The mechanism of insulin action on islet amyloid polypeptide fiber formation

The pathology of type II diabetes includes the presence of cytotoxic amyloid deposits in the islets of Langerhans. The main component of these deposits, islet amyloid polypeptide (IAPP), is a hormone involved in glucose metabolism and is normally co-secreted with insulin by the beta-cells of the pancreas. Here, we perform in vitro IAPP fibrillogenesis experiments in the presence and in the absence of insulin to elucidate the mechanism by which insulin acts on fiber formation. We find that insulin is an exceptionally potent inhibitor. In contrast to the vast excess of insulin over IAPP in vivo, substoichiometric amounts of insulin inhibit seeded and unseeded reactions by more than tenfold in vitro. Unusually, the magnitude of the inhibitory effect is dependent on the concentration of insulin, yet independent of the concentration of IAPP. In addition, insulin appears to bind non-specifically to fiber surfaces, giving rise to altered morphology. IAPP fiber formation in vitro requires a minimum of three steps: fiber-independent nucleation, elongation, and fiber-dependent nucleation. Furthermore, these steps are attenuated by the presence of a dispersed-phase transition. We interpret these data in the context of the phase-mediated fibrillogenesis model (PMF) and conclude through experiment and kinetic simulation that the dominant effect of insulin is to act on the elongation portion of the reaction. These results suggest that amyloid formation in type II diabetes involves either an additional agent that acts as an accelerant, or a step that segregates IAPP from insulin.     

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.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

Structure-activity relationship of amyloid fibrils

Protein aggregation is a process in which proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as either amorphous or highly ordered, the most common form of the latter being amyloid fibrils. Amyloid fibrils composed of cross-β-sheet structure are the pathological hallmarks of several diseases including Alzheimer’s disease, but are also associated with functional states such as the fungal HET-s prion. This review aims to summarize the recent high-resolution structural studies of amyloid fibrils in light of their (potential) activities. We propose that the repetitive nature of the cross-β-sheet structure of amyloids is key for their multiple properties: the repeating motifs can translate a rather non-specific interaction into a specific one through cooperativity.     

Recovery and purification of highly aggregation-prone disulfide-containing peptides: application to islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a 37-residue pancreatic hormone. It is responsible for the formation of islet amyloid in vivo and is very insoluble and aggregation-prone in vitro, particularly at basic pH. The peptide contains a disulfide bridge between residues two and seven and an amidated C terminus. There is no reported expression system for the production of amidated IAPP. The peptide is difficult to synthesize and formation of the disulfide by traditional methods is problematic. We have found that the use of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or dimethyl sulfoxide (DMSO) significantly improves disulfide formation and purification of highly aggregation-prone IAPP sequences. The use of these organic solvents increases the solubility of the hydrophobic peptides, avoids the use of aqueous basic solutions, and eliminates the need for continuous stirring during oxidation to form the Cys-2 to Cys-7 disulfide bridge. Elimination of the stirring step and basic solution helps to reduce aggregation and allows for more consistent high-performance liquid chromatography (HPLC) retention times. Formation of the intramolecular disulfide using DMSO was found to be the most effective method for IAPP oxidation, reducing the reaction time from 24 to 5 h. Aggregated IAPP can be resolubilized by HFIP or DMSO and recovered by HPLC with very good yield.     

Identification and characterization of a novel molecular-recognition and self-assembly domain within the islet amyloid polypeptide

