Islet Amyloid: An Enigma of Type 2 Diabetes Anne Clark

Diabetes Research Laboratories, Radcliffe lnfirmary and Department of Human Anatomy, University of Oxford, U.K.

1. INTRODUCTION

Pancreatic islets have only recently reemerged as a site of a causative pathology in type 2 diabetes as a result of the identification in 1987-1988 of the peptide which is the principal component of islet amyloid.’,2 Early in the twentieth century, histopathological studies identified pancreatic lesions in d i a b e t e ~and ,~~ more than 30 years before the biochemical identification of two separate forms of diabetes, a far-sighted statement by Wright in 1927 described islet amyloid (hyalinosis) as a possible diagnostic feature.6 He suggested that ”insular changes. . . in younger individuals consist chiefly of inflammatory lesions,. . . hyaline change is characteristic of a group of cases in which the duration of the disease has been of some length,. . . and in which the patients are middle aged or older.” Since that time, islet amyloid has been shown unequivocally to be an important diagnostic feature for type 2 diabetes at postmortem, but the roles of the islet deposits and of the hormonal action of the amyloid peptide in the onset and progression of the disease are still unclear.

11. WHAT IS AMYLOID? Amyloid is the name given to amorphous masses of fibrils found in the body tissues Addressee for correspondence: Anne Clark, Diabetes Research Laboratories,Radcliffe Infirmary, Woodstock Road, Oxford OX3 6HE, U.K.

which have characteristic histological staining properties. The cotton dye Congo red binds to the fibrils, staining them pink. When viewed in polarized light, the stained fibrils exhibit a green/yellow birefringence due to the regular repeating pattern of Congo red binding sites.7 The term amyloid was coined in 1853 by Virchow, who found that amyloid deposits stained black with iodine, suggesting a “starchlike” material. This staining characteristic results both from polyiodide complexes which attach to the fibrils and from glycosylated compounds which are an integral part of amyloid deposits.8 However, the major fibrillar component is proteinaceous and each amyloid dyscrasia is characterized by a specific ~ e p t i d e Systemic .~ amyloidoses are commonly related to a generalized systemic disease. For example, in chronic inflammation, fibrils are formed from serum amyloid A protein,1° whereas in localized amyloidoses, deposits are restricted to the site of origin of the component peptide. Furthermore, amyloid which develops in medullary carcinoma of the thyroid is restricted to the thyroid and derived from calcitonin-related peptides;” Alzheimer’s amyloid in the brain is formed from A4 protein and found only in the brain;I2 islet amyloid is found only in the pancreas and is formed from islet amyloid polypeptide (IAPP), a peptide component of the islet B - ~ e l l s . ’ ~ , ’ ~ Amyloid fibrils are formed from two or more twisted filaments composed of stacked sheets of the peptide in a P-pleated conformation held together by hydrogen bonding. This structure confers a considerable degree of insolubility to the amyloid deposits.

Diabetes/Metabolism Reviews, Vol. 8, No. 2, 117-132 (1992)

0 1992 by John Wiley & Sons, Ltd.

07424221/92/020117- 16$8.00

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111. IS ISLET AMYLOID SPECIFIC FOR TYPE 2 DIABETES?

