hIAPP Fibril Formation Research Articles

The islet tissue plasminogen activator/plasmin system is upregulated with human islet amyloid polypeptide aggregation and protects beta cells from aggregation-induced toxicity

Aims/hypothesisApart from its fibrinolytic activity, the tissue plasminogen activator (tPA)/plasmin system has been reported to cleave the peptide amyloid beta, attenuating brain amyloid deposition in Alzheimer’s disease. As aggregation of human islet amyloid polypeptide (hIAPP) is toxic to beta cells, we sought to determine whether activation of the fibrinolytic system can also reduce islet amyloid deposition and its cytotoxic effects, which are both observed in type 2 diabetes.MethodsThe expression of Plat (encoding tPA) and plasmin activity were measured in isolated islets from amyloid-prone hIAPP transgenic mice or non-transgenic control islets expressing non-amyloidogenic mouse islet amyloid polypeptide cultured in the absence or presence of the amyloid inhibitor Congo Red. Plat expression was also determined in hIAPP-treated primary islet endothelial cells, bone marrow-derived macrophages (BMDM) and INS-1 cells, in order to determine the islet cell type(s) producing tPA in response to hIAPP aggregation. Cell-free thioflavin-T assays and MS were used to respectively monitor hIAPP aggregation kinetics and investigate plasmin cleavage of hIAPP. Cell viability was assessed in INS-1 beta cells treated with hIAPP with or without plasmin. Finally, to confirm the findings in human samples, PLAT expression was measured in freshly isolated islets from donors with and without type 2 diabetes.ResultsIn isolated islets from transgenic mice, islet Plat expression and plasmin activity increased significantly with the process of amyloid deposition (p≤0.01, n=5); these effects were not observed in islets from non-transgenic mice and were blocked by Congo Red (p≤0.01, n=4). In response to hIAPP exposure, Plat expression increased in BMDM and INS-1 cells vs vehicle-treated cells (p≤0.05, n=4), but not in islet endothelial cells. Plasmin reduced hIAPP fibril formation in a dose-dependent manner in a cell-free system, and restored hIAPP-induced loss of cell viability in INS-1 beta cells (p≤0.01, n=5). Plasmin cleaved monomeric hIAPP, inducing a rapid decrease in the abundance of full-length hIAPP and the appearance of hIAPP 1–11 and 12–37 fragments. hIAPP 12–37, which contains the critical amyloidogenic region, was not toxic to INS-1 cells. Finally, PLAT expression was significantly increased by 2.4-fold in islets from donors with type 2 diabetes (n=4) vs islets from donors without type 2 diabetes (n=7) (p≤0.05).Conclusions/interpretationThe fibrinolytic system is upregulated in islets with hIAPP aggregation. Plasmin rapidly degrades hIAPP, limiting its aggregation into amyloid and thus protecting beta cells from hIAPP-induced toxicity. Thus, increasing islet plasmin activity might be a strategy to limit beta cell loss in type 2 diabetes.Graphical

TSPO in pancreatic beta cells and its possible involvement in type 2 diabetes

Amyloidosis forms a large family of pathologies associated with amyloid deposit generated by the formation of amyloid fibrils or plaques. The amyloidogenic proteins and peptides involved in these processes are targeted against almost all organs. In brain they are associated with neurodegenerative disease, and the Translocator Protein (TSPO), overexpressed in these inflammatory conditions, is one of the target for the diagnostic. Moreover, TSPO ligands have been described as promising therapeutic drugs for neurodegenerative diseases. Type 2 diabetes, another amyloidosis, is due to a beta cell mass decrease that has been linked to hIAPP (human islet amyloid polypeptide) fibril formation, leading to the reduction of insulin production. In the present study, in a first approach, we link overexpression of TSPO and inflammation in potentially prediabetic patients. In a second approach, we observed that TSPO deficient rats have higher level of insulin secretion in basal conditions and more IAPP fibrils formation compared with wild type animals. In a third approach, we show that diabetogenic conditions also increase TSPO overexpression and IAPP fibril formation in rat beta pancreatic cell line (INS-1E). These data open the way for further studies in the field of type 2 diabetes treatment or prevention.