The islet amyloid polypeptide (hIAPP) is a 37 amino acid residue polypeptide that was found to accumulate as amyloid fibrils in the pancreas of individuals with type II diabetes. Previous studies identified various fragments of hIAPP that can form amyloid fibrils in vitro (e.g. hIAPP(8-20), hIAPP(23-27), and hIAPP(30-37)). However, no comparative and systematic information was available on the role of these structural domains (or others) in the process of molecular recognition that mediates fibrillization, in the context of the full-length polypeptide. To systematically map and compare potential recognition domains, we studied the ability of hIAPP to interact with an array of 28 membrane-spotted overlapping peptides that span the entire sequence of hIAPP (i.e. hIAPP(1-10), hIAPP(2-11...), hIAPP(28-37)). Our study clearly identified a major domain of molecular recognition within hIAPP, as the polypeptide was found to bind with high affinity to a defined linear group of peptides ranging from hIAPP(7-16) to hIAPP(12-21). The maximal binding of the full-length polypeptide was to the hIAPP(11-20) peptide fragment (with the sequence RLANFLVHSS). In order to define the minimal fragment, within this apparent recognition motif, that is capable of self-association and thus may serve as the core molecular recognition motif, we examined the ability of truncated analogs of the recognition sequence to self-assemble into amyloid fibrils. The shortest active fragments capable of self-assembly were found to be the pentapeptides FLVHS and NFLVH. The apparent role of this motif in the process of hIAPP self-assembly is consistent with the profile of the hIAAP-binding distribution to the peptide array. The identification of such short recognition motifs is extremely useful in the attempts to develop means to block amyloid fibril formation by hIAPP. It is worth mentioning that this is only the second time in which peptides as short as a pentapeptide were shown to form amyloid fibrils (the other pentapeptide is FGAIL).     

Full-length rat amylin forms fibrils following substitution of single residues from human amylin

Pancreatic amyloid deposits, composed of the 37 amino acid residue peptide amylin, represent an integral part of type 2 diabetes mellitus pathology. Human amylin (hA) forms fibrils in vitro and is toxic to cultured pancreatic islet beta-cells. In contrast, rat amylin (rA) which differs from hA by only six amino acid residues in the central region of the peptide, residues 18-29, does not form fibrils and is not cytotoxic. To elucidate the role of individual residues in fibril formation, we have generated a series of full-length rA variants and examined their ability to form fibrils in vitro. Single-residue substitutions with amino acids from corresponding positions of the hA sequence, i.e. R18H, L23F, or V26I, were sufficient to render rA competent for fibril formation albeit at a small yield. Combining two or three of these substitutions generally increased the ability to produce fibrils. Variant rA fibril morphologies were examined by negative stain electron microscopy and found to be similar to those generated by hA itself. Bulk assays, i.e. involving thioflavin-T fluorescence and sedimentation, showed that the amount of fibril formation was relatively small for these rA variants when compared to hA under the same conditions. Fibril growth was demonstrated by time-lapse atomic force microscopy, and MALDI-TOF mass spectrometry was used to verify that fibrils consisted of full-length peptide. Our observations confirm previous reports that the three proline residues play a dominant negative role in fibril formation. However, their presence is not sufficient to completely abolish the ability of rA to form fibrils, as each of the other three implicated residues (i.e. R18, L23 and V26) also has a dominant modulating effect.     

Identification of minimal peptide sequences in the (8-20) domain of human islet amyloid polypeptide involved in fibrillogenesis

We have examined a series of overlapping peptide fragments from the 8-20 region of human islet amyloid polypeptide (IAPP) with the objective of defining the smallest fibril-forming domain. Peptide fragments corresponding to LANFLV (residues 12-17) and FLVHSS (residues 15-20) were strong enhancers of beta-sheet transition and fibril formation. Negative stain electron microscopy illustrated the ability of these peptide fragments to form fibrils independently when incubated alone in solution. Circular dichroism analysis revealed that when full-length human IAPP was incubated in the presence of these two fragments, fibrillogenesis was accelerated. While the two fragments, LANFLV and FLVHSS, were able to enhance the recruitment of additional IAPP molecules during fibril formation, the "seeding" activity of these peptides had no effect on altering IAPP-induced cytotoxcity as determined by cell culture studies. Therefore, this study has identified two internal IAPP peptide fragments within the 8-20 domain that may have a role in enhancing the folding and aggregation of human IAPP. These fragments are the smallest sequences identified, within the 8-20 region of hIAPP, that can independently form fibrils, and that can interact with IAPP to assemble into fibrils with characteristics similar as those formed by human IAPP alone.     

