Islet Amyloid Formation Research Articles

Suppression by polycyclic compounds of the conversion of human amylin into insoluble amyloid.

There is a significant correlation between the occurrence of pancreatic islet amyloid and beta-cell failure in advanced type II diabetes mellitus. Islet amyloid is composed primarily of the fibrillar form of the pancreatic hormone, amylin. Using thioflavin-T fluorescence binding and radioprecipitation assays, we investigated whether or not a series of small tricyclic compounds, tetracycline or Congo Red could interfere with the conversion of synthetic human amylin into its insoluble amyloid form. Of the compounds investigated, incubation of human amylin with a 20-fold molar excess of either Congo Red or Acridine Orange resulted in significant inhibition in the rate of amyloid formation. With Congo Red, maximal inhibition effectively occurred at a 1:1 molar ratio or greater over human amylin, whereas inhibition by Acridine Orange was dose-dependent. A 20-fold molar excess of the compound tetracycline also decreased insoluble amyloid content after extended incubation periods of approx. 20 h. Amyloid fibril morphology in the presence of tetracycline, as measured by transmission electron microscopy, was characterized by short fragmented fibrils compared with the longer and denser appearance of fibrils formed by amylin alone. These findings show that polycyclic compounds can suppress the formation of amyloid by human amylin, providing support for an alternative approach to peptide-based strategies by which islet amyloid formation could be modulated.

Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formation rather than direct action of amyloid.

Nondiabetic obese humans adapt to insulin resistance by increasing beta-cell mass. In contrast, obese humans with type 2 diabetes have an approximately 60% deficit in beta-cell mass. Recent studies in rodents reveal that beta-cell mass is regulated, increasing in response to insulin resistance through increased beta-cell supply (islet neogenesis and beta-cell replication) and/or decreased beta-cell loss (beta-cell apoptosis). Prospective studies of islet turnover are not possible in humans. In an attempt to establish the mechanism for the deficit in beta-cell mass in type 2 diabetes, we used an obese versus lean murine transgenic model for human islet amyloid polypeptide (IAPP) that develops islet pathology comparable to that in humans with type 2 diabetes. By 40 weeks of age, obese nontransgenic mice did not develop diabetes and adapted to insulin resistance by a 9-fold increase (P < 0.001) in beta-cell mass accomplished by a 1.7-fold increase in islet neogenesis (P < 0.05) and a 5-fold increase in beta-cell replication per islet (P < 0.001). Obese transgenic mice developed midlife diabetes with islet amyloid and an 80% (P < 0.001) deficit in beta-cell mass that was due to failure to adaptively increase beta-cell mass. The mechanism subserving this failed expansion was a 10-fold increase in beta-cell apoptosis (P < 0.001). There was no relationship between the extent of islet amyloid or the blood glucose concentration and the frequency of beta-cell apoptosis. However, the frequency of beta-cell apoptosis was related to the rate of increase of islet amyloid. These prospective studies suggest that the formation of islet amyloid rather than the islet amyloid per se is related to increased beta-cell apoptosis in this murine model of type 2 diabetes. This finding is consistent with the hypothesis that soluble IAPP oligomers but not islet amyloid are responsible for increased beta-cell apoptosis. The current studies also support the concept that replicating beta-cells are more vulnerable to apoptosis, possibly accounting for the failure of beta-cell mass to expand appropriately in response to obesity in type 2 diabetes.

Proteoglycans synthesized and secreted by pancreatic islet β-cells bind amylin

Pancreatic islet amyloid deposits in type 2 diabetes are associated with decreased islet β-cell function. They contain both amylin (islet amyloid polypeptide), the β-cell-derived unique fibrillogenic component, and heparan sulfate proteoglycans (HSPGs). We hypothesized that β-cell HSPGs contribute to islet amyloidogenesis. [ 35 S ]Sulfate-labeled proteoglycans from islet-derived β-TC3 cell cultures eluted from diethylaminoethyl Sephacel at 0.35 M NaCl. Chromatography on Sepharose CL-4B and SDS–PAGE analysis revealed distinct populations of proteoglycans. Medium HSPGs eluted at K av∼0.18 and 0.50 with glycosaminoglycan chains of ∼28 and 19 kDa, respectively. A third population containing chondroitin/dermatan sulfate eluted at K av∼0.70 with glycosaminoglycan chains of ∼10 kDa. A single size class of heparan and chondroitin/dermatan sulfate proteoglycans in the cell layer eluted at K av∼0.40 with glycosaminoglycan chains of ∼19 kDa. Medium and cell layer proteoglycans bound exclusively to fibrillogenic amylin, as determined by gel mobility shift assays, indicating a possible role for β-cell-derived proteoglycans in islet amyloid formation.

