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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.
Key Words: Islet amyloid; Islet amyloid polypeptide; IAPP; ProIAPP, ER-Stress;
Apoptosis; Posttranslational processing.
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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 (7-10), 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,
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originally described in brain, is also a regulator of glucagon expression in alpha-cells
(17).
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 enterochromaffine 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 dibasic 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 Cterminally 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 (29-31) . 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 (32-34). 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
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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
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, 43-46). 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 (49-52). 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
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IAPP belongs to the calcitonin family of peptides also including calcitonin (57),
calcitonin gene-related peptides (CGRP) (58), adrenomedullin (59) and intermedin
(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
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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 (69-71). Glucose stimulated insulin secretion from perfused rat 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.
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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, shortterm 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
long-term exposure. IAPP can regulate glycogen synthesis by activation of glycogen
phosphorylase and inactivation of glycogen synthase (77-79), 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 (86-88). 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
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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
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 (93-96) and peripherally (97-99)
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.
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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
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 (106-108) 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 wild type 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),
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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
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
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believed to cause the specific tinctorial characteristic of amyloid, affinity for Congo
red and green birefringence in cross polarized light.
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%
(118-122). 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
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insulin treatment all had islet amyloid (119). This points to an association between
the severity of the disease and the prevalence of islet amyloid .
In addition to man, islet amyloid occurs in primates (123-125) 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
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to determine the extent of amyloid (123). This latter is different from studies on
human where islet amyloid was significantly associated with a higher mean HbA 1c 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 intramolecular 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 sixfold 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 nondiabetic 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 BCL2associated 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 ERstress 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).
Genome-wide associated (GWA) studies performed in Caucasian (179, 180) and in
Han Chinese (181) populations have until know identified 20 different polymorphisms
with shifting associations to type 2 diabetes (T2D), and hitherto, neither of the
pinpointed loci include IAPP.
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 (182). 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
22
implants and amyloid developed to the same degree in human islets implanted to the
spleen or liver (183).
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.
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% (184).
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 (185). 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 (186). 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.
23
32-33 split proinsulin, 19
AC187, 9
adrenomedullin, 6
alpha-cell, 4
amylin, 1, 3, 24
amyloid fibrils, 12, 16, 19
anorectic effects, 9
anorexia nervosa, 10
AP/NST, 9
apoptosis, 16, 17, 20
autocrine, 7
beta-cell, 2, 4, 5, 16, 17, 18, 22, 24
beta-sheet structures, 14
bone, 10
brain, 3, 6, 9, 10, 16
brain-4, 3
Brochmann body, 6
calcitonin, 3, 6, 24
cat, 8, 24
chaperones, 17, 19
chicken, 6,
CHOP, 18
clearance of IAPP, 5
C-peptide, 4
crinophagy,. 17
dexamethasone, 15
diabetes mellitus,, 8
dog, 8
enteroendocrine cells, 6
ER-stress, 18, 20
fish, 3, 6
gastric emptying, 8
gastrin, 6
gastrointestinal tract, 6
glycogen phosphorylase, 8,
glycogen synthase, 8
growth hormone, 15
HbA1c, 11, 14
HSPA5, 18
hyperglycemia, 15,
hyperinsulinemic, 19
hypothalamus, 10
IAPP, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15,
16, 17, 18, 19, 20, 21, 22, 24
IAPP gene, 3, 5, 15, 20
IAPP in the secretory granule, 18
insulin, 3, 4, 5, 6, 7, 8, 11, 13, 14, 15, 17,
19, 21, 22
intermedin, 6
islet amyloid, 1, 2, 12, 13, 14, 15, 18, 21,
24
islet amyloid polypeptide, 1, 24
Macaca mulatta, 13
Macaca nigra, 13
misfolding diseases, 11
monkey, 9, 29
mouse, 6, 9, 14, 15, 18, 21, 22
N-IAPP, 19
Octodon degu, 14
oligomers, 2, 16, 18, 20, 24
osteoblasts, 10
osteoclasts, 10
paracrine, 7
PC1/3, 3, 4, 19
PC2, 4, 19
pdx-1, 3
peptide YY, 6
plasma levels of IAPP, 4
postprandial glucose, 8
posttranslational processing, 17
Pramlintide/symlin, 10
preproIAPP, 5
primates, 13
proIAPP, 4, 18, 19, 22
proinsulin, 19
proline substitutions, 10, 14
rat, 6, 7, 8, 9, 10, 14, 15, 17, 18, 21
regulation of food intake, 10
S20G, 20
satiety factor, 9
secretory granules, 4, 17, 18
short interference (si) RNA, 22
somatostatin, 5, 6
streptozotocin, 22
transgenic mouse, 15, 22
transplanted islets, 21, 22
type 1 diabetes, 8, 20, 21
type 2 diabetes, 2, 3, 11, 12, 13, 18, 20,
21, 24
UPR, 17
24
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.