The effect of islet amyloid polypeptide (amylin) and calcitonin gene-related peptide on glucose removal in the anaesthetized rat and on insulin secretion from rat pancreatic isletsin vitro

The effect of intravenous infusion of islet amyloid polypeptide (IAPP/amylin) and calcitonin gene-related peptide (CGRP) on blood glucose and plasma insulin in the basal and glucose-stimulated state was investigated in the anaesthetized rat. Both peptides had no effect on basal blood glucose or plasma insulin but following an intravenous bolus of glucose, CGRP-treated rats were hyperglycaemic and hyperinsulinaemic compared with control animals which were similar to IAPP-treated rats. IAPP had no effect on glucose-stimulated islet insulin secretion. These results suggest that CGRP, but not IAPP, alters glucose removalin vivo.     

Optimized experimental pre-treatment strategy for temporary inhibition of islet amyloid polypeptide aggregation

Islet amyloid polypeptide (IAPP) is a neuroendocrine hormone from pancreatic β-cells. Misfolded, aggregated IAPP is believed to be toxic to islet cells and amyloid deposits in the pancreas are pathological hallmarks of type 2 diabetes. Rapid fibrillization of this peptide makes it difficult to study in its soluble form, impeding a better understanding of its role. In this study, a variety of popular pretreatment methods were tested for their ability to delay aggregation of IAPP, including solutions of hexafluoroisopropanol, sodium hydroxide, hydrochloric acid, phosphate buffered saline, ammonium hydroxide, as well as tris buffer at different pH and containing either calcium (II), zinc (II), or iron (II). Aggregation was assessed using the thioflavin T fluorescence assay as well as by transmission electron microscopy. Tris buffer at pH 8.1 containing Zn(II) was found to have the best balance of temporary inhibition of aggregation and biological relevance.     

Elongated oligomers assemble into mammalian PrP amyloid fibrils

In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly alpha-helical state into beta-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a beta form of recombinant mouse PrP (residues 91-231). The beta-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrP Sc. Using electron microscopy and image processing, we obtained three-dimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of approximately 60 A. The repeating unit along each protofilament can be accounted for by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-beta amyloid fold.     

Pituitary adenylate cyclase-activating polypeptide and islet amyloid polypeptide in primary sensory neurons

Primary sensory neurons serve a dual role as afferent neurons, conveying sensory information from the periphery to the central nervous system, and as efferent effectors mediating, e.g., neurogenic inflammation. Neuropeptides are crucial for both these mechanisms in primary sensory neurons. In afferent functions, they act as messengers and modulators in addition to a principal transmitter; by release from peripheral terminals, they induce an efferent response, “neurogenic inflammation,” which comprises vasodilatation, plasma extravasation, and recruitment of immune cells. In this article, we introduce two novel members of the sensory neuropeptide family: pituitary adenylate cyclase-activating polypeptide (PACAP) and islet amyloid polypeptide (IAPP). Whereas PACAP, a vasoactive intestinal polypeptide-resembling peptide, predominantly occurs in neuronal elements, IAPP, which is structurally related to calcitonin gene-related peptide, is most widely known as a pancreatic β-cell peptide; as such, it has been recognized as a constituent of amyloid deposits in type 2 diabetes. In primary sensory neurons, under normal conditions, both peptides are predominantly expressed in small-sized nerve cell bodies, suggesting a role in nociception. On axotomy, the expression of PACAP is rapidly induced, whereas that of IAPP is reduced. Such a regulation of PACAP suggests that it serves a protective role during nerve injury, but that of IAPP may indicate that it is an excitatory messenger under normal conditions. In contrast, in localized adjuvant-induced inflammation, expression of both peptides is rapidly induced. For IAPP, studies in IAPP-deficient mice support the notion that IAPP is a pronociceptive peptide, because these mutant mice display a reduced nociceptive response when challenged with formalin.     