The effect of apolipoprotein E deficiency on islet amyloid deposition in human islet amyloid polypeptide transgenic mice.

Islet amyloid deposits are present in over 85% of Type 2 diabetic patients and have been suggested to be pathogenic. The mechanism that converts islet amyloid polypeptide (IAPP), the unique component of these deposits, into amyloid fibrils in vivo is not known. The amino acid sequence of IAPP is critical but insufficient for beta-pleated sheet formation. As apolipoprotein E (apoE), another component of islet amyloid deposits, plays a critical role in amyloid formation in Alzheimer's disease, we hypothesised that apoE could play an important role in islet amyloid formation. Transgenic mice expressing the human form of IAPP ( hIAPP (+/0)) were crossbred with apoE deficient ( apoE (-/-)) mice and followed for 12 months, at which time the prevalence and severity of islet amyloid, as well as plasma glucose, hIAPP, immunoreactive insulin (IRI) and lipid concentrations were measured. The prevalence and severity of islet amyloid after one year of follow up were comparable among hIAPP (+/0) mice that were apoE (+/+), apoE (+/-) or apoE (-/-). Differences in glucose tolerance, lipid abnormalities or changes in pancreatic content or plasma concentrations of hIAPP and/or IRI did not account for these findings. Our data shows that, unlike in the localized amyloidosis in the brain characteristic of Alzheimer's disease, apoE is not critical for islet amyloid formation in a transgenic mouse model of Type 2 diabetes mellitus. These results indicate that the mechanisms of localised amyloid formation probably vary among different amyloid-associated disorders. Therefore, therapeutic strategies targeting apoE might not apply equally to patients with different amyloid associated diseases.

Immunohistochemical localization of amylin in human pancreas, thyroid, pituitary and their tumors

The aim of the present study was to investigate immunohistochemically expression of amylin, a 37 amino acid peptide, cosecreted with insulin by beta cells in pancreatic islets in 12 non-tumorous pancreatic tissues, 22 pancreatic islet tumors, 14 non-tumorous thyroids, 14 medullary carcinomas of the thyroid, 10 non-tumorous pituitaries and 50 pituitary adenomas including 10 amyloid-forming prolactin-cell adenomas using the streptavidin-biotin-peroxidase complex method. Amylin was expressed in non-tumorous pancreatic islets but not in non-tumorous thyroids and pituitaries. Since amylin plays an important role in amyloid formation in pancreatic islets, those tumor types were selected to study which may produce amyloid. Amylin was widely expressed in one insulin producing beta cell tumor. Few tumor cells were immunopositive in 8 islet-cell tumors and in 5 medullary thyroid carcinomas. Immunostaining was not found in pituitary adenomas, including those which produced amyloid. It can be concluded that amylin is not a satisfactory immunohistochemical marker to identify pancreatic islet tumors, medullary thyroid carcinomas and pituitary adenomas.

Amylin in the periphery.

Amylin (islet amyloid polypeptide) is a peptide synthesized principally in the b-cells of the pancreatic islets together with insulin and has actions as a hormone, growth factor, and modifier of behavior. As a hormone, amylin acts to modify gastric motility, renal resorption, and has metabolic actions. It is postulated that the principal function of amylin as a hormone is the activation of physiological processes associated with feeding. As a growth factor, amylin acts on bone cells, renal proximal tubular cells, and islet b-cells. Amylin has important targets in the brain that mediate its actions in the modification of behavior, including thirst and satiety. In man, amylin can form islet amyloid deposits, an event linked to the reduction of b-cell mass and loss of signal-secretion coupling. Recent evidence has defined a new role for monomeric amylin as a growth factor and regulator of b-cell mass that is postulated to be a key factor in pathophysiological processes that result in overt diabetes.

