Establishment of radioimmunoassay for human islet amyloid polypeptide and its tissue content and plasma concentration

Using a synthetic C- terminal tetradecapeptide of human islet amyloid polypeptide (IAPP), we prepared an antiserum for human IAPP [24-37] and established a highly sensitive radioimmunoassay (RIA) for human IAPP. Analyses of human pancreatic extract using reverse-phase high performance liquid chromatography coupled with the RIA revealed that the antiserum specifically detects human IAPP. The content of IAPP in the pancreas of two non-diabetic patients was 604.0 and 1447.7 pg/mg wet weight, and a small amount of IAPP-immunoreactivity was detected in the stomach, duodenum, and jejunum. The mean plasma concentration of IAPP in 10 normal individuals was 13.5 +/- 4.8 (SD) pg/ml. The RIA established in this study provides a useful tool to elucidate the physiological function of IAPP and its pathophysiological significance in non-insulin-dependent diabetes mellitus (NIDDM).     

Sequence divergence in a specific region of islet amyloid polypeptide (IAPP) explains differences in islet amyloid formation between species

Amyloid deposits in the islets of Langerhans occur in association with type 2 diabetes mellitus (DM) in humans and cats and consist of a 37-amino-acid polypeptide known as islet amyloid polypeptide (IAPP). In order to find an explanation for the situation that islet amyloid (IA) does not develop in common rodent species, we have deduced the amino acid sequence of the IAPP molecule in mouse, rat and hamster. We find that a specific region of the molecule diverges to a high degree. Synthetic peptides corresponding to this region of human and hamster IAPP were compared for their ability to form amyloid fibrils in vitro. Whereas the human peptide readily formed fibrils with amyloid character, the hamster peptide completely lacked this property. We suggest this to be a likely explanation for the differences in IA formation between humans and rodents and discuss our findings in relation to the type 2 DM syndrome.     

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.     

胰岛淀粉样多肽的研究进展

由蛋白错误折叠后聚集所产生的淀粉样蛋白沉积是导致老年痴呆症、疯牛病、2型糖尿病等多种疾病的重要因素。由胰岛淀粉样多肽(islet amyloid polypeptide,IAPP)所形成的淀粉样蛋白沉积,具有破坏胰岛β细胞膜结构、诱导β细胞凋亡和损伤β细胞功能的作用,被认为是2型糖尿病的重要致病原因之一。对IAPP的聚集性、聚集体的结构,以及其对β细胞的毒性作用研究,不但有助于明确2型糖尿病的发病机制,而且最新研究也表明抑制IAPP的聚集可有效减少β细胞的凋亡,提高胰岛移植的成功率。因此,IAPP已成为2型糖尿病治疗中一个具有良好前景的靶点。该文对IAPP研究的最新进展进行了简要介绍。     

Membrane interaction of islet amyloid polypeptide

Increasing evidence suggests that the misfolding and deposition of IAPP plays an important role in the pathogenesis of type II, or non-insulin-dependent diabetes mellitus (T2DM). Membranes have been implicated in IAPP-dependent toxicity in several ways: Lipid membranes have been shown to promote the misfolding and aggregation of IAPP. Thus, potentially toxic forms of IAPP can be generated when IAPP interacts with cellular membranes. In addition, membranes have been implicated as the target of IAPP toxicity. IAPP has been shown to disrupt membrane integrity and to permeabilize membranes. Since disruption of cellular membranes is highly toxic, such a mechanism has been suggested to explain the observed IAPP toxicity. Here, we review IAPP-membrane interaction in the context of (1) catalyzing IAPP misfolding and (2) being a potential origin of IAPP toxicity.     

Membrane interaction of islet amyloid polypeptide

Increasing evidence suggests that the misfolding and deposition of IAPP plays an important role in the pathogenesis of type II, or non-insulin-dependent diabetes mellitus (T2DM). Membranes have been implicated in IAPP-dependent toxicity in several ways: Lipid membranes have been shown to promote the misfolding and aggregation of IAPP. Thus, potentially toxic forms of IAPP can be generated when IAPP interacts with cellular membranes. In addition, membranes have been implicated as the target of IAPP toxicity. IAPP has been shown to disrupt membrane integrity and to permeabilize membranes. Since disruption of cellular membranes is highly toxic, such a mechanism has been suggested to explain the observed IAPP toxicity. Here, we review IAPP-membrane interaction in the context of (1) catalyzing IAPP misfolding and (2) being a potential origin of IAPP toxicity.     