Inhibitors of islet amyloid polypeptide fibrillogenesis, and the treatment of type-2 diabetes

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. Although it may be a secondary event in the etiology of diabetes, the accumulation of insoluble IAPP fibrils is considered to be a primary cause of β-cell failure in affected individuals. A possible means of inhibiting this process is through the use of small peptides that bind to IAPP and prevent fibril polymerization. This approach has been examined using a series of overlapping hexamers that target the known amyloidogenic regions of IAPP. Peptides were examined usingin vitroassays and active inhibitors were identified by their ability to prevent amyloid-related conformational transitions and IAPP aggregation. Fragments such as those corresponding to the IAPP-derived sequences, SNNFGA (residues 20–25) and GAILSS (residues 24–29), were potent inhibitors ofβ-sheet folding and amyloid fibril formation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP significantly decreased the density of fibrils and any remaining structures displayed altered morphology. In some, but not all cases, inhibition of amyloid fibrils also correlated with an ability to reduce IAPP-mediated cytotoxicity as determined in cell culture studies. The results from these studies suggest that these two peptide inhibitors differ in their mechanisms of action possibly due to unique interactions with the full-length IAPP molecule. These inhibitors form the basis of a therapeutic strategy to prevent amyloid accumulation leading to improved islet survival and a potentially novel treatment for type-2 diabetes.     

Human pro-islet amyloid polypeptide (ProIAPP1-48) forms amyloid fibrils and amyloid spherulites in vitro

Deposition of β sheets of islet amyloid polypeptide (IAPP) in pancreatic tissue is implicated in the aetiology of type 2 diabetes mellitus (T2DM). IAPP is cleaved from its precursor protein, pro-islet amyloid polypeptide (ProIAPP) and incomplete cleavage results in ProIAPP_1-48, which is found co-deposited with IAPP. Cu(II) prevents IAPP from forming amyloid and herein we investigated if it would also prevent ProIAPP_1-48 from forming β sheets. Excess Cu(II) prevented ProIAPP_1-48 from forming amyloid and additionally reversed the formation of β sheets in pre-formed fibrils of the peptide. The latter was also true for ProIAPP_1-48 fibrils formed in the presence of Al(III). An unexpected finding was the formation of spherulites of ProIAPP_1-48 which were only observed in preparations which included Al(III). The spherulites were 40-100 μm in diameter and stained positively for Al(III) suggesting a role for this metal in their formation.The abolition by Cu(II) of the propensity of ProIAPP_1-48 to form amyloid may have important implications for the treatment of T2DM. The immediate significance for diabetes of the equally novel observation of spherulites of ProIAPP_1-48 is unknown though, as with spherulites of Aβ₄₂ in Alzheimer's disease, there may be implications for the aetiology of the disease.     

Conformational studies of human islet amyloid peptide using molecular dynamics and simulated annealing methods

Molecular dynamics simulations and simulated annealing in vacuum, model aqueous solution, and simulated membrane were used to analyze the conformational preferences of a segment spanning 20–29 residues of human islet amyloid polypeptide, [referred to as IAPP^H(20–29)]. Molecular dynamics simulations were conducted at 300 K on IAPP^H(20–29). The minimum energy conformers obtained in model aqueous solution and vacuum exhibited similar structures. Even in the absence of any constraints on peptide bonds, trans conformation was preferred consistently by all the peptide bonds. Analysis of the minimum energy conformers indicated that IAPP^H(20–29) showed a strong preference for turn structures in all the environments. These turn structures were stabilized by the formation of hydrogen bonds between the backbone amide and carbonyl groups. A good agreement was found between the results obtained from the molecular dynamics simulation and solid-state nmr experimental studies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 9–20, 1998     