Identification of a Heparin Binding Domain in the N-terminal Cleavage Site of Pro-islet Amyloid Polypeptide: IMPLICATIONS FOR ISLET AMYLOID FORMATION

Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin) as well as the basement membrane heparan sulfate proteoglycan perlecan. Impaired processing of the IAPP precursor has been implicated in the mechanism of islet amyloid formation. The N- and C-terminal cleavage sites where pro-IAPP is processed by prohormone convertases contain a series of basic amino acid residues that we hypothesized may interact with heparan sulfate proteoglycans. This possibility was tested using affinity chromatography by applying synthetic fragments of pro-IAPP to heparin-agarose and heparan sulfate-Sepharose. An N-terminal human pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose and heparan sulfate-Sepharose, eluting at 0.18 m NaCl at pH 7.5. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. At pH 5.5, the affinity of the wild-type peptide for heparin/heparan sulfate was increased, implying a role for histidine residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP fragment (residues 41-67) had no specific affinity for either heparin or heparan sulfate, and the N- or C-terminal fragments had only weak affinity for chondroitin sulfate. These data suggest that monomeric N-terminal human pro-IAPP contains a heparin binding domain that is lost during normal processing of pro-IAPP.

The prohormone convertase enzyme 2 (PC2) is essential for processing pro-islet amyloid polypeptide at the NH2-terminal cleavage site.

Impaired processing of pro-islet amyloid polypeptide (proIAPP), the precursor of the beta-cell peptide islet amyloid polypeptide (IAPP) (amylin), has been implicated in islet amyloid formation in type 2 diabetes. The prohormone convertase enzymes PC3 (also known as PC1) and PC2 are localized to beta-cell secretory granules with proIAPP and proinsulin and are responsible for proinsulin processing. To determine whether PC2 might be essential for proIAPP processing, we performed Western blot analysis of freshly isolated islets from normal mice and mice lacking active PC2. As expected, the primary species of IAPP immunoreactivity in islets from wild-type mice was fully processed (4-kDa) IAPP, with only small amounts of the 8-kDa precursor (unprocessed proIAPP) present. Islets from heterozygous PC2 null mice were identical to wild-type animals, suggesting that half the normal complement of PC2 is sufficient for normal proIAPP processing. By contrast, in islets from homozygous PC2 null mice, the predominant IAPP-immunoreactive form was of intermediate size (approximately 6 kDa), with no detectable mature IAPP and slightly elevated amounts of the 8-kDa precursor form present. Thus, in the absence of PC2, proIAPP processing appears to be blocked at the level of a proIAPP conversion intermediate. Immunofluorescence of pancreas sections and immunoblotting using antisera raised to the NH2- and COOH-terminal flanking regions of mouse proIAPP demonstrated that the 6-kDa intermediate form was an NH2-terminally extended proIAPP conversion intermediate (processed only at the COOH-terminus). These data indicate that PC2 is essential for processing of proIAPP at the NH2-terminal cleavage site in vivo and that PC3 is likely only capable of processing proIAPP at the COOH-terminal cleavage site.

Oophorectomy promotes islet amyloid formation in human islet amyloid polypeptide transgenic mice.

Islet amyloid polypeptide (IAPP) (amylin) is the unique peptide component of the amyloid deposits found at autopsy in >90% of subjects with type 2 diabetes (1). These amyloid deposits are thought to replace islet mass and to thereby contribute to the � -cell dysfunction of the disease. The mechanism(s) underlying islet amyloid deposition is unclear, but an amyloidogenic sequence in IAPP is required. Islet amyloid can develop in humans, nonhuman primates, and cats, but it never develops in rodents because their IAPP lacks the critical amino acid sequence and is thus nonamyloidogenic. Several groups have generated transgenic mice expressing human IAPP (hIAPP) (2). We successfully induced amyloid formation in our hIAPP transgenic mice by feeding a diet containing an increased quantity of fat (9% wt/wt) (3). Interestingly, we noted a gender-dependent difference, as islet amyloid deposits were found in 81% of male transgenic mice but in only 11% of female transgenic mice. This observation suggested that either testicular products promote or ovarian products protect against islet amyloid deposition. Recently, another group has observed a similar gender-dependent difference in islet amyloid deposition in double-transgenic hIAPP ob/ob mice, with 83% of male mice compared with 20% of female mice developing islet amyloid deposits (4). To test the hypothesis that ovarian products protect against the development of islet amyloid in hIAPP transgenic mice, we performed a bilateral oophorectomy (n = 11) or a sham procedure (n = 6) in female hIAPP transgenic mice at 6‐8 weeks of age. The animals were followed for 1 year on a 9% fat (wt/wt) diet. Body weight was measured every 2 weeks throughout the study. At 57 weeks, an intraperitoneal glucose tolerance test was performed after an overnight fast to determine whether glucose tolerance or � -cell function were altered and contributed to amyloidogenesis. When the animals were killed, 4-h fasting plasma glucose, immunoreactive insulin (IRI), and hIAPP levels were measured. The pancreas was harvested and used to quantify IRI and hIAPP content and to determine the prevalence and severity of islet amyloid by thioflavin S staining. The severity of amyloid deposition was determined using an arbitrary scale of 0‐3, where 0 denoted no amyloid and 3 denoted extensive amyloid deposits. A minimum of six islets in each section were examined, and the mean score was calculated. No amyloid was detected in the six sham-operated mice. In sharp contrast, 7 of 11 (64%) oophorectomized mice developed islet amyloid (P < 0.05), with the mean (± SE) severity of amyloid in this group being 0.40 ± 0.18 arbitrary units (range 0‐2.00). Incremental body weight

