises partly with gastrin, somatostatin and peptide YY in

enteroendocrine cells in the gastrointestinal tract [65; 66] and in pancreas IAPP is

present in beta and delta cells [67]. IAPP is expressed in the rat brain, and

sometimes with a different distribution of that shown for CGRP [68].

In chicken the IAPP immunoreactivity was co-localised with insulin in the small islets,

but mRNA expression analysis revealed higher signals from intestines and brain [11].

In fish, IAPP immunoreactivity is present in the islet organ Brochmann body [12] and

in the intestinal tract .



Physiology of IAPP



Glucose regulation

Taken together the results from a large group of researchers suggest that IAPP exert

an autocrine or paracrine effect on beta cells and act as a modulator of insulin

secretion [69; 70; 71]. Glucose stimulated insulin secretion from perfused rat
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pancreas can be inhibited by IAPP at 75pmol/l, a concentration equal to that

determined in the effluent from rat pancreas. The inhibitory effect on insulin secretion

is limited to physiological changes of glucose and no effect remains when glucose

levels are augmented from 5.5 mmol/l to 16.6 mmol/l [72] . Insulin secretion in

response to other secretagogues such as sulfonylurea that block ATP dependent K

channels or KCl that depolarized beta cells are also markedly reduced by IAPP [73].

IAPP infusions in rats with hyperglycaemia clamped at 11 mmol/l showed a dose

dependent reduction in insulin secretion, and 8.5 pmol/min and 85 pmol/min reduced

plasma insulin by 31% and 53%, respectively [74]. The inhibitory effect on insulin

secretion ceased by time and IAPP is suggested to be a short-time regulator of

insulin secretion [74]. Immunoneutralisation of intra-islet IAPP by specific antibodies

or by the IAPP inhibitor IAPP 8-37 potentiates both glucose and arginine stimulated

insulin release [71]. This is in accordance with the finding that IAPP null mice have a

more rapid glucose clearance in response to both oral and intravenous administrated

glucose [75]. This phenotype was reversed by the introduction of human IAPP in the

IAPP null mice.



Peripheral effects of IAPP

There are great differences in the reported in vitro and in vivo effects on IAPP and

some of these differences could be ascribed to the use of pharmacological levels of

IAPP, the solubility of IAPP or other not yet known circumstances.

    An early reported effect for IAPP was the reduction of basal and insulin-stimulated

glycogen synthesis in rat skeletal muscle [76]. In the work by Furnsinn et al, short-

term IAPP infusion reduced glycogen content in the hindlimb rat muscle [74]. Again,

this effect was only seen after short-time IAPP exposure and did not remain after
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long-term exposure. IAPP can regulate glycogen synthesis by activation of glycogen

phosphorylase and inactivation of glycogen synthase [77; 78; 79], effects

antagonised by IAPP 8-37 [79]. IAPP has also been shown to cause peripheral

insulin resistance in vivo in cat [80], rat [81] and dogs [82]. In contrary, Kassir et al.

failed to measure any change in the insulin-stimulated glucose disposal rate in dog

[83].

    A single injection of IAPP was shown to partly inhibit glucagon release in freely

feed mice while IAPP had no effect after a glucose load. Glucagon secretion

stimulated by L-arginine was reduced by IAPP while glucagon stimulated by

hypoglycaemia was unaffected [84]. In cat, rat-IAPP injection 5 minutes prior to

intravenous administration of arginine or glucose lowered plasma glucagon levels

and reduced also the insulin levels [85]. Therefore, one effect for IAPP secreted

together with insulin in response to a rise in blood glucose may be to modulate

postprandial glucagon secretion.



Gastric emptying

Administration of IAPP has been shown to delay gastric emptying and thereby

reduced the increase in postprandial glucose [86; 87; 88]. Due to the absence of

endogenous IAPP in type 1 diabetes gastric emptying is expected to be accelerated.

When this was monitored in 21 patients with type 1 diabetes mellitus, no significant

difference in mean or median time compared to the controls could be detected.

However, it should be pointed out that a large variation occurred among the

individuals with type 1 diabetes and increased emptying occurred in a sub-group

without secondary complication [89]. However, in a recent study by Heptulla et al, it

was hypothesised that accelerated gastric emptying should occur in children with
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complication-naive type 1diabetes. Instead they found delayed gastric emptying

when compared to controls [90]. Therefore, administration of IAPP in individuals

lacking the hormone seems to have a different effect than expected on gastric

mobility.