have untested glucose intolerance. Furthermore, islet amyloid deposits are not uniformly distributed throughout the organ and therefore multiple tissue sampling is The much quoted papers of Opie'5,'6 reported Islet amyloid is a characteristic of type 2 pathological "hyalinosis" in the pancreas of two diabetes in every ethnic group examined to date, diabetic patients. Although one subject was a including Pima Indians,25 Asian Indians,2h and middle-aged patient with a history of glycosuria, the Japanese.27 Furthermore, islet amyloid has the second subject was only 17 years old and been identified in older patients with diabetes post-morterm autolysis was so far advanced as to arising secondary to cystic f i b r o ~ i s . ' ~Extensive ,~~ obscure definition of the i ~ 1 e t s . lMore ~ rigorous amyloid deposits are found in many cases of large post-mortem studies showed the association polypeptide-producing tumours including insuof islet hyalinosis with d i a b e t e ~ . ~ ~BelP7 ,'~,~~ l i n ~ m a s . ~A ~ , peptide ~' component common to reported that the prevalence of islet amyloid was both islet and insulinoma amyloid was proposed greater than 40% in more than 1000 diabetic from early histochemical studies31 and subpatients over the age of 40 compared with 8 % in sequently identified as islet amyloid polypeptide. an age-matched non-diabetic group. However, this group of diabetic patients must have included some individuals who would today be classified as type 1 diabetic subjects, since diagnosis was IV. WHAT IS ISLET AMYLOID based only on the results of a urinary glucose POLYPEPTIDE? test. The reported prevalence of islet amyloid in post-mortem studies is very variable-between 25 and 92% in type 2 diabetic patients over the Histochemical and biochemical studies pionage of 40 years.1a22 However, in all reports, the eered by Westermark have shown that the amyloid occurrence of amyloid is less (between 0 and of islets and insulinomas differs biochemically 54%) in age-matched non-diabetic subjects'a22 from that formed in other systemic amyloidoses, (Figure 1). Accurate diagnosis of patients and being insoluble in distilled water and most organic solvents and lacking tryptophan.32 In sampling of pancreatic tissue are of critical 1986-1987 a novel peptide was extracted from importance, since many elderly patients dying in pancreatic tissue of type 2 diabetic patients and hospital of cardiovascular-related disease may

0Non-diabetic

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40

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Figure 1. Prevalence of islet amyloid in post-mortem pancreatic specimens from diabetic (B) and non-diabetic (Cl) subjects in different ethnic groups. The numbers of patients examined are indicated above the histograms. Data derived from published studies, refs 1&23 (Caucasian), 27 (Japanese), 26 (Asian), and 25 (Pima Indian).

ISLET AMYLOID: AN ENIGMA OF TYPE 2 DIABETES

from an insulinoma and shown to be composed This , ~ ~discovery was made of 37 amino a ~ i d s . ' ~ by two separate groups in Sweden and the U.K. with the consequent result of multiple, and sometimes confusing, terminology for the same peptide: diabetes associated peptide (DAP), insulinoma amyloid polypeptide, islet amyloid polypeptide (IAPP), and "amylin". Subsequently, the terms IAPP and "amylin" have been retained and IAPP will be used throughout this review. Human IAPP (hIAPP) has a 46% structural homology with human calcitonin gene-related peptide (hCGRP-2), a neuropeptide produced largely by the C-cells of the t h y r ~ i d , ' ~the ,~~ homologous sequences being largely situated at the N- and C-terminal ends of the peptide. Although there are some similarities with insulin, particularly the N-terminal Cys bridge,35 IAPP has a closer association with the CGRP family of peptides than with insulin. The solubility of synthetic IAPP is low due to the primary and secondary structure of the peptide. The central region of hIAPP, particularly residues 20-24 (SNNFG) (Figure Z), is more hydrophobic than the comparable region ( G G W K ) of the more soluble CGRP.36 Secondary

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structure predictions from circular dichroism spectroscopy data indicate that the hCGRP-1 molecule contains a greater OL helical component than IAPP but similar amounts of the p protein c o n f ~ r m a t i o n The . ~ ~ amount of p structure is an important factor in the amyloidogenic potential of a peptide: secondary structure predictions indicate that over 50% of the molecular structure of the amyloidogenic proteins tra n ~ th y r e tin ~' and P 2 - m i ~ r o g l o b u l i nis~ ~in the form of a P-pleated sheet. Aggregation of IAPP in vivo is critically dependent on the amino acid sequence of IAPP2(r2q, which varies between specie^.^^,^^ IAPP has been identified in every species examined to date: in the monkey, cat,44hamster,4scougar,"rat, mouse, g ~ i n e a - p i g , and ~ ~ -degu5I ~ ~ (Figure 2). The sequence of IAPP,-,, is highly conserved with a minimum of 78% homology present in the guinea pig and degu (Figure 2). IAPP also aggregates to form fibrils in vitro, a feature shared by other amyloidogenic proteins including residues 18-28 of the Alzheimer A4 p r ~ t e i n . ~The ' sequence Ala-Ile-Leu-Ser (hIAPP,,,,) appears to be critical , ~ ~ sequence is for fibrillogenesis in v i f r ~ . * ~This also important for amyloid formation in vivo since