Enhanced in vitro production of amyloid-like fibrils from mutant (S20G) islet amyloid polypeptide

Islet amyloid polypeptide (IAPP, “amylin”) is the amyloid-fibril-forming polypeptide in the islets of Langerhans associated with type 2 diabetes mellitus. A missense mutation in the IAPP gene associated with early-onset type 2 diabetes has been identified in the Japanese population. This mutation results in a glycine for serine substitution at position 20 of the mature IAPP molecule. Whether or not formation of islet amyloid with resulting destruction of islet tissue is the cause of this diabetes is yet not known. The present in vitro study was performed in order to investigate any influence of the amino acid substitution on the fibril formation capacity. Synthetic full-length wild type (lAPPwt) and mutant (IAPPS20G) as well as corresponding truncated peptides (position 18-29) were dissolved in dimethylsulfoxide (DMSO) or in 10% acetic acid at a concentration of 10 mg/mL and their fibril forming capacity was checked by Congo red staining, electron microscopy, a Congo red affinity assay and Thioflavine T fluorometric assay. It was found that full-length and truncated IAPPS20G both formed more amyloid-like fibrils and did this faster compared to IAPPwt. The fibril morphology differed slightly between the preparations. Conclusion: The amino acid substitution (S20G) is situated close to the region of the IAPP molecule implicated in the IAPP fibrillogenesis. The significantly increased formation of amyloid-like fibrils by IAPPS20G is highly interesting and may be associated with an increased islet amyloid formation in vivo and of fundamental importance in the pathogenesis of this specific form of diabetes.

Beta-cell glucokinase deficiency and hyperglycemia are associated with reduced islet amyloid deposition in a mouse model of type 2 diabetes.

Type 2 diabetes is characterized by impaired beta-cell function, hyperglycemia, and islet amyloid deposition. The primary constituent of islet amyloid is the 37-amino acid beta-cell product called islet amyloid polypeptide (IAPP) or amylin. To study mechanisms of islet amyloid formation, we developed a transgenic mouse model that produces and secretes the amyloidogenic human IAPP (hIAPP) molecule and have shown that 81% of male transgenic mice develop islet amyloid after 14 months on a high-fat diet. To test whether impaired beta-cell function and hyperglycemia could enhance islet amyloid formation, we cross-bred our hIAPP transgenic mice with beta-cell glucokinase-knockout mice (GKKO) that have impaired glucose-mediated insulin secretion and fasting hyperglycemia. The resulting new (hIAPPxGKKO) line of mice had higher basal plasma glucose concentrations than the hIAPP transgenic mice at 3, 6, and 12 months of age (P < 0.05), as did GKKO mice compared with hIAPP transgenic mice at 6 and 12 months of age (P < 0.05). Basal plasma immunoreactive insulin (IRI) levels were lower in hIAPP x GKKO mice than in hIAPP transgenic mice at 6 months of age (P < 0.05). The area under the glucose curve in response to an intraperitoneal glucose challenge (1 g/kg body weight) was larger in hIAPPxGKKO mice than in hIAPP transgenic mice at 3, 6, and 12 months of age (P < 0.005) and in GKKO mice compared with hIAPP transgenic mice at 6 and 12 months of age (P < 0.005). The area under the IRI curve was lower in hIAPPxGKKO mice at 6 and 12 months of age (P < 0.05) than in hIAPP transgenic mice and in GKKO mice compared with hIAPP transgenic mice at 12 months of age (P < 0.05). Despite the presence of hyperglycemia, hIAPPxGKKO mice had a lower incidence (4 of 17 vs. 12 of 19, P < 0.05) and amount (0.40 +/- 0.24 vs. 1.2 +/- 0.3 arbitrary units, P < 0.05) of islet amyloid than hIAPP transgenic mice had. As expected, no islet amyloid was observed in GKKO mice lacking the hIAPP transgene (0 of 13). There was no difference in pancreatic content of IRI and hIAPP among the three groups of mice. Thus, despite the presence of impaired islet function and hyperglycemia, hIAPPxGKKO mice had a decreased incidence and quantity of islet amyloid. Therefore, our data suggest that impaired beta-cell glucose metabolism or hyperglycemia are not likely to contribute to islet amyloid formation in diabetes. Furthermore, this finding may explain the lack of progression of glycemia in patients with maturity-onset diabetes of the young.