Antibodies specific for the pancreatic islet amyloid polypeptide associated with type 2 diabetes mellitus

Antibodies raised to a lysine solubilized peptide composed of residues 20-29 of the pancreatic islet amyloid polypeptide react selectively and specifically with this polypeptide and with islet amyloid deposits in Type 2 diabetes mellitus. These antibodies may prove useful in studies employing radioimmunoassay of body fluids and islet cell cultures in order to define if a pathogenic relationship exists between the islet amyloid polypeptide and Type 2 diabetes mellitus.     

Fibril structure of human islet amyloid polypeptide

Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two β-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing β-sheets that twist around each other. The two β-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (∼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding.     

Structural characterisation of islet amyloid polypeptide fibrils

Islet amyloid is found in many patients suffering from type 2 diabetes. Amyloid fibrils found deposited in the pancreatic islets are composed of a 37-residue peptide, known as islet amyloid polypeptide (IAPP) (also known as amylin) and are similar to those found in other amyloid diseases. Synthetic IAPP peptide readily forms amyloid fibrils in vitro and this has allowed fibril formation kinetics and the overall morphology of IAPP amyloid to be studied. Here, we use X-ray fibre diffraction, electron microscopy and cryo-electron microscopy to examine the molecular structure of IAPP amyloid fibrils. X-ray diffraction from aligned synthetic amyloid fibrils gave a highly oriented diffraction pattern with layer-lines spaced 4.7 A apart. Electron diffraction also revealed the characteristic 4.7 A meridional signal and the position of the reflection could be compared directly to the image of the diffracting unit. Cryo-electron microscopy revealed the strong signal at 4.7 A that has been previously visualised from a single Abeta fibre. Together, these data build up a picture of how the IAPP fibril is held together by hydrogen bonded beta-sheet structure and contribute to the understanding of the generic structure of amyloid fibrils.     

Amyloidogenicity and cytotoxicity of islet amyloid polypeptide

Insoluble amyloid formation by islet amyloid polypeptide (IAPP) in the islets of Langerhans of the pancreas is a major pathophysiological feature of noninsulin dependent diabetes mellitus (NIDDM) or type II diabetes. Because in vivo formed amyloid colocalizes with areas of cell degeneration and IAPP amyloid aggregates are cytotoxic per se, the process of IAPP amyloid formation has been strongly associated with the progressive pancreatic cell degeneration and thus much of the pathology of type II diabetes. IAPP is a pancreatic polypeptide of 37 residues that, in its soluble form, is believed to play a role as a regulator of glucose homeostasis. The molecular cause and mechanism of the conversion of soluble IAPP into insoluble amyloid aggregates in vivo and its role in disease progress still remain to be clarified. Nevertheless, in the past few years significant progress has been made in understanding the amyloidogenesis pathway of IAPP in vitro and gaining insight into the structural and conformational "requirements" of IAPP amyloidogenesis and related cytotoxic effects. Importantly, several of the studies have revealed significant similarities of the above features of IAPP to other amyloidogenic polypeptides such as the beta-amyloid polypeptide Abeta. This suggests that, at the molecular level, amyloidogenesis, and possibly related cell degeneration and disease pathogenesis by completely different polypeptide sequences, may obey to common structural and conformational "rules" and follow similar molecular pathways. This review describes studies on the structural and conformational features of IAPP amyloid formation and cytotoxicity, and the application of the obtained knowledge for the understanding of the molecular mechanism of the IAPP amyloidogenesis pathway and the related cytotoxicity.     

Structure of alpha-helical membrane-bound human islet amyloid polypeptide and its implications for membrane-mediated misfolding

Human islet amyloid polypeptide (hIAPP) misfolding is thought to play an important role in the pathogenesis of type II diabetes mellitus. It has recently been shown that membranes can catalyze the misfolding of hIAPP via an alpha-helical intermediate of unknown structure. To better understand the mechanism of membrane-mediated misfolding, we used site-directed spin labeling and EPR spectroscopy to generate a three-dimensional structural model of this membrane-bound form. We find that hIAPP forms a single alpha-helix encompassing residues 9-22. The helix is flanked by N- and C-terminal regions that do not take up a clearly detectable secondary structure and are less ordered. Residues 21 and 22 are located in a transitional region between the alpha-helical structure and C terminus and exhibit significant mobility. The alpha-helical structure presented here has important implications for membrane-mediated aggregation. Anchoring hIAPP to the membrane not only increases the local concentration but also reduces the encounter between peptides to essentially a two-dimensional process. It is significant to note that the alpha-helical membrane-bound form leaves much of an important amyloidogenic region of hIAPP (residues 20-29) exposed for misfolding. Misfolding of this and other regions is likely further aided by the low dielectric environment near the membrane that is known to promote secondary structure formation. Based upon these considerations, a structural model for membrane-mediated aggregation is discussed.     

Presence of islet amyloid polypeptide in rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content.