NH2- terminal Signal peptide

propep t ide

CKIH-terminal I s l e t amyloid polypeptide

propeptide 37

p-pleated sheet forming region Figure 2. Amino acid sequence of the islet amyloid precursor protein, prepro-IAPP, in nine species of mammal. Whereas prepro-IAPP contains 89 amino acids in man, monkey, cat and dog, other species have 91-93 amino acids. The amino acid sequence which is critical for fibrillogenesis, hIAPP24_28(GAILS), is conserved in species in which amyloid develops in islets or insulinomas (human, cat, and dog). Data derived from refs 42-51. Amino acids are indicated by a single letter code: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; Y, tyrosine.

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in embryologial d e ~ e l o p m e n tIn . ~ addition, ~ islets it is common to man, domestic cat, and the cougar containing amyloid may be restricted to one or (Figure 2), which are all species in which diabetes two exocrine lobules. A lobular distribution of develops later in life. Diabetes-related amyloid pathological change is also a characteristic of the also occurs in older non-human primate^.^^,^^ early phases of autoimmune destruction of B-cells IAPP in macaque monkeys (Macaca nemestrina) in type 1 diabetes.59 Why this should occur is differs from hIAPP and feline IAPP in having unclear, but islets originating from different threonine at position 25,42 but since islet amyloid is present in other closely related macaque pancreatic duct stem cells could have different species (Macaca mufatta and Macaca n i g ~ a ) ~this ~ , ~ ~ ,susceptibilities to pathological processes. substitution is unlikely to affect the potential for amyloid formation. Residue 25 is proline in mice, B. Does Amyloid Originate Inside or rats, and hamsters, and Westermark et aL40 have Outside the B-Cells? suggested that this particular residue is a critical determinant for fibrillogenesis. However, analysis Electron microscopy has demonstrated that the margins of the B-cells adjacent to the amyloid of canine IAPP has shown that the amino acid show invaginations filled with fibrils.22,h"The sequence cannot be the only determining factor for islet amyloid formation: whereas the feline marginal B-cell plasma membrane is not clearly and canine IAPP2P29 sequence is identical, islet defined in the region of these invaginations, and amyloid is associated with diabetes in the cat cellular debris, including apparently isolated pockets of cytoplasm and insulin granules, are but not in the dog, even though amyloid formed from IAPP is present in some canine present within the amyloid masses (Figure 3b)." i n s ~ l i n o m a s . This ~ ~ , ~suggests ~ that in addition These observations suggest that amyloid could be formed at an intracellular site; accumulation to the IAPP sequence, physiological and/or biochemical factors, possibly determined by of insoluble IAPP within the cell could disrupt genetic or temporal features, are involved in the normal metabolism and lead to B-cell death amyloid formation. resulting in apparently extracellular masses of amyloid. Further support for this hypothesis comes from the finding of immunoreactive amyloid fibrils, apparently within the B-cell, in islets V. WHERE ARE AMYLOID AND IAPP of diabetic cats62and in human insulinoma tissue FOUND? (Figure 4).63In addition, many B-cells in amyloidcontaining islets show degenerative changes. A. Amyloid Deposits in the Pancreas IAPP is a normal constituent of B-cells in nondiabetic and diabetic individuals, where it is Islet amyloid deposits are situated within the boundaries of the islet between the endocrine located in insulin granules, and lysosomes.64~65 cells and islet capillaries and show immunoreactivThe highest density of labelling in human and ity for IAPP (Figure 3a). Islet amyloid in the South monkey B-cells was found in secondary lysosomes and in lipofuscin bodies, which are storage American rodent, the degu, is formed from insulin rather than from IAPP.s6 Islet amyloid, in common organelles for insoluble, partially degraded material. If polymerization of IAPP into fibrils with other forms of amyloid, contains other substances: notably, heparan sulphate and amyoccurred as a result of accumulations of the loid P protein. The role of these amyloidophilic peptide, lipofuscin bodies would represent a potential intracellular site for the initiation of compounds in the process of deposition of the amyloidosis. Abnormal cleavage of IAPP,_,, from fibrils is not ~ l e a r . Calcification, ~~,~~ in the form the N-terminal flanking peptide has been proof calcium phosphate crystals, is often associated posed in amyloidosis" but there is no evidence with extensive amyloid deposits, particularly those in monkey and cats. for involvement of the C-terminal flanking peptide The distribution of amyloid is far from in amy10id.~~ uniform throughout all of the islets of the pancreas, which makes accurate identification of C. IAPP in Non-8-Cells islet amyloidosis a tedious procedure. Amyloid has not been found in islets in the head of the Although IAPP is localized to B-cells in the human pancreas which contain few B-cells and pancreas of adult cats, monkeys, and man, which are derived from the ventral primordium expression during development (and possibly