Islet Amyloid Development in a Mouse Strain Lacking Endogenous Islet Amyloid Polypeptide (IAPP) but Expressing Human IAPP

Several mouse strains expressing human islet amyloid polypeptide (IAPP) have been created to study development of islet amyloid and its impact on islet cell function. The tendency to form islet amyloid has varied strongly among these strains by factors that have not been elucidated. Because some beta cell granule components are known to inhibit IAPP fibril formation in vitro, we wanted to determine whether a mouse strain expressing human IAPP but lacking the nonamyloidogenic mouse IAPP is more prone to develop islet amyloidosis. Such a strain was created by cross-breeding a transgenic mouse strain and an IAPP null mouse strain. When fed a fat-enriched diet, male mice expressing only human IAPP developed islet amyloid earlier and to a higher extent than did mice expressing both human and mouse IAPP. Supporting these results, we found that mouse IAPP dose-dependently inhibits formation of fibrils from human IAPP. Female mice did not develop amyloid deposits, although small extracellular amorphous IAPP deposits were found in some islets. When cultivated in vitro, amyloid deposits occurred within 10 days in islets from either male or female mice expressing only human IAPP. The study shows that formation of islet amyloid may be dependent on the environment, including the presence or absence of fibril inhibitors or promoters.

Degradation of Amylin by Insulin-degrading Enzyme

A pathological feature of Type 2 diabetes is deposits in the pancreatic islets primarily composed of amylin (islet amyloid polypeptide). Although much attention has been paid to the expression and secretion of amylin, little is known about the enzymes involved in amylin turnover. Recent reports suggest that insulin-degrading enzyme (IDE) may have specificity for amyloidogenic proteins, and therefore we sought to determine whether amylin is an IDE substrate. Amylin-degrading activity co-purified with IDE from rat muscle through several chromatographic steps. Metalloproteinase inhibitors inactivated amylin-degrading activity with a pattern consistent with the enzymatic properties of IDE, whereas inhibitors of acid and serine proteases, calpains, and the proteasome were ineffective. Amylin degradation was inhibited by insulin in a dose-dependent manner, whereas insulin degradation was inhibited by amylin. Other substrates of IDE such as atrial natriuretic peptide and glucagon also competitively inhibited amylin degradation. Radiolabeled amylin and insulin were both covalently cross-linked to a protein of 110 kDa, and the binding was competitively inhibited by either unlabeled insulin or amylin. Finally, a monoclonal anti-IDE antibody immunoprecipitated both insulin- and amylin-degrading activities. The data strongly suggest that IDE is an amylin-degrading enzyme and plays an important role in the clearance of amylin and the prevention of islet amyloid formation.

Oophorectomy promotes islet amyloid formation in a transgenic mouse model of Type II diabetes.