Regulation of food intake

The central high affinity binding sites for IAPP are concentrated to nucleus

accumbens, area postrema and in the immediate adjacent nucleus of the solitary

tract in rat [91] and monkey [92]. Both centrally [93; 94; 95; 96] and peripherally [97;

98; 99] administered IAPP reduces food intake and produces anorexia in mouse and

rat. Chronic subcutaneous infusion of IAPP, at concentrations kept within the

pathophysiological range, causes a dose-dependent reduction of food intake and

body weight gain by lowering the adiposity [99].The anorectic effect from chronic

peripheral infusion was abolished in rats after AP/NST lesion [100] and the effects of

intraperitoneal injections of IAPP was reduced by direct injections of IAPP receptor

antagonist AC187 into the area postrema [95]. Chronic intraperitoneal infusions of

AC187 increased the total food intake in genetically obese fa/fa rat but were

ineffective on lean littermates.

    IAPP can enter the blood brain barrier [101] and the anorectic effects exerted by

peripheral IAPP indicate that the molecule might be a satiety factor. One side-effect

found during the clinical trials with the IAPP analogue pramlintide was a slight weight

reduction in the study group.

    The expression pattern of intracranial IAPP is somewhat unclear and local

expression was reported to occur at multiple sites in the rat brain [68] [102]. If IAPP
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produced at this site participates in the regulation of food intake has yet to be

resolved.

     In a study on postpartum mRNA regulation a 25 fold increase of IAPP was

detected in the preoptic area of the hypothalamus. The increase was verified at the

peptide level and suggests that IAPP plays part in maternal regulation [103].



Calcium metabolism

IAPP participates in the regulation of total bone mass and stimulates osteoblast

proliferation and bone formation, in both rodent and human [104; 105] cultured

osteoblasts. Bone absorption is reduced because IAPP slower the mobility of

osteoclasts [106; 107; 108] and prevents the fusion of the preosteoclasts into

multinucleated osteoclasts shown in rodent cell culture [109]. IAPP null mice have a

50 % reduction in bone mass when compared to witdtype mice [110]. It still needs to

be elucidated if IAPP has any significance for the development of osteopenia, but

IAPP fasting levels are reported to be significantly lower in patients with osteoporosis

and in women with anorexia nervosa, a disease frequently associated with

osteoporosis [111] .



IAPP as a drug in obesity and diabetes treatment

Pramlintide/symlin is a synthetic analogue of IAPP with three structure-breaking

proline substitutions inserted at position 25, 28 and 29 to inhibit aggregation of the

peptide (Figure 1). This exchange of residues makes pramlitide more like the rat

IAPP. Symlin was approved by US food and drug administration (FDA) in 2005 [112],

but not elsewhere, to be given together with insulin to control post meal blood

glucose in patients with type 1 and type 2 diabetes. Over the recent years, multiple
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clinical trials on the effects of Pramlintide have been undertaken. All in all the

reported biological effects including an often mild weight decrease, significant

reduction in HbA1c and a decrease in insulin dose. The drawback was the

experience of a transient nausea.

     More interesting is the finding from ongoing clinical studies where a combination

of pramlintide and the leptin analoge metreleptin were given and resulted in an

approximately 13% weight loss after 24 weeks, a reduction significantly more than

after treatment with pramlintide or metreleptin alone [113].



Amyloid

Amyloid in general

Amyloidoses constitute the largest group among the protein misfolding diseases, and

today, thirty different proteins have been characterized from amyloid deposits in

human [114]. The proteins are unrelated and each protein is linked to a specific

amyloid disease. Based on the distribution pattern, the diseases are divided into two

forms, systemic amyloidosis where deposits are present throughout the body and

where the precursor proteins most often are plasma proteins, and localised

amyloidosis where the deposits mainly are restricted to the site of production and

where not all but many of the precursors are polypeptide hormones.

Amyloid is often referred to as an amorphous material, but it consists of fibrilar

structures with a diameter of 7-10 nm and of indefinite length. The protein molecules

that make up the fibrils are aligned perpendicular to the fibrilar axis and this is

believed to cause the specific tinctorial characteristic of amyloid, affinity for Congo

red and green birefringence in cross polarized light.
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            Formation of amyloid can be separated into three separate phases, a lag

phase, an elongation phase and a plateau phase. The lag phase is of undefined

length and can last from minutes up to a life time. It is during this period the monomer

unfolds and forms small amyloid aggregates. These aggregates can act as templates

and amyloid fibrils extend from these during the elongation phase. The elongation of

fibrils will continue until the plateau phase is reached, dependent on the equilibrium

for the specific peptide [115; 116]. In experimental in vivo and in vitro models for

amyloid formation, the introduction of a minute amount of preformed amyloid fibrils

can dramatically shorten the lag phase and cause rapid amyloid formation. It is

evident that extracellular amyloid can be degraded and cleared, but often the

formation of amyloid exceeds the resolution and therefore the amyloid mass will

continue to grow as long as the precursor is supplied.