ISLET AMYLOID: AN ENIGMA OF TYPE 2 DIABETES

121 ^---

-_I

- -

_ I.L

~ " -

Figure 3. Amyloid deposits in islets of type 2 diabetic patients showing immunoreactivity for IAPP. (a) Immunoperoxidase-labelled amyloid deposits (arrows) within the boundaries of the islet. Amyloid is located as large deposits in the centre of the islet or adjacent to islet capillaries (arrow-heads). Scale bar = 100 pm. (b) Immunogold-labelled islet amyloid (AM) adjacent to an insulin-containing B-cell (B) and a glucagon-containing Acell (A). Cellular debris (C) including nuclei (N) and granules (I) appear to be engulfed by the amyloid. Scale bar = 1.0 pm.(c) Immunogold-labelled (anti-IAPP) islet amyloid fibrils in extracellular islet deposits. Scale bar = 0.1 pm.

during ontogeny) may not be so specific. During human foetal development, IAPP appears in Bcells at 10 weeks' gestation together with insulin.68 However, IAPP and insulin expression may be less tightly linked in foetal endocrine cells than in adult tissue: immunoreactivity has been identified in pluripotent foetal islet cells6' and IAPP can be co-expressed with glucagon (A-cells) or somatostatin (D-cells) in cell lines transformed from neonatal rat islet cells.70 Furthermore, IAPP has been identified in the stomach and duodenum of man and the rat; mRNA IAPP in extracts of rat stomach were approximately 5% of that found in the pancreas of and cells immunoreactive for IAPP but not for insulin have been localized to the mucosa of the pyloric antrum in rat and man.71

VI. IS ISLET AMYLOID A PRIMARY OR A SECONDARY FEATURE OF DIABETES? Diabetes-associated amyloid is found only in humans, non-human primates, and cats.7' Islet amyloid is not found in rodent models of obesity and diabetes, even though the physiological parameters may be similar to those of the syndrome of type 2 diabetes in man. Amyloid deposits can be detected before the onset of hyperglycaemia in the course of development of spontaneous diabetes in ~ a t sand ~ ~monkey^.^' , ~ ~ Whereas the amyloid deposits in the "prediabetic" period are relatively small and restricted to a few islets, the degree of amyloid formation and the number of islets affected increase with

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Figure 4. Fibrils immunoreactive for IAPP within B-cells of human insulinomas. (a) Radiating circular pattern of fibrils (arrows) within the cytoplasm adjacent to insulin granules (I), but within the cell membrane (CM). Scale bar = 1.0 pm. (b) Parallel arrays of immunogold-labelled fibrils not enclosed by detectable membranes (arrows). I = Insulin granule; M = Mitochondrion. Scale bar = 1.0 pm.