The islet amyloid polypeptide (IAPP) immunoreactivity of the adult rat pancreas is located in insulin-containing B cells as well as in somatostatin-containing D cells. In both cell types, the IAPP immunoreactivity is identical to rat synthetic IAPP in terms of its elution position after reversed phase HPLC and its binding to IAPP antibodies. The IAPP content per 10(6) B-cells is more than 100 fold lower than the corresponding insulin content, but comparable to the IAPP content of D cells. After induction of diabetes by streptozotocin, pancreatic IAPP seems predominantly located in somatostatin-containing cells. In normal rats, pancreatic insulin and IAPP content increase 20 fold from birth to 12 weeks of age; beyond week 12, the further rise in pancreatic insulin was not paralleled by an increase in IAPP content.     

Identifying structural features of fibrillar islet amyloid polypeptide using site-directed spin labeling

Pancreatic amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are a characteristic feature found in more than 90% of patients with type II diabetes. Although IAPP amyloid deposits are associated with areas of pancreatic islet beta-cell dysfunction and depletion and are thought to play a role in disease, their structure is unknown. We used electron paramagnetic resonance spectroscopy to analyze eight spin-labeled derivatives of IAPP in an effort to determine structural features of the peptide. In solution, all eight derivatives gave rise to electron paramagnetic resonance spectra with sharp lines indicative of rapid motion on the sub-nanosecond time scale. These spectra are consistent with a rapidly tumbling and highly dynamic peptide. In contrast, spectra for the fibrillar form exhibit reduced mobility and the presence of strong intermolecular spin-spin interactions. The latter implies that the peptide subunits are ordered and that the same residues from neighboring peptides are in close proximity to one another. Our data are consistent with a parallel arrangement of IAPP peptides within the amyloid fibril. Analysis of spin label mobility indicates a high degree of order throughout the peptide, although the N-terminal region is slightly less ordered. Possible similarities with respect to the domain organization and parallelism of Alzheimer's amyloid beta peptide fibrils are discussed.     

A single mutation in the nonamyloidogenic region of islet amyloid polypeptide greatly reduces toxicity

Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide secreted with insulin by beta-cells in the islets of Langerhans. The aggregation of the peptide into either amyloid fibers or small soluble oligomers has been implicated in the death of beta-cells during type 2 diabetes through disruption of the cellular membrane. The actual form of the peptide responsible for beta-cell death has been a subject of controversy. Previous research has indicated that the N-terminal region of the peptide (residues 1-19) is primarily responsible for the membrane-disrupting effect of the hIAPP peptide and induces membrane disruption to a similar extent as the full-length peptide without forming amyloid fibers when bound to the membrane. The rat version of the peptide, which is both noncytotoxic and nonamyloidogenic, differs from the human peptide by only one amino acid residue: Arg18 in the rat version while His18 in the human version. To elucidate the effect of this difference, we have measured in this study the effects of the rat and human versions of IAPP(1-19) on islet cells and model membranes. Fluorescence microscopy shows a rapid increase in intracellular calcium levels of islet cells after the addition of hIAPP(1-19), indicating disruption of the cellular membrane, while the rat version of the IAPP(1-19) peptide is significantly less effective. Circular dichroism experiments and dye leakage assays on model liposomes show that rIAPP(1-19) is deficient in binding to and disrupting lipid membranes at low but not at high peptide to lipid ratios, indicating that the ability of rIAPP(1-19) to form small aggregates necessary for membrane binding and disruption is significantly less than hIAPP(1-19). At pH 6.0, where H18 is likely to be protonated, hIAPP(1-19) resembles rIAPP(1-19) in its ability to cause membrane disruption. Differential scanning calorimetry suggests a different mode of binding to the membrane for rIAPP(1-19) compared to hIAPP(1-19). Human IAPP(1-19) has a minimal effect on the phase transition of lipid vesicles, suggesting a membrane orientation of the peptide in which the mobility of the acyl chains of the membrane is relatively unaffected. Rat IAPP(1-19), however, has a strong effect on the phase transition of lipid vesicles at low concentrations, suggesting that the peptide does not easily insert into the membrane after binding to the surface. Our results indicate that the modulation of the peptide orientation in the membrane by His18 plays a key role in the toxicity of nonamyloidogenic forms of hIAPP.     