Islet amyloid polypeptide inserts into phospholipid monolayers as monomer

Amyloid deposits in the pancreatic islets of Langerhans are thought to be a main factor responsible for death of the insulin-producing islet beta-cells in type 2 diabetes. It is hypothesized that beta-cell death is related to interaction of the 37 amino acid residue human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid, with cellular membranes. However, the mechanism of hIAPP-membrane interactions is largely unknown. Here, we study the nature and the molecular details of the initial step of hIAPP-membrane interactions by using the monolayer technique. It is shown that both freshly dissolved hIAPP and the non-amyloidogenic mouse IAPP (mIAPP) have a pronounced ability to insert into phospholipid monolayers, even at lipid packing conditions that exceed the conditions that occur in biological membranes. In contrast, the fibrillar form of hIAPP has lost the ability to insert. These results, combined with the observations that both the insertion kinetics and the dependence of insertion on the initial surface pressure are similar for freshly dissolved hIAPP and mIAPP, indicate that hIAPP inserts into phospholipid monolayers most likely as a monomer. In addition, our results suggest that the N-terminal part of hIAPP, which is nearly identical with that of mIAPP, is largely responsible for insertion. This is supported by experiments with hIAPP fragments, which show that a peptide consisting of the 19 N-terminal residues of hIAPP efficiently inserts into phospholipid monolayers, whereas an amyloidogenic decapeptide, consisting of residues 20-29 of hIAPP, inserts much less efficiently. The results obtained here suggest that hIAPP monomers might insert with high efficiency in biological membranes in vivo. This process could play an important role as a first step in hIAPP-induced membrane damage in type 2 diabetes.     

Synergistic action of polyanionic and non-polar cofactors in fibrillation of human islet amyloid polypeptide

Recent studies have led us to suppose that synergistic action of multiple solute cofactors could play substantial roles in amyloid-type fibrillogenesis of pathogenic polypeptides. To support this view, we performed aggregation experiments of human islet amyloid polypeptide (IAPP) in media containing both polyanions and non-polar solvents. The results demonstrated that the fibrillation at sub-micromolar IAPP occurred only when polyanionic and non-polar solutes coexist. A simple sum of two independent cofactor's effects could not account for the synergistic action. We propose that this synergy of polyanionic and nonpolar milieus could substantially modify the amyloidgenesis in the human body.     

Destabilization of human IAPP amyloid fibrils by proline mutations outside of the putative amyloidogenic domain: is there a critical amyloidogenic domain in human IAPP?

Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.     

Islet amyloid polypeptide forms rigid lipid-protein amyloid fibrils on supported phospholipid bilayers

Islet amyloid polypeptide (IAPP) forms fibrillar amyloid deposits in the pancreatic islets of Langerhans of patients with type 2 diabetes mellitus, and its misfolding and aggregation are thought to contribute to β-cell death. Increasing evidence suggests that IAPP fibrillization is strongly influenced by lipid membranes and, vice versa, that the membrane architecture and integrity are severely affected by amyloid growth. Here, we report direct fluorescence microscopic observations of the morphological transformations accompanying IAPP fibrillization on the surface of supported lipid membranes. Within minutes of application in submicromolar concentrations, IAPP caused extensive remodeling of the membrane including formation of defects, vesiculation, and tubulation. The effects of IAPP concentration, ionic strength, and the presence of amyloid seeds on the bilayer perturbation and peptide aggregation were examined. Growth of amyloid fibrils was visualized using fluorescently labeled IAPP or thioflavin T staining. Two-color imaging of the peptide and membranes revealed that the fibrils were initially composed of the peptide only, and vesiculation occurred in the points where growing fibers touched the lipid membrane. Interestingly, after 2-5 h of incubation, IAPP fibers became “wrapped” by lipid membranes derived from the supported membrane. Progressive increase in molecular-level association between amyloid and membranes in the maturing fibers was confirmed by Förster resonance energy transfer spectroscopy.