the progression of symptoms which lead to d i a b e t e ~ . The ~ ~ ,onset ~ ~ of hyperglycaemia could be closely related to the degree of B-cell loss which correlates with the progressive increase in amyloid deposition (Figure 5a). In man, it is not possible to study such a progression of islet pathology. The extent of islet amyloid formation at post-mortem in type 2 diabetic subjects who had received diet or oral hypoglycaemic agent therapy is very variable: these patients have less than 5% or more than 60% of islets affected with minimal reduction in islet cell population.zz Although the amyloid is located between the islet cells and capillaries in affected islets, it seems unlikely that this could seriously disrupt total pancreatic insulin production. However, if amyloid formation is initiated intracellularly, this could have a considerable effect on the normal function of the B-cell. In severe islet amyloidosis in type 2 diabetic

man, amyloid deposits can be found in up to 90% of the pancreatic islets, occupying up to 80% of the islet spacezz and in diabetic cats and monkeys, 60-100% of islets are affected.7677 In this severe pathological state, the few remaining islet cells are completely enclosed within the mass of amyloid (Figure 5b). The time course and extent of glucose stimuli and the resulting insulin release in such conditions are likely to be severely compromised by the physical barrier between the islet cells and capillaries. In spontaneously diabetic monkeys, fasting insulin levels are closely correlated with the proportion of the islet occupied by amy10id.~~ Furthermore, in type 2 diabetic man, the extent of islet amyloidosis has been shown to relate to the severity of the diabetic symptoms as defined by the need for insulin therapy.” Thus, is is likely that, once initiated, polymerization of IAPP to form fibrils continues over periods of years, causing progressive deposition of islet amyloid. This clinical undetectable process dis-

-

ISLET AMYLOID: AN ENIGMA OF TYPE 2 DIABETES Islet

123 Islet Amyloid %

B-cell %

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* * * * * * diabetic *** **

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Figure 5. Islet amyloid and 8-cells in islets of spontaneously diabetic monkeys. (a) Morphometric data of the proportion of the islet occupied by amyloid ( 1 and B-cells (W in post-mortem tissue of 26 Macaca mulutta monkeys examined at different stages in progression to hyperglycaemia. Amyloid deposits (*) were found in some islets of 3/9 obese animals older than 12 years, 4/6 obese hyperinsulinaemic animals, and in all islets of 8/8 diabetic monkeys. B-cells occupied 4.540% of the islet area in non-diabetic animals but this area was reduced to less than 10% in most diabetic animals. (b) Pancreatic islet from a diabetic monkey. B-cells, immunoperoxidase labelled for insulin, (arrows) are severely reduced in number and enclosed in amyloid (arrow-heads). Scale bar = 100 pm. (c) Adjacent histological section immunoperoxidase-labelled for IAPP. Large amyloid deposits (arrow-heads) which occupy most of the islet area could severely compromise insulin secretion. (Studies in collaboration with Barbara Hansen, Baltimore USA.)

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rupts the islets and destroys the B-cells, resulting in diminishing islet function.

VII. IS THERE A GENETIC DEFECT IN ISLET AMYLOID POLYPEPTIDE IN DIABETES? Type 2 diabetes is a familial disease with a high concordance rate in identical twins.78 Although the glucokinase gene has been implicated in mild beta-cell d y ~ f u n c t i o nthe , ~ ~genetic basis of more severe type 2 diabetes has not been identified. The discovery of IAPP and the high prevalence of islet amyloid in type 2 diabetes offered an attractive candidate for a genetic defect. Following the identification of the amino acid sequence of the amyloid peptide, the cDNA encoding IAPP was determined from a h phage library of human genomic DNA and from an insulinoma cDNA library.80,8' The 37-amino acid peptide is derived from a larger precursor of 89 amino acids in man (molecular weight 9808 D).83 The hIAPP gene consists of three exons: exon 1 (104 bp) encodes most of the 5' untranslated part of the mRNA; exon 2 (95 bp) encodes 15 untranslated nucleotides, the signal sequence (22 residues), and the first nine amino acids of the N-terminal flanking peptide (N-IAPP); and exon 3 (1059 bp) encodes the dibasic processing site between N-IAPP and IAPPI-37, the C-terminal peptide, and the remainder of the 3' untranslated region (Figure 2).80-83 At least two sites for polyadenylation of the hIAPP and mRNA have been identified in extracts of human pancreas and i n s u l i n ~ m a . ~ ~ The IAPP gene has been located as a single copy gene on the short arm of chromosome 12.81,83 This contrasts with the related CALC-1,CALC-11, and CALC-I11 genes encoding CGRP and calcitonin which are assigned to chromosome 11. However, chromosomes 11 and 12 are thought to be related in evolution, and a possible common ancestral gene for IAPP, CGRP, and calcitonin has been proposed.84 Compared with IAPP, the CALC genes show a greater degree of complexity with five or six exons and alternative RNA processing in the CALC-I gene. Other forms of amyloid disease are associated with genetic abnormalities which result in single or multiple amino acid substitutions in the protein that forms the a m y l ~ i d . ~The ~ . ~amino ~ acid sequence for hIAPP deduced from cDNA was