Islet amyloid

Islet amyloidosis is a localised form of amyloid disease and was described by Opie in

1901 [117]. Islet amyloid is the main islet pathology present in individuals diagnosed

with type 2 diabetes, but the reported frequency of amyloid varies from 40 -100%

[118; 119; 120; 121; 122]. This rather large discrepancy in amyloid frequency

between reports and the presence of amyloid in islets of non-diabetic subjects has

questioned the importance for islet amyloid as a cause of type 2 diabetes. In a study

by Maloy et al., amyloid was present in 59% of the subjects with diabetes but when

the group was subdivided dependent on treatment, it was shown that the patients

that received insulin treatment all had islet amyloid [119]. This points to an

association between the severity of the disease and the prevalence of islet amyloid .
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     In addition to man, islet amyloid occurs in primates [123; 124; 125] and cats [126].

In primates, there is an increased risk for spontaneous diabetes when kept in

captivity and the disease development includes obesity and hyperinsulinaemia, a

disease pattern that resembles the type 2 diabetes that develops in humans. There

are three studies on different monkeys that connect islet amyloid with the

development of diabetes. In Macaca nigra, the amyloid area was determined in

pancreas biopsies and at autopsy in 18 monkeys, some followed for 10 years [127].

The amyloid area was determined and compared to the result of an intravenous

glucose tolerance test. In non-diabetic monkeys the amyloid area did not exceed 3%

and no abnormalities in insulin secretion or glucose clearance was detected. When

the amyloid load progressed and affected 20-40% of the islet area both insulin

secretion and glucose clearance was decreased. Diabetes shown by hyperglycaemia

developed when the amyloid area exceeded 50-60 %. In Macaca mulatta the

progression of the metabolic deterioration was correlated to the islet morphology

present in autopsy biopsies [125]. Animals were divided into 4 different groups:

1;lean young monkeys, 2; monkeys > 10 years old, 3; monkeys with

normoglycaemia and hyperinsulinaemia and 4; diabetic animals. In group 3 the beta

cell volume was increased while group 4 animals had a reduced beta cell volume.

Amyloid deposits were present to a varying degree in 4 of 6 group 3 animals

replacing 0.03- 45 % of the islet mass. In the diabetic group amyloid was present in 8

of 8 animals and the affected area varied between 37-81 % of total islet area. In the

third study, performed on 150 baboons the metabolic state was correlated to the islet

amyloid mass and the result thereof showed that the levels of fasting plasma glucose

was sensitive and specific enough to determine the extent of amyloid [123]. This
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latter is different from studies on human where islet amyloid was significantly

associated with a higher mean HbA1c but not with fasting blood glucose levels [128].

     Islet amyloid does not develop in mouse or rat. This depends on the amino acid

composition and especially the three proline substitutions present at position 25, 28

and 29 in rodent IAPP are assumed to prevent amyloid aggregation [129]. In the

model for human IAPP fibril formation presented by Jaikaran et al. the regions made

up by residues 1 to17, 18 to 27 and 30 to 37 form strands that fold and form intra-

molecular beta-sheet structures while the residues at position 17-19 and 28 and 29

form the beta-turns. The presence of proline residues, which are known beta-strand

breakers, at position 28 and 29 will disrupt the structure and prevent fibril formation

(Figure 1) [130].

     CD analysis of human IAPP in monomeric form revealed mainly random coil

structure [131; 132], and NMR analysis on human IAPP and rat IAPP when bound to

membrane, showed alpha-helical content in the N-terminus [133].