Structural Insights into Seeding Mechanisms of hIAPP Fibril Formation.

The deposition of islet amyloid polypeptide (hIAPP) fibrils is a hallmark of β-cell death in type II diabetes. In this study, we employ state-of-the-art MAS solid-state spectroscopy to investigate the previously elusive N-terminal region of hIAPP fibrils, uncovering both rigidity and heterogeneity. Comparative analysis between wild-type hIAPP and a disulfide-deficient variant (hIAPPC2S,C7S) unveils shared fibril core structures yet strikingly distinct dynamics in the N-terminus. Specifically, the variant fibrils exhibit extended β-strand conformations, facilitating surface nucleation. Moreover, our findings illuminate the pivotal roles of specific residues in modulating secondary nucleation rates. These results deepen our understanding of hIAPP fibril assembly and provide critical insights into the molecular mechanisms underpinning type II diabetes, holding promise for future therapeutic strategies.

Regulation of oxaliplatin and carboplatin on the assembly behavior and cytotoxicity of human islet amyloid polypeptide

Human islet amyloid polypeptide (hIAPP) is associated with the pathology of Type II diabetes (T2DM) due to its misfolding and amyloid deposition. The peptide is widely concerned as a potential drug target, and the prevention of hIAPP fibrillation is a rational therapeutic strategy for T2DM. Platinum complexes are promising anticancer agents with good biocompatibility, they can resist the aggregation of amyloid peptides, while the effects of oxaliplatin and carboplatin on hIAPP fibrillation are unknown. In the present work, we selected the two platinum drugs to reveal their inhibition and disaggregation against hIAPP fibrillation by various biophysical methods. The two complexes impeded hIAPP fibril formation and dispersed the aggregates into small oligomers and most monomers. They also reduced peptides oligomerization and promoted rat insulinoma β-cells viability. They bound to hIAPP mainly through metal coordination and hydrophobic interactions. Moreover, oxaliplatin showed better inhibition and regulation on peptides aggregation and cytotoxicity than carboplatin. This work is of important biomedical values for clinical platinum drugs against T2DM and other amyloidosis related diseases.

Inhibitory activities of flavonoids from Scutellaria baicalensis Georgi on amyloid aggregation related to type 2 diabetes and the possible structural requirements for polyphenol in inhibiting the nucleation phase of hIAPP aggregation

Human islet amyloid polypeptide (hIAPP)-mediated cytotoxicity is identified as a potential target for developing new anti-diabetic molecules. Herein, we investigated the effect of the major bioactive compounds of Scutellaria baicalensis Georgi (S. baicalensis), including baicalein, baicalin, wogonin and oroxylin A, on hIAPP aggregation. We found that all of these compounds inhibited hIAPP fibril formation in a dose-dependent manner. But baicalein and baicalin, especially baicalein are more effective than wogonin and oroxylin A in stabilizing hIAPP monomers and eliminating toxic hIAPP assembly, suggesting that flavonoids with ortho-hydroxyl group on the A-ring exhibited higher anti-hIAPP nucleation potential than those without this structure. This stimulated our interest in further studying the possible structure-activity relationship between polyphenol and hIAPP aggregation inhibition. Our results demonstrated that flavonoids with ortho-hydroxyl group on the B-ring are also more effective against hIAPP nucleation than those without this structure. These results suggest that the ortho-hydroxybenzene structure is a key structural feature required for polyphenols to effectively inhibit hIAPP nucleation. This was further confirmed by the effects of polyphenol and phenols in inhibiting hIAPP nucleation. The conclusion that pyrogallol-type polyphenols are potential lead inhibitors may provide a valuable structural template for the further development of polyphenol-based inhibitor of amyloid peptides.

HIAPP-Amyloid-Core Derived d-Peptide Prevents hIAPP Aggregation and Destabilizes Its Protofibrils.