identical to the peptide structure identified in extracts from a type 2 diabetic pancreas and from an i n ~ u l i n o m a , ' ~suggesting ,~~ that there is no causative structural change. Although abnormalities of the IAPP gene would be a convenient explanation for type 2 diabetes, extensive analyses have shown no variations in cDNA in diabetes: IAPP cDNA was examined in 25 well-defined type 2 diabetic individuals and found to be identical in each case, with the same encoding sequence on both alleles which matched that identified in the genomic library.87 Sequence polymorphisms do exist in the hIAPP cDNAX8 and these have been informative for linkage studies. It has been suggested that sequence polymorphisms are present in the 3' untranslated region of the gene.82 A population study of 88 type 2 diabetic and 67 non-diabetic age-matched subjects showed no linkage between diabetes and IAPP cDNA restriction fragment length p o l y r n o r p h i ~ m sAlthough .~~ subtle changes in the untranslated region cannot be excluded, it now seems unlikely that an abnormal IAPP gene plays a major role in the aetiology of type 2 diabetes. Whereas the nucleotide sequences in the IAPP,_,, domain show a high degree of conservation, there are considerable differences in the regions of the N- and C-flanking peptides."&" In man and cat, the N-terminal propeptide consists of 11 residues, but in the rat, mouse, and guineapig, three additional residues are encoded by nucleotides at the junction of exon 2 and the adjacent intron. Sizes of mRNA transcripts which have been reported indicate variation among species, suggesting that there may be multiple sites for initiation of transcription and/or termination or, possibly, alternative RNA splicing.8283 Studies on the transcriptional control of the human IAPP gene have identified a mechanism of negative control.93 Transcription of IAPP and insulin may be regulated by similar mechanisms. However, to date, quite extensive analyses of the IAPP gene have not provided any information which can explain islet amyloid formation.

VIII. IS ISLET AMYLOID FORMED AS A RESULT OF OVERPRODUCTION OF IAPP? Expression of mRNA and secretion of IAPP and insulin are closely linked in physiological responses to B-cell secretagogues and in animal