     The presence of amyloid in the islets of Langerhans in the South American rodent

Octodon degus was surprising since the predicted IAPP sequence after cDNA

analysis from degu revealed a non-amyloidogenic IAPP sequence with protective

proline residues at position 28 and 29 [134]. Interestingly, an insulin sequence was

obtained when the degu islet amyloid was sequenced [135]. Degu insulin sequence

diverges from human and rat insulin at 32 out of 53 positions [134] and these

differences could result in a potentiated amyloidogeneity. The degu develops

diabetes when kept in captivity, and therefore, despite the different origin of the

amyloid in the islets of Langerhans in degu it points clearly to the importance for

amyloid in the islets.
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Amyloid in transgenic animal models

The original data on islet amyloid derive from studies performed on material

recovered post-mortem and we are still waiting for new methodology that will allow in

vivo studies on islet amyloid in humans. Meanwhile studies have been performed on

transgenic animals which have been very useful and facilitated a large number of

studies on IAPP cell toxicity and amyloid formation and allowed the exploration of

different pathways role in amyloidogenesis.

     Several transgenic mouse strains that express the human IAPP gene linked to the

rat insulin I or II promoter [136; 137], cDNA for human IAPP associate with the rat

insulin II promoter, cDNA for human IAPP linked to human insulin promoter [138]

and a transgenic rat strain expressing the cDNA encoding human IAPP driven by rat

insulin II promoter (HIP rat) have been established [139]. A strain that expresses

human IAPP, but made deficient for endogenous IAPP expression was made by

crossing a transgenic mouse with an IAPP deficient strain. Expression of the human

IAPP gene in the IAPP null mice ameliorated the defect insulin secretion detected in

this strain. Formation of amyloid caused solely by over-expression of human IAPP

was only found in one mouse strain [140]. In other strains, amyloid occurred in mice

fed a diet high fat [141; 142] after treatment with dexamethasone or growth hormone

[143] or when introduced into a diabetogenic trait [144]. In human IAPP transgenic

ob/ob mice the extensive IAPP production caused amyloid to form in parallel with the

development of insulin deficiency and persisting hyperglycaemia [144]. In the HIP rat,

over-expression of human IAPP lead to spontaneous development of hyperglycemia

in transgenic rats by the age of 4 months and overt diabetes was present in all rats

by the age of 10 months. In these animals the amyloid amount did not correlate to
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the fasting blood glucose. Instead a positive relationship between beta-cell apoptosis

and fasting blood glucose was reported.

     An earlier prerequisite in the definition of amyloid was that it should be present

extracellularly and this was also the main finding in the post-mortem material, often

affected with massive amyloid load and autolysis. However, some amyloid present in

insulinoma [145] and human islets transplanted to mice [146] appeared to be

present intracellularly. In transgenic mice or in cultured islets isolated from such

animals, it was shown that initial amyloid formation occurs intracellularly [140; 142;

147]. The amount of amyloid deposited in cultured islets was clearly dependent on

the glucose concentration.



Oligomers and cell toxicity

In some amyloid diseases it has been clear that the massive amyloid burden does

not always correlate to the clinical picture. Instead, the attention was drawn to the

fibril formation process and it was shown that aggregation to amyloid fibrils involves

formation of intermediates, and these oligomeric assemblies are ascribed the cell

toxic effect. The term oligomer is still a matter of debate. It does not define a

homogenous population of aggregates and the number of monomers varies.

Most of the results on oligomers arise from studies on A-beta, the amyloid protein

deposited in the Alzheimer brain where soluble oligomers have been implicated as

the toxic species, responsible for cell death [148] and [149]. When Lorenzo et al.

added mature IAPP fibrils to beta-cells in culture they detected apoptosis. With

today’s knowledge, it is most likely that oligomers were present in the solution and

the propagation of amyloid fibrils induced apoptosis [150]. The general mechanism is

supported by the existence of antibodies that recognize cell toxic oligomers
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independent of the nature of amyloid protein [151]. Different models for how

oligomers exert their toxic activity exist. An early finding [150] was that A-beta can

form ion leaking channels in lipid layers [152; 153]. Human IAPP was also shown to

form active channel structures while this was not seen by rat IAPP [154]. Atomic

force microscopy studies on channel structures suggest that the IAPP channel

consists of five IAPP molecules [155]. A second model for IAPP toxicity is membrane

permeabilization during fibril elongation [156; 157]. The N-terminal part of human and

rat IAPP contains alpha-helical structures and can interact with the membrane, but

only human IAPP can aggregate and form the amyloid fibrils that disrupt the

membrane. The result of this model fits well with the electron microscopical picture

on amyloid interaction with beta-cells (Figure 2).

     Being a secretory protein IAPP will after synthesis enter the secretory pathway

starting with the endoplasmic reticulum where the SS-bond is form and eventual

further folding is assisted by chaperons, transported to golgi and finally to the

secretory granule where the main part of the posttranslational processing occurs. The

mature proteins are stored in the secretory granules, waiting for secretion. If not used

the granule content will be degraded by crinophagy.