The aberrant misfolding of human islet amyloid polypeptide into cytotoxic amyloid aggregates is the hallmark of type II diabetes. In order to avert the formation of amyloid aggregation, a variety of peptides has been used as inhibitors. Recently, a peptide derived from the amyloidogenic core of hIAPP (hIAPP22-27) and consisting of all d-amino acid residues (D-nl), was found to efficiently prevent hIAPP fibril formation. To investigate the mechanism via which D-nl inhibits hIAPP aggregation, we have carried out all-atom molecular dynamics simulations, where we observe that the ordered β-sheet structure of hIAPP22-27 is completely destabilized when D-nl is incorporated in it. The formation of β-sheet structures by full-length hIAPP is also not favored in the presence of D-nl peptides, due to which hIAPP tends to attain a random loosely packed conformation. As a control, we also study the influence of hIAPP22-27 (L-nl) on the aggregation propensity of full length hIAPP. While L-nl supports the aggregation of hIAPP by stabilizing the β-sheet rich aggregates, D-nl interrupts hIAPP-hIAPP interactions via hydrogen bonding and hydrophobic interactions, thus obstructing the self-aggregation of hIAPP. Further, D-nl also partially dissolves the preformed hIAPP protofibrils. This work provides new insight into the activity of peptide inhibitors against amyloid aggregation at a molecular level and can be exploited to advance the field of diabetes treatment.

Metastable intermediate during hIAPP aggregation catalyzed by membranes as detected with 2D IR spectroscopy.

The aggregation of human islet amyloid polypeptide (hIAPP) into amyloid fibrils involves formation of oligomeric intermediates that are thought to be the cytotoxic species responsible for β-cell dysfunction in type 2 diabetes. hIAPP oligomers permeating or disrupting the cellular membrane may be one mechanism of toxicity and so measuring the structural kinetics of aggregation in the presence of membranes is of much interest. In this study, we use 2D IR spectroscopy and 13C18O isotope labeling to study the secondary structure of the oligomeric intermediates formed in solution and in the presence of phospholipid vesicles at sites L12A13, L16V17, G24A25 and V32G33. Pairs of labels monitor the couplings between associated polypeptides and the dihedral angles between adjacent residues. In solution, the L12A13 residues form an oligomeric β-sheet in addition to an α-helix whereas with the phospholipid vesicles they are α-helical throughout the aggregation process. In both solution and with DOPC vesicles, L16V17 and V32G33 have disordered structures until fibrils are formed. Similarly, under both conditions, G24A25 exhibits 3-state kinetics, created by an oligomeric intermediate with a well-defined β-sheet structure. Amyloid fibril formation is often thought to involve intermediates with exceedingly low populations that are difficult to detect experimentally. These experiments establish that amyloid fibril formation of hIAPP when catalyzed by membranes includes a metastable intermediate and that this intermediate has a similar structure at G24A25 in the FGAIL region as the corresponding intermediate in solution, thought to be the toxic species.

Investigation of the effects of two major secretory granules components, insulin and zinc, on human-IAPP amyloid aggregation and membrane damage

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic peptide found in pancreatic islets of type-2 diabetes (T2D) patients. Under certain conditions, hIAPP is able to form amyloid fibrils that play a role in the progression of T2D. hIAPP is synthesized in the β-cell of the pancreas and stored in the secretory granules before being released into the extracellular compartment. It has been suggested that natural stabilizing agents, such as insulin or zinc present in the secretory granules with hIAPP could prevent hIAPP fibril formation. The difference in the amino acid sequences of IAPP among species strongly correlates with amyloidogenicity and toxicity. The residue histidine at position 18 is known to be important in modulating the fibril formation, membrane leakage and toxicity. In this study, we have synthesized four analogues of hIAPP (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and characterized their aggregation with either insulin or zinc in order to determine the effect of the residue-18 on the insulin-IAPP and zinc-IAPP interactions using a variety of biophysical experiments including thioflavin-T fluorescence, transmission electron microscopy imaging, circular dichroism, and NMR spectroscopy. We show that insulin reduced hIAPP fibril formation both in solution and in the presence of membrane and hIAPP-membrane damage and that the interactions are somewhat mediated by the residue-18. In addition, our results reveal that zinc affects the process of hIAPP fibril formation in solution but not in the presence of membrane. Our results indicate that the nature of the residue-18 is important for zinc binding. Based on this observation, we hypothesize that zinc binds to the residues in the N-terminal region of hIAPP, which is not accessible in the presence of membrane due to its strong interaction with lipids.