In Type II (non-insulin-dependent) diabetes mellitus, amyloid depletes islet mass. We previously found that 81% of male human islet amyloid polypeptide (IAPP) transgenic mice but only 11% of female mice developed islet amyloid, suggesting that either testosterone promotes or ovarian products protect against amyloid deposition. We did a bilateral oophorectomy or sham procedure in female human IAPP transgenic mice (n = 11 and n = 8, respectively) and in female non-transgenic mice (n = 7 and n = 9, respectively) at 6-8 weeks of age. Animals were followed for 1 year on a 9% fat (w/w) diet. Before we killed them we measured, fasting plasma human IAPP and did an intraperitoneal glucose tolerance test. Pancreatic content of IAPP and immunoreactive insulin (IRI) were estimated and pancreata were analysed for islet amyloid. No amyloid was detected in either the sham-operated transgenic mice or, as expected, in both groups of non-transgenic mice. In strong contrast, 7 of 11 (64%) oophorectomized mice developed islet amyloid (p < 0.05). Amyloid deposition in the oophorectomized transgenic mice was not associated with any differences in incremental body weight, fasting human IAPP concentrations or glucose tolerance between the groups. Furthermore, pancreatic content of mouse IAPP, human IAPP and immunoreactive insulin did not differ between groups. Oophorectomy is associated with an enhancement of islet amyloid formation in the absence of changes in glucose tolerance, circulating IAPP or pancreatic content of IRI, mouse or human IAPP. Thus, the early stages of islet amyloidogenesis seem to be independent of glucose tolerance, with ovarian products having a protective role.

Expression of Islet Amyloid Polypeptide. Localization and regulation in the pancreatic islets, gastrointestinal tract and sensory nervous system

Islet amyloid polypeptide (IAPP; also named ”amylin”) is a member of the calcitonin superfamily of peptides, displaying the closest structural relationship to the neuropeptide calcitonin gene-related peptide (CGRP). It is expressed in islet beta-cells and forms islet amyloid in type 2 diabetic patients. IAPP may restrain release and peripheral actions of insulin. Thus, IAPP has been implicated in the pathogenesis of type 2 diabetes. By examinations of tissue-specific expression of IAPP and its regulation under normal and experimental conditions, we aimed to illuminate a possible role of IAPP. IAPP was expressed in islet beta-cells in nearly all species examined; in some species IAPP occurred also in delta-cells. IAPP expression was of early onset during embryonic islet development. In vivo, we found a parallel regulation of IAPP and insulin expression by administration of glucose. These findings agree with a role for IAPP in glucose homeostasis by autocrine/paracrine regulation of islet function as well as by endocrine secretion. Following dexamethasone treatment of rats, a model for type 2 diabetes, we found a non-parallel expression of IAPP and insulin; in relative terms, IAPP was over-expressed compared with insulin. In rodents given streptozotocin or alloxan, conferring beta-cell depletion reminiscent of type 1 diabetes, IAPP expression was consistently less impaired than that of insulin. These studies show that IAPP and insulin gene expression are differentially regulated. Apparently, beta-cell stress, regardless of ethiology, results in over-expression of IAPP. IAPP was also found to be expressed in the gastrointestinal tract in a region- and species-dependent manner; when present, IAPP expression was most abundant proximally, occurring in somatostatin and gastrin cells. Also gastrointestinal IAPP expression was of early embryonic onset. In vitro, IAPP relaxed pre-contracted gut musculature. In the sensory nervous system IAPP was expressed in CGRP neurons and displayed a distribution typical of sensory neuropeptides. Upon sciatic axotomy, IAPP and CGRP expression were co-ordinately downregulated. In contrast, following adjuvant-induced rat paw inflammation, IAPP expression was upregulated in innervating neurons, indicating that IAPP may play a role in neurogenic inflammation. In conclusion, our studies have expanded the view of IAPP as an exclusive islet hormone to become a ”classical” neurohormonal peptide, with several endocrine and neuronal expression sites. The studies on the regulation of islet IAPP and insulin expression in response to glucose and in experimental diabetes may form the basis for an understanding of the role of IAPP in diabetes. Provided that the amyloidogenic mechanisms and metabolic effects of IAPP are elucidated, an over-expression of IAPP may prove to be relevant for diabetes development.

Two novel immortal pancreatic beta-cell lines expressing and secreting human islet amyloid polypeptide do not spontaneously develop islet amyloid.