     Type 2 diabetes is often preceded by peripheral insulin resistance that is

compensated for by an increased insulin biosynthesis. This increase in the demand

on the secretory machinery in the beta-cells can cause endoplasmic reticulum (ER)-

stress which can induce apoptosis if not compensated by activation of the unfolding

protein response (UPR). The UPR response includes upregulation of ER-resided

chaperones to assist folding of aggregated proteins, a selective inhibition of protein

synthesis to reduce ER workload in favour for synthesis of proteins that augment

UPR and transport of mis-folded proteins to the ubiqutine-proteosome system (UPS)
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for degradation. It has been shown that over-expression of IAPP in cell lines and in

the HIP rat activates apoptosis and reduces the beta-cell number [139; 158]. A six-

fold increase of positive islet cell nuclei was detected in human pancreatic sections

from patients with type 2 diabetic subjects not present in non-obese or obese non-

diabetic patients [159]. The stress inducible transcription factor CHOP is present in

the ER and if activated during ER-stress, it will translocate to the cell nucleus. An

increased production of the ER-stress markers HSPA5, CHOP, DNAJC3 and BCL2-

associated X protein was detected in human pancreatic islets recovered from diabetic

subjects [160]. However, in this immunological study CHOP reactivity appeared to be

restricted to the cytosole without translocation to the cell nucleus.

     The association between human IAPP expression and ER stress induction is still

contradictory. Hull et al. failed to detect changes in the mRNA expression of the ER-

stress markers Bip, Atf4 and CHOP and splicing of Xbp1 mRNA in mouse islets

expressing human IAPP after culture in 11.1, 16.7 and 33.3 mmol/l glucose [161].

The islet amyloid that developed was associated with reduced beta-cell area in a

glucose- and time-dependent manner. In a recent paper from Peter Butlers research

team, where the commercially available oligomer antibody A11 was used, oligomers

were found intracellularly in human islets from patients with type 2 diabetes [162].

The oligomers disrupted the membranes of the secretory pathway and entered the

cytosol. Oligomers were also found in close association to mitochondria.



IAPP in the secretory granules

IAPP is present in the halo region of the secretory granules and a fibrilar material,

recognised by proIAPP specific antibodies are present in beta-cells affected by small

amounts of amyloid [163; 164]. When the intracellular amyloid mass expands,
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granule-sized aggregates fuse and replace the cytosol. Cells stained for intracellular

amyloid are also recognised by the apoptos-marker M-30 [164]. During the

hyperinsulinemic period that precedes diabetes there is an increase in secretion in

proinsulin and partially processed proinsulin (32-33 split proinsulin) [165; 166].

Because proIAPP is processed by the same convertases a similar change of

processing of proIAPP is expected with an increase in secretion of IAPP bound to the

N-terminal propeptide (N-IAPP). When human beta-cells were incubated in 20mmol/l

glucose the cellular content of insulin was decreased without a concomitant decrease

of IAPP resulting in a shift in IAPP to insulin ratio. Western blot analysis of cell

content showed a raise in proIAPP and an intermediate that in size corresponded to

N-IAPP [167]. Expression of human proIAPP in B-TC 6 cells that express PC2 and

PC1/3 and where proIAPP is expected to be processed into IAPP, failed to show

amyloid formation. Expression of proIAPP in GH4C1 cells that lack PC2 and PC1/3

or AtT-20 cells that lack PC1/3 and where aberrant processing of proIAPP occurs,

lead to amyloid formation [168].

     IAPP is known to be one of the most amyloidogenic peptides and is readily

assembled into amyloid fibrils, and the absence of fibrillar aggregates in the granules

during non-pathological condition raises the question if an endogenous inhibitor is

present in the secretory granule. It was shown that IAPP aggregation was in a

concentration dependent manner inhibited by insulin [32; 169]. Therefore, a change

in the intragranular milieu may be enough to facilitate aggregation of proIAPP/IAPP.

When the composition of endocrine granules was determined it was shown that

chaperones were present. This shows that assisted folding may be of importance

also at this site [170].
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     It is possible that two different ways exist for IAPP to reduce the beta cell number

in islets of Langerhans in patients with type 2 diabetes. One is through formation of

oligomers that induce ER-stress ultimately leading to apoptosis. Amyloid formation

has been suggested to primarily constitute a surviving pathway where formation of

fibrils is a way to neutralize toxic oligomers. However, intracellular growth of amyloid

which replaces the cytoplasm may also induce apoptosis.