Tetracycline derivatives resist the assembly behavior of human islet amyloid polypeptide

The misfolding of amyloid proteins is closely correlated with the pathogenesis of protein conformation-related diseases, such as Alzheimer’s disease (AD), prion disease, and type 2 diabetes mellitus (T2DM). The deposition of human islet amyloid polypeptide (hIAPP) and amyloid-β (Aβ) protein is entangled in AD and diabetes mellitus. The development of potential inhibitors is a feasible therapeutic strategy to treat these diseases by resisting peptide aggregation. Doxycycline is a typical clinical antibiotic that has been utilized in neurodegenerative studies. However, the roles of tetracyclines in hIAPP aggregation remain unclear. Herein, we studied the inhibitory effects of three tetracycline derivatives, namely, minocycline hydrochloride (1), methacycline hydrochloride (2), and doxycycline (3), on the fibril formation and cytotoxicity of hIAPP and compared with that of Aβ. The well-known 3 was selected and compared with 1 and 2. Tetracycline derivatives acted as effective inhibitors to reverse the self-assembly of hIAPP and Aβ, and disaggregate the aged peptides fibrils into mostly monomers. Tetracycline derivatives also reduced the cytotoxicity induced by amyloid peptide oligomerization. Further molecular mechanism studies revealed hydrophobic and hydrogen bond interactions as the primary binding pattern between tetracycline derivatives and peptides. A good bioactivity against amyloidosis was demonstrated by three tetracyclines. This work provided a basis for using tetracycline antibiotics as potential inhibitors against hIAPP aggregation.

The flanking peptides issue from the maturation of the human islet amyloid polypeptide (hIAPP) slightly modulate hIAPP-fibril formation but not hIAPP-induced cell death

Type 2 diabetes mellitus is a disease characterized by the formation of amyloid fibrillar deposits consisting mainly in human islet amyloid polypeptide (hIAPP), a peptide co-produced and co-secreted with insulin. hIAPP and insulin are synthesized by pancreatic β cells initially as prehormones resulting after sequential cleavages in the mature peptides as well as the two flanking peptides (N- and C-terminal) and the C-peptide, respectively. It has been suggested that in the secretory granules, the kinetics of hIAPP fibril formation could be modulated by some internal factors. Indeed, insulin is known to be a potent inhibitor of hIAPP fibril formation and hIAPP-induced cell toxicity. Here we investigate whether the flanking peptides could regulate hIAPP fibril formation and toxicity by combining biophysical and biological approaches. Our data reveal that both flanking peptides are not amyloidogenic. In solution and in the presence of phospholipid membranes, they are not able to totally inhibit hIAPP-fibril formation neither hIAPP-membrane damage. In the presence of INS-1 cells, a rat pancreatic β-cell line, the flanking peptides do not modulate hIAPP fibrillation neither hIAPP-induced cell death while in the presence of human islets, they have a slightly tendency to reduce hIAPP fibril formation but not its toxicity. These data demonstrate that the flanking peptides do not strongly contribute to reduce mature hIAPP amyloidogenesis in solution and in living cells, suggesting that other biochemical factors present in the cells must act on mature hIAPP fibril formation and hIAPP-induced cell death.