ISLET AMYLOID: A N ENIGMA OF TYPE 2 DIABETES

models of B-cell dysfunction. In the rat pancreas, the amount of pancreatic IAPP mRNA is approximately 5-10% that of mRNA for insulin and is reduced in parallel with insulin in streptozotocininduced diabetes, h y p ~ g l y c a e m i a , ~and ~ , ~ in ~,~~ spontaneously diabetic (BB) Wistar rats.95 IAPP is co-secreted with insulin in response to B-cell secretagogues. Studies on B-cells, islets and pancreas in vitro have indicated that, under most conditions, changes in IAPP secretion parallel changes in insulin secretion and represent 1-20% of the insulin production on a molar In non-diabetic man and animals, in U I Z I O plasma IAPP concentrations are low in comparison with insulin: basal levels in man vary from 2 to 13 pmol/l and are elevated in parallel with insulin secretion to 5-17 pmol/l after a glucose load (Figure 6).'00-106 Obese individuals in whom basal and stimulated insulin levels are elevated have proportionally higher levels of IAPP compared with normal-weight subject^.^^^-"^ However, there is no evidence for elevated or reduced basal or stimulated IAPP concentrations in type 2 diabetic subjects or in subjects with impaired glucose tolerance who may be at risk of developing diabetes. The highest reported plasma concentrations of IAPP (25 pmol/l) occurred in patients with chronic renal insufficiency. Following dialysis, the IAPP levels in these patients were reduced by SO%, suggesting that, like C-peptide, IAPP is normally excreted via the kidney.l" There are considerable differences in the reported absolute levels (Figure 6) and the molar ratio of secreted IAPP/insulin, and this area continues to be controversial since elevated IAPP secretion in the prediabetic period or in diabetes could contribute to amyloid formation. The measurement of IAPP in plasma samples by radioimmunoassay is difficult. The iodinated IAPP tracer is relatively unstable and the low plasma concentrations of IAPP require that plasma samples be concentrated and extracted before assay.'12 Furthermore, more than one molecular form of IAPP has been detected in the circulation: both intact IAPPl-,7 and a shorter form, IAPP17-37,have been identified, in variable ratios, in rat and human p1a~rna.l'~ If amyloid formation was caused by overexpression of IAPP, elevated IAPP concentrations would be expected not only in the islets but also in the general circulation, where the IAPP concentration would be dependent on both secretion and clearance. The lack of elevated circulating IAPP levels in type 2 diabetic subjects

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with or without insulin resistance suggests that plasma levels of IAPP, as measured by the present methods, are not robust markers for amyloid formation. In non-diabetic patients with endstage renal failure, which is associated with elevated plasma concentrations of IAPP,"' there is a higher prevalence of islet amyloid compared with age-matched non-diabetic subjects without renal disease.l13 This could result from the higher than normal plasma IAPP concentration in the islet circulation, undiagnosed prediabetes, or the insulin resistance that often accompanies renal insufficiency. Secretion of incompletely processed B-cell products in the form of proinsulin have been implicated in the pathophysiology of type 2 diabete~,"~ and has been associated with islet amyloidosis in one patient.115

IX. WHAT IS THE FUNCTION OF IAPP? A. Peripheral Effects The C-terminal amidation of IAPP suggests that it has endocrine activity, and effects of IAPP have been studied in a variety of tissues, most notably, skeletal muscle."~"2" High concentrations of IAPP have been shown to reduce insulin-stimulated uptake of glucose and incorporation of glucose into glycogen, and to stimulate production of phosphorylase-a in isolated human and rat skeletal muscle strips.l2' It was therefore proposed that IAPP acts to modulate insulin resistance and, as such, would be a significant factor in the decreased insulin sensitivity of obesity and type 2 diabetes. However, confirmation of the in vitro findings in intact animals and in man has not been universal. Concentrations of IAPP required to inhibit insulin action on skeletal muscle in vitro range between 10 and 100 nM. Infusions of high concentrations of IAPP significantly reduced insulin-dependent disposal of a glucose load and suppressed hepatic glucose output in some experiments and showed no effects in Ho wever, lower plasma concentrations within the range of measured IAPP levels in man (5-50 pM) showed no effects on glucose handling in intact rats or dogs.'22,123,12x It has been suggested that effects of IAPP on glucose metabolism are critically dependent on the molecular conformation (intact cysteine bridge and C-terminal amidation) of the synthetic peptide and that these factors are not necessarily reflected in the measurements of plasma IAPP. However,

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st i mu1at ed

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la n 15 0

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Figure 6. Measurements of plasma IAPP concentrations (pmol/l) in man under basal conditions and peak values following an oral glucose tolerance test (stimulated). Data are derived from seven published studies: N = ref. 103; H = ref. 109; B = ref. 105; M = ref. 104; L = ref. 108; S = ref.107; J = ref.106. + = Non-diabetic patients; 0 = type 1 diabetic subjects; 0 = type 2 diabetic subjects, diet or oral therapy; = type 2 diabetic subjects, insulin therapy; A = obese, normal glucose tolerance test; A = obese glucose-intolerant non-diabetic patients. There is an increase in circulating IAPP levels following glucose stimulation, but no clear indication of elevated basal concentrations in any of the patient groups examined.