Type 2 diabetes is characterized by islet amyloid deposits, which are primarily composed of the amyloidogenic human form of islet amyloid polypeptide (IAPP, amylin). The mechanism of islet amyloido-genesis is not known, but other products (e.g., apolipoprotein E and perlecan) contained within islet amyloid may be necessary. Because rodent IAPP does not form islet amyloid, the currently available beta-cell lines are not useful for studying processes involved in amyloid formation. To develop a suitable in vitro cell system for the study of islet amyloid formation, we generated two new beta-cell lines that express the amyloidogenic human IAPP. We did this by crossbreeding human IAPP transgenic mice with RIP-Tag mice that develop islet tumors and then culturing one of these islet tumors from two separate offspring of this cross. The resultant 2350-2C0 and 2511 cell lines produce human as well as mouse IAPP-like immunoreactivity (IAPP-LI) and immunoreactive insulin (IRI). Incubation of both these cell lines with 16.7 mmol/l glucose resulted in a two- to fourfold increase in human IAPP-LI, mouse IAPP-LI, and IRI secretion compared with 1.67 mmol/l glucose and the combination of 16.7 mmol/l glucose and 10 mmol/l arginine, 0.1 mmol/l 3-isobutyl-1-methylxanthine (IBMX), and 5 micromol/l carbachol induced a >50-fold increase in the release of these peptides. The omission of calcium from the above secretagogue cocktail reduced secretion of all three peptides to only two- to sixfold higher than the 16.7 mmol/l glucose condition. Perifusion with 16.7 mmol/l glucose plus 0.1 mmol/l IBMX caused a biphasic secretion of human IAPP-LI and mouse IAPP-LI, as well as IRI, in both cell lines, with the peak of the first phase being five- to sixfold higher than the prestimulated 1.67 mmol/l glucose condition. Immunoelectron microscopic inspection of both 2350-2C0 and 2511 cells after 7 days of culture did not reveal the presence of amyloid fibrils, suggesting the need for other critical components. We conclude that we have established two novel beta-cell lines that produce and secrete human IAPP in a regulated manner. These cell lines will be a useful tool to investigate the secretion of human IAPP as well as the necessity of other components for islet amyloid formation.

Extensive islet amyloid formation is induced by development of Type II diabetes mellitus and contributes to its progression: pathogenesis of diabetes in a mouse model.

Type II (non-insulin-dependent) diabetes mellitus is a multifactorial disease in which pancreatic islet amyloid is a characteristic histopathological finding. Islet amyloid fibrils consist of the beta-cell protein "islet amyloid polypeptide" (IAPP)/"amylin". Unlike human IAPP (hIAPP), mouse IAPP cannot form amyloid. In previously generated transgenic mice, high expression of hIAPP as such did not induce islet amyloid formation. To further explore the potential diabetogenic role of amyloidogenic IAPP, we introduced a diabetogenic trait ("ob" mutation) in hIAPP transgenic mice. Plasma concentrations of IAPP, insulin and glucose were determined at 3.5 (t1), 6 (t2), and 16-19 months of age (t3). At t3, the mice were killed and the pancreas was analysed (immuno)histochemically. In non-transgenic ob/ob mice, insulin resistance caused a compensatory increase in insulin production, normalizing the initial hyperglycaemia. In transgenic ob/ob mice, concurrent increase in hIAPP production resulted in extensive islet amyloid formation (more often and more extensive than in transgenic non-ob/ob mice), insulin insufficiency and persistent hyperglycaemia: At t3, plasma insulin levels in transgenic ob/ob mice with amyloid were fourfold lower than in non-transgenic ob/ob mice (p < 0.05), and plasma glucose concentrations in transgenic ob/ ob mice were almost twofold higher (p < 0.05). In addition, the degree of islet amyloid formation in ob/ob mice was positively correlated to the glucose:insulin ratio (r(s) = 0.53, p < 0.05). Islet amyloid is a secondary diabetogenic factor which can be both a consequence of insulin resistance and a cause of insulin insufficiency. [Diabetol

Islet amyloid: a long-recognized but underappreciated pathological feature of type 2 diabetes.