The small molecule inhibitor anle145c thermodynamically traps human islet amyloid peptide in the form of non-cytotoxic oligomers

Type 2 diabetes (T2DM) is associated with aggregation of the human islet amyloid polypeptide (hIAPP) into cytotoxic amyloid species. Here we tested the effect of a diphenylpyrazole (DPP)-derived small molecule inhibitor, anle145c, on cytotoxicity and on aggregation properties of hIAPP. We demonstrate that incubation of hIAPP with the inhibitor yields ~10 nm-sized non-toxic oligomers, independent of the initial aggregation state of hIAPP. This suggests that anle145c has a special mode of action in which anle145c-stabilized oligomers act as a thermodynamic sink for the preferred aggregation state of hIAPP and anle145c. We also demonstrate that the inhibitor acts in a very efficient manner, with sub-stoichiometric concentrations of anle145c being sufficient to (i) inhibit hIAPP-induced death of INS-1E cells, (ii) prevent hIAPP fibril formation in solution, and (iii) convert preformed hIAPP fibrils into non-toxic oligomers. Together, these results indicate that anle145c is a promising candidate for inhibition of amyloid formation in T2DM.

2124-P: The Tissue Plasminogen Activator (tPA)/Plasmin System Reduces Amyloid Formation by Cleaving Human Islet Amyloid Polypeptide (hIAPP)

Type 2 diabetes is characterized by islet amyloid deposition, which is associated with β-cell loss. hIAPP is the unique peptide component of these deposits. We previously found that expression of tPA, the initiator of fibrinolysis, is upregulated in islets by amyloid formation in vitro. As plasmin, the proteolytic product of tPA activity, cleaves Aβ and attenuates amyloid deposition in Alzheimer’s disease, we hypothesized that the increase in islet tPA by amyloid deposition serves a protective function by generating plasmin to limit further accumulation of hIAPP. We therefore sought to determine whether the tPA/plasmin system reduces amyloid formation by hIAPP and, if so, what islet cell type produces tPA. Using a thioflavin T assay, we found 4 µM plasmin abrogated 20 µM hIAPP fibril formation by 91.6±0.9% (n=5; p<0.01). To determine whether this could be by plasmin proteolysis of hIAPP, 20 µM hIAPP was incubated with 4 µM plasmin or buffer and then mass spectrometry was performed. After 10 minutes of incubation with plasmin, intact hIAPP was absent and expected cleavage products were present. As tPA is produced by macrophages and endothelial cells, we next determined whether tPA expression is increased by hIAPP in either or both of these cell types. In rat bone-marrow-derived macrophages treated for 24 h with 10 µM hIAPP, non-amyloidogenic 10 µM rat IAPP (rIAPP) or vehicle, tPA expression was increased only by hIAPP (4.2±1.0 vs. 1.5±0.6 vs. 1.0±0.5 fold, respectively; n=4; p<0.05 for hIAPP vs. rIAPP and vehicle). In contrast, in primary rat islet endothelial cells, tPA expression was not increased by 20 µM hIAPP (0.7±0.3 vs. 1.0±0.4 fold; n=3; p=0.2 for hIAPP vs. vehicle). In summary, plasmin rapidly degrades hIAPP, curbing its aggregation into amyloid. As tPA is expressed in macrophages, enhancing its activity or its production by islet macrophages may represent a novel strategy to generate plasmin, cleave hIAPP and thus reduce islet amyloid formation and β-cell loss. Disclosure N. Esser: None. M.F. Hogan: None. A. Aplin: None. A.T. Templin: None. S. El-Osta: None. D. Raleigh: None. J.S. Edgar: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. R.L. Hull: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk A/S. Consultant; Self; Neurimmune. Other Relationship; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk A/S. Funding U.S. Department of Veterans Affairs (BX001060); National Institutes of Health (GM078114); Belgian American Educational Foundation; Société Francophone du Diabète

Influence of methionine-ruthenium complex on the fibril formation of human islet amyloid polypeptide.