Cleavage agents for soluble oligomers of human islet amyloid polypeptide

Soluble oligomers of human islet amyloid polypeptide (h-IAPP) are implicated in the initiation of beta-cell apoptosis leading to type 2 diabetes mellitus (T2DM). Cleavage of the h-IAPP included in an oligomer may provide a novel method for reducing the level of h-IAPP oligomers, offering a new therapeutic option for T2DM. From the combinatorial library of triazine derivatives prepared by exploiting the Co(III) complex of cyclen as the cleavage center for peptide bonds, eight compounds were selected as cleavage agents for oligomers of h-IAPP. After reaction with cleavage agents for 36 h at 37 degrees C and pH 7.50, up to 20 mol% of h-IAPP (initial concentration: 4.0 microM) was cleaved, although the target oligomers existed as transient species. Considerable activity was manifested at agent concentrations as low as 100 nM.     

Genetic background determines the extent of islet amyloid formation in human islet amyloid polypeptide transgenic mice

Genetic background is important in determining susceptibility to metabolic abnormalities such as insulin resistance and beta-cell dysfunction. Islet amyloid is associated with reduced beta-cell mass and function and develops in the majority of our C57BL/6J x DBA/2J (F(1)) male human islet amyloid polypeptide (hIAPP) transgenic mice after 1 yr of increased fat feeding. To determine the relative contribution of each parental strain, C57BL/6J (BL6) and DBA/2J (DBA2), to islet amyloid formation, we studied male hIAPP mice on each background strain (BL6, n = 13; and DBA2 n = 11) and C57BL/6J x DBA/2J F(1) mice (n = 17) on a 9% (wt/wt) fat diet for 1 yr. At the end of 12 mo, islet amyloid deposition was quantified from thioflavin S-stained pancreas sections. The majority of mice in all groups developed islet amyloid (BL6: 91%, F(1): 76%, DBA2: 100%). However, the prevalence (%amyloid-positive islets; BL6: 14 +/- 3%, F(1): 44 +/- 8%, DBA2: 49 +/- 9%, P < 0.05) and severity (%islet area occupied by amyloid; BL6: 0.03 +/- 0.01%, F(1): 9.2 +/- 2.9%, DBA2: 5.7 +/- 2.3%, p < or = 0.01) were significantly lower in BL6 than F(1) and DBA2 mice. Increased islet amyloid severity was negatively correlated with insulin-positive area per islet, in F(1) (r(2) = 0.75, P < 0.001) and DBA2 (r(2) = 0.87, P < 0.001) mice but not BL6 mice (r(2) = 0.07). In summary, the extent of islet amyloid formation in hIAPP transgenic mice is determined by background strain, with mice expressing DBA/2J genes (F(1) and DBA2 mice) being more susceptible to amyloid deposition that replaces beta-cell mass. These findings underscore the importance of genetic and environmental factors in studying metabolic disease.     

Lipid membranes modulate the structure of islet amyloid polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is thought to play an important role in the pathogenesis of type II diabetes. Despite a growing body of evidence implicating membrane interaction in IAPP toxicity, the membrane-bound form has not yet been well characterized. Here we used circular dichroism (CD) and fluorescence spectroscopy to investigate the molecular details of the interaction of IAPP with lipid membranes of varying composition. In the presence of membranes containing negatively charged phosphatidylserine (PS), we observed significant acceleration in the formation of IAPP aggregates. This acceleration is strongly modulated by the PS concentration and ionic strength, and is also observed at physiologically relevant PS concentrations. CD spectra of IAPP obtained immediately after the addition of membranes containing PS revealed features characteristic of an alpha-helical conformation approximately approximately 15-19 residues in length. After a longer incubation with membranes, IAPP gave rise to CD spectra characteristic of a beta-sheet conformation. Taken together, our CD and fluorescence data indicate that conditions that promote weakly stable alpha-helical conformations may promote IAPP aggregation. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.     

Secretion of islet amyloid polypeptide in response to glucose

The content of islet amyloid polypeptide (IAPP) in isolated rat pancreatic islets was determined by a radioimmunoassay. Reverse-phase high-performance liquid chromatography analysis revealed that a main peak of IAPP immunoreactivity in the extracts from the islets corresponded to a synthetic rat IAPP. Secretion of IAPP from the cells is regulated by the extracellular glucose concentration. Thus, IAPP may be a novel regulator for glucose homeostasis and changes in the secretion perhaps relate to insular amyloid deposits and impaired glucose tolerance in type 2 diabetes mellitus.     

Isolation and sequence determination of rat islet amyloid polypeptide

Rat islet amyloid polypeptide (IAPP) was isolated from the pancreata of normal rats by utilizing cross-reactivity of a radioimmunoassay system for human IAPP with rat IAPP. Rat IAPP was a 37-amino acid polypeptide with tyrosine amide at the C-terminus, as was the case with human IAPP. Amino acid sequences of rat and human IAPPs were 84% identical, and the most highly conserved sequences were found in the N- and C-terminal regions. Rat IAPP sequence was also 51% identical to those of alpha and beta rat calcitonin gene-related peptide sequences.