Islet amyloid has been recognized as a pathological entity in type 2 diabetes since the turn of the century. It has as its unique component the islet beta-cell peptide islet amyloid polypeptide (IAPP), or amylin, which is cosecreted with insulin. In addition to this unique component, islet amyloid contains other proteins, such as apolipoprotein E and the heparan sulfate proteoglycan perlecan, which are typically observed in other forms of generalized and localized amyloid. Islet amyloid is observed at pathological examination in the vast majority of individuals with type 2 diabetes but is rarely observed in humans without disturbances of glucose metabolism. In contrast to IAPP from rodents, human IAPP has been shown to form amyloid fibrils in vitro. Because all human subjects produce and secrete the amyloidogenic form of IAPP, yet not all develop islet amyloid, some other factor(s) must be involved in islet amyloid formation. One hypothesis is that an alteration in beta-cell function resulting in a change in the production, processing, and/or secretion of IAPP is critical to the initial formation of islet amyloid fibrils in human diabetes. This nidus of amyloid fibrils then allows the progressive accumulation of IAPP-containing fibrils and the eventual replacement of beta-cell mass by amyloid and contributes to the development of hyperglycemia. One factor that may be involved in producing the changes in the beta-cell that result in the initiation of amyloid formation is the consumption of increased dietary fat. Dietary fat is known to alter islet beta-cell peptide production, processing, and secretion, and studies in transgenic mice expressing human IAPP support the operation of this mechanism. Further investigation using this and other models should provide insight into the mechanism(s) involved in islet amyloidogenesis and allow the development of therapeutic agents that inhibit or reverse amyloid fibril formation, with the goal being to preserve beta-cell function and improve glucose control in type 2 diabetes.

Contribution of advanced glycosylation to the amyloidogenicity of islet amyloid polypeptide.

The formation of amyloid within the islets of Langerhans is associated with the development of type II diabetes mellitus and occurs by the aggregation and insolubilization of islet amyloid polypeptide (IAPP). Recent in vitro studies suggest that amyloid formation follows a nucleation-dependent polymerization mechanism, i.e. aggregation is initiated by pre-formed aggregates or nucleation seeds. Modification of the Alzheimer's disease amyloid peptide by advanced glycosylation end products (AGEs), which form spontaneously by the non-enzymatic addition of glucose to protein amino groups, has been shown to enhance peptide aggregation in vitro. To explore the possibility that AGEs contribute to islet amyloid formation, we prepared AGE-modified IAPP (AGE-IAPP) in vitro and studied its properties by biochemical and biophysical techniques. AGE modification induced the formation of high-molecular-mass IAPP aggregates and amyloid formation was demonstrated by Congo red green-gold birefringence and by the presence of a characteristic fibrillar structure by electron microscopy. AGE-IAPP also showed an increase in cytotoxicity toward the astroglioma cell line HTB14. When added to soluble IAPP, AGE-IAPP seeds accelerated IAPP aggregation and abolished the nucleation period required for the polymerization of unseeded IAPP. Circular dichroism spectropolarimetry indicated that AGE-IAPP seeds may act as a template to stabilize the beta-sheet conformation of IAPP, thereby promoting its aggregation. Our studies demonstrate that AGE modification of IAPP results in high-molecular mass, fibrillar amyloid structures that nucleate IAPP amyloid formation and suggest a model for intra-islet amyloid deposition that may occur by the progressive advanced glycosylation of IAPP in vivo.

Transgenic overproduction of islet amyloid polypeptide (amylin) is not sufficient for islet amyloid formation.

Islet amyloid polypeptide forms islet amyloid deposits in non-insulin-dependent diabetes mellitus. We have generated transgenic mice which express human islet amyloid polypeptide in their pancreatic beta cells yet do not develop islet amyloid deposits despite producing levels of the amyloidogenic human peptide 2 - 3 fold higher than the native (mouse) peptide. To determine whether marked overproduction of islet amyloid polypeptide is a potential cause of islet amyloid formation, we increased expression of this transgene by producing homozygous transgenic animals and by making heterozygous mice experimentally insulin resistant with nicotinic acid. Pancreatic content of islet amyloid polypeptide-like immunoreactivity in homozygous and nicotinic acid-treated mice was 2-fold (25 +/- 7 fmol/microg; n = 6) and 3.5-fold (47 +/- 20 fmol/microg; n = 3) higher, respectively, than that of untreated heterozygous animals (13+/-2 fmol/microg; n = 11; both p < 0.05). Despite this marked increase in production of islet amyloid polypeptide, neither group of mice developed gross islet amyloid deposits even after 16 months of age. We conclude that overproduction of islet amyloid polypeptide, even as produced by extreme insulin resistance, is not in itself sufficient for islet amyloid formation.