The abnormal aggregation and deposition of human islet amyloid polypeptide (hIAPP) are implicated in the pathogeny of type 2 diabetes mellitus (T2DM). Many aromatic ring-containing Ru complexes inhibit the aggregation of hIAPP. A new Ru complex Ru(bipy)(met)2·3H2O (1), where bipy is 2,2'-bipyridine and met is methionine, was synthesized and employed to resist the fibril formation of hIAPP and to promote the biocompatibility of metal complexes. Two polypyridyl Ru complexes, namely [Ru(bipy)3]Cl2(2) and Ru(bipy)2Cl2(3), were used for comparison. Results reveal that the three Ru complexes can inhibit hIAPP aggregation and depolymerize mature hIAPP fibrils. Interaction studies show that Ru complexes bind to hIAPP through metal coordination, hydrophobic interaction, and other intermolecular forces. The binding of the three compounds is spontaneous and exothermic. The compounds also rescue peptide-induced cytotoxicity to some extent. Similar to 3, the novel methionine-Ru complex 1 exhibits an enhanced inhibitory effect and binding affinity to hIAPP possibly because of the smaller steric hindrance and more profitable molecular configuration of 1 than those of 2. The newly designed amino acid-Ru complex may provide new insights into the treatment of T2DM and related amyloidosis diseases. Methionine-Ru complex effectively impedes the fibril formation of human islet amyloid polypeptide.

Regulation of heteronuclear Pt–Ru complexes on the fibril formation and cytotoxicity of human islet amyloid polypeptide

The deposition of human islet amyloid polypeptide (hIAPP) is considered as a causative factor of type 2 diabetes mellitus (T2DM). Developing effective inhibitors against the fibril formation of hIAPP is a potential way to treat T2DM. Recent studies indicate that various metal complexes including homo-binuclear Ru complexes can inhibit hIAPP aggregation. Hetero-multinuclear PtRu metal complexes exhibit multiple bioactivities, but their roles in reversing amyloidosis remain unclear. In this work, we synthesized and identified a new hetero-binuclear PtRu metal complex Na{[RuCl4(DMSO-S)](bpy)[Pt(DMSO-S)Cl2]} (bpy: 4,4′-bipyridyl). We studied the inhibitory effect of the compound on hIAPP aggregation together with K{[RuCl4(DMSO-S)](pyz)[Pt (DMSO-S)Cl2]} (pyz: pyrazine) through diverse biophysical methods. Results showed that two PtRu metal complexes can remarkably reverse hIAPP aggregation and scatter the fibrils into nanoscale particles. Thermodynamic and spectrometric studies revealed that the binding of metal complexes with hIAPP was a spontaneous, enthalpy-driven process resulting from the predominant hydrophobic interaction and metal coordination. Two hetero-binuclear PtRu metal complexes showed stronger binding affinity and better inhibitory effects against peptide fibril formation than homo-binuclear Ru complexes and corresponding mononuclear Ru complexes. The compounds also regulated the peptide-induced cytotoxicity against Insulinoma β-cells and significantly increased the cell viability. This work shed light on a potential strategy for designing hetero-multinuclear metal complexes against amyloidosis-related diseases.

Schiff base oxovanadium complexes resist the assembly behavior of human islet amyloid polypeptide

The misfolding and fibrillation of human islet amyloid polypeptide (hIAPP) is related to the pathologic process of type II diabetes mellitus (T2DM). The inhibitors of hIAPP aggregation include aromatic organic molecules, short peptides, and metal complexes. Vanadium complexes have been applied for the treatment of diabetes since the 19th century. However, the antidiabetes mechanism remains unclear. In this work, we used four Schiff base oxidovanadium(IV) complexes, namely VO(bhbb)·H2O (1, and ligand 1 H2bhbb, 2-(5-bromo-2-hydroxylbenzylideneamino) benzoic acid), VO(nhbb)·H2O (2, and lignad 2 H2nhbb, 2-(5-nitro-2-hydroxylbenzylideneamino) benzoic acid), VO(cpmp)2 (3, and ligand 3 Hcpmp, 4-chloro-2-(phenylimino) methyl) phenol), and VO(bpmp)2 (4, and ligand 4 Hbpmp, 4-bromo- 2-(phenylmino) methyl) phenol) to inhibit the fibril formation of hIAPP and reduce peptide-induced cytotoxicity. Results indicated that the four Schiff base oxidovanadium complexes effectively impeded hIAPP aggregation and disaggregated mature fibrils into monomers or oligomers. These V complexes also decreased hIAPP-induced cytotoxicity. Among the four V complexes, 1 is a promising candidate metallodrug considering its inhibitory effect, disaggregation ability, regulation of peptide-induced cytotoxicity, and binding affinity to the peptide. Our research provides a new outlook for the design of oxidovanadium complexes as effective inhibitors of hIAPP against T2DM.