even with intact IAPP.NH2, concentrations of 10 nM are required to induce effects in vitro,"* whereas the highest concentration recorded in vivo is 25 pmol/l.lll CGRP and IAPP have similar biological effects and both peptides act via CGRPbinding sites in skeletal muscle and hepatic cells'29 possibly involving activation of adenylate c y ~ 1 a s e . The l ~ ~ hypothesis of a specific peripheral action of IAPP on carbohydrate metabolism in diabetes therefore remains attractive, but unproven. An action of IAPP on calcium metabolism has been proposed because infusions of IAPP decrease plasma calcium concentration^.^^^

IAPP also affects bone resorption, although, compared with calcitonin, IAPP is 10 to 100 times less potent in this r e ~ p e c t . ' ~ l , l ~ ~ B. Effects within the islet CGRP affects insulin and glucagon secretion under various physiological conditions in rats and mice,133,134 pigs,13' and dogs.136It is therefore possible that the structurally homologous IAPP molecule could act as a paracrine regulator within the islets. Although high concentrations of IAPP (10-500 pM) inhibit insulin secretion, IAPP concen-

ISLET AMYLOID: AN ENIGMA OF TYPE 2 DIABETES

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trations less than 5 p M failed to reduce stimulated insulin production from either isolated rat islets137 or the more sensitive perfused pancreas preparation or in intact Furthermore, 10 pM amidated IAPP had no effect on proinsulin biosynthesi~.'~~ A paracrine/autocrine regulatory role for IAPP within the islet is equally attractive but remains unproven. IAPP partially shares the potent vasodilator properties of CGRP,143,144and CGRP immunoreactive nerves are present in pancreatic i ~ 1 e t s . A I ~possibility ~ therefore exists that IAPP, co-secreted with insulin, and CGRP from nerves could facilitate insulin delivery to the circulation by dilatation of vessels in the islets or pancreas.

remains a marker for an unidentified dysfunction of 8-cells associated with type 2 diabetes but may also provide valuable information about the clinical progression of the disease to insulin requirement. To understand the genetically determined abnormalities which result in a predisposition for type 2 diabetes we need to identify the physiological and biochemical factors involved in the accumulation of IAPP as islet amyloid.

X. CONCLUSION

The physiological function of IAPP remains unresolved partially since its effects and the receptors specific for IAPP have yet to be identified. The proposal to regulate hepatic glycogen stores in type l diabetes by combined therapeutic use of IAPP and insulin'46 may be premature whilst a physiological role for IAPP remains unclear. However, even though the cause of islet amyloid formation remains unknown, the effects of the deposits in the islets are clearly associated with decreased islet function and, in severe amyloidosis, destruction of 8-cells. In sumary, islet amyloid is a characteristic feature of type 2 diabetes and forms a unifying pathological marker for the heterogeneous clinical features of the disease. IAPP, the peptide component of islet amyloid, is a normal constituent of islet B-cells. However, unlike in many systemic forms of amyloidosis, there is no evidence for a molecular abnormality of IAPP in amyloid and little evidence for significant hypersecretion of the peptide in type 2 diabetes. Although amyloid formation precedes the onset of hyperglycaemia in animals, it seems unlikely that small amounts of extracellular amyloid would have a causative role in the onset of the disease. However, as in other amyloidoses, progressive accumulation of the deposits results in dysfunction of the affected organ, which in type 2 diabetes is manifested by a progressive decrease in insulin secretion. The physiological role of IAPP remains unclear although extensive experimental investigations have revealed pharmacological actions of IAPP which are similar to those of CGRP. Islet amyloid

Acknowledgements I am very grateful to John Morris, Eelco d e Koning, and my collegues at the Diabetes Research Laboratories for their helpful discussions and to Victoria Summers for her assistance in the preparation of the manuscript. I thank the British Diabetic Association and Novo Nordisk for financial support.

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