Mutations in the IAPP gene and amyloid

Mutations in the IAPP gene occur both in the coding region and in the regulatory part

of the gene. The most studied mutation is the S20G, present in the Asian population

[171]. In a search for mutations within the coding region of IAPP, 294 patients with

type 2 diabetes were analysed and the S20G mutation was found in 4.1 %, but was

absent in the control group and in patients with type 1 diabetes. In a more

comprehensive study that included >1500 Japanese subjects with type 2 diabetes

the mutation was found in 2.6 % and it was concluded that IAPPS20G is linked to an

increased risk for the development of this disease [172]. A study performed on a

Chinese population identified the mutation in 2.6 % of the individuals with early-onset

type 2 diabetes but in none of the control subjects. Screening for the mutation in

other populations failed to identify the S20G variant [173].There is an increase in the

fibrillation propensity of S20G IAPP in vitro [174; 175] and expression of the mutant in

Cos-1 cells induced more apoptosis [175]. The in vitro findings indicate that S20G

may form more cell toxic amyloid in vivo. A gene promoter polymorphism in the

region -132 G/A of IAPP has been identified in a Spanish population. The frequency

of the G/A genotype was 9.7% in the studied 186 individuals with type 2 diabetes and

1.5% in the non-diabetic control group [176]. The presence of the mutation has been

shown to increase the basal transcriptional rate of the IAPP promoter [177]. This is
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interesting for the amyloidogenesis since over expression and increase of the

amyloid precursors is believed trigger amyloid formation. However, the search for the

promoter mutations in other countries have failed to show association to type 2

diabetes or islet amyloid load [178].



Importance of amyloid in transplanted islets

Impact of amyloid in transplanted human islets is a fairly new field. Islet

transplantation as a possible strategy to restore or improve the glucose homeostasis

in patients with type 1 diabetes was tried out already in the 1970s, but with low

success [179]. Despite major changes in e.g. islet isolation protocols, transplantation

procedure and immunesuppression regime few recipients remained insulin

independent 1 year after transplantation. Over the years many experimental

transplant studies have been performed with rat and mouse islets which are

protected against islet amyloid formation (see above). In a study from 1995, human

islets were implanted under the kidney capsule of nude mice which were either

normoglycaemic or made diabetic with alloxan [146]. The implants were recovered

after 2 weeks and, surprisingly, amyloid was detected in 16 out of 22 transplants

(73%) after Congo red staining or by immune electron microscopy. There was no

difference between diabetic and nondiabetic recipients. Further studies on

transplanted human islets showed that amyloid formation was not restricted to kidney

implants and amyloid developed to the same degree in human islets implanted to the

spleen or liver [180].

     Experimental studies with transgenic mouse islets, expressing human IAPP have

verified the findings. A graft containing 100 islets isolated from transgenic mice were

implanted under the kidney capsule on mice with streptozotocin induced diabetes.
22

The graft was sufficient for adjusting the blood glucose level, but over the 6 following

weeks an increase in plasma glucose concentration was detected but was not seen

in mice transplanted with non-transgenic mouse islets. The implants were recovered

after 6 weeks and amyloid was found in 92 % of the transplants with transgenic islets

and the beta-cell volume was reduced by 30% [181].

     Studies in human material have of natural reasons been very limited. We have,

hovever, studied the amyloid content in human islets implanted to the liver of a type 1

diabetic man, dying from a myocardial infarction [182]. The recipient received three

different grafts and was off insulin treatment for a period between transplantations.

Amyloid was found in about 50 % of the islets identified in the liver. This finding

clearly points to amyloid as important factor for loss of graft survival. Is it possible to

extend the survival of transplanted islets? Marzban et.al. reduced the proIAPP

expression by 75% through the introduction of short interference (si) RNA in human

islets kept in culture [183]. The reduction of proIAPP synthesis reduced the amyloid

load by 63% in islets cultured for 10 days. The results indicate that the proIAPP

synthesis most likely must be abolished if amyloid formation should be prevented.



Conclusion
Taken together the results from the animal studies show that amyloid develops
before beta-cell deficiency and the occurrence of oligomers and amyloid intracellular
induce beta cell death. Prevention of islet amyloid will save beta-cells and extend
hormone secretion.


Acknowledgements
I thank Per Westermark for valuable suggestions. Supported by The Swedish Research
Council, the European Framework 6 Program to EURAMY, the Swedish Diabetes
Association and Family Ernfors Fund.
23




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