Amyloid-induced β-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment.

Human islet amyloid polypeptide (hIAPP) aggregation is associated with β-cell dysfunction and death in type 2 diabetes (T2D). we aimed to determine whether in vivo treatment with chemical chaperone 4-phenylbutyrate (PBA) ameliorates hIAPP-induced β-cell dysfunction and islet amyloid formation. Oral administration of PBA in hIAPP transgenic (hIAPP Tg) mice expressing hIAPP in pancreatic β cells counteracted impaired glucose homeostasis and restored glucose-stimulated insulin secretion. Moreover, PBA treatment almost completely prevented the transcriptomic alterations observed in hIAPP Tg islets, including the induction of genes related to inflammation. PBA also increased β-cell viability and improved insulin secretion in hIAPP Tg islets cultured under glucolipotoxic conditions. Strikingly, PBA not only prevented but even reversed islet amyloid deposition, pointing to a direct effect of PBA on hIAPP. This was supported by in silico calculations uncovering potential binding sites of PBA to monomeric, dimeric, and pentameric fibrillar structures, and by in vitro assays showing inhibition of hIAPP fibril formation by PBA. Collectively, these results uncover a novel beneficial effect of PBA on glucose homeostasis by restoring β-cell function and preventing amyloid formation in mice expressing hIAPP in β cells, highlighting the therapeutic potential of PBA for the treatment of T2D.-Montane, J., de Pablo, S., Castaño, C., Rodríguez-Comas, J., Cadavez, L., Obach, M., Visa, M., Alcarraz-Vizán, G., Sanchez-Martinez, M., Nonell-Canals, A., Parrizas, M., Servitja, J.-M., Novials, A. Amyloid-induced β-cell dysfunction and islet inflammation are ameliorated by 4-phenylbutyrate (PBA) treatment.

Residue specific effects of human islet polypeptide amyloid on self-assembly and on cell toxicity

Type 2 diabetes mellitus is characterized histopathologically by the presence of fibrillary amyloid deposits in the pancreatic islets of Langerhans. Human islet amyloid polypeptide (hIAPP), the 37-residue pancreatic hormone, is the major constituent of these amyloid deposits. The propensity of IAPP to form amyloid fibrils is strongly dependent on its primary sequence. An intriguing example is His at residue 18. Although H18 is located outside the amyloidogenic region, it has been suggested that this residue and its charge state play an important role in the kinetics of conformational changes and fibril formation as well as in mediating cell toxicity. To gain more insight into the importance of this residue, we have synthesized four analogues (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and we performed a full biophysical study on the properties of these peptides. Kinetic experiments as monitored by thioflavin-T fluorescence, transmission electron microscopy, circular dichroism and cell toxicity assays revealed that all variants are less fibrillogenic and less toxic than native hIAPP both at neutral pH and at low pH. This demonstrates that the effect of H18 in native IAPP is not simply determined by its charge state, but rather that residue 18 is important for specific intra- and intermolecular interactions that occur during fibril formation and that may involve charge, size and hydrophobicity. Furthermore, our results indicate that H18R-IAPP has a strong inhibiting effect on native hIAPP fibril formation. Together these results highlight the large impact of modifying a single residue outside the amyloidogenic domain on fibril formation and cell toxicity induced by IAPP, opening up new avenues for design of inhibitors or modulators of IAPP aggregation.

Molecular Structure, Membrane Interactions, and Toxicity of the Islet Amyloid Polypeptide in Type 2 Diabetes Mellitus.

Human islet amyloid polypeptide (hIAPP) is the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Mature hIAPP, a 37-aa peptide, is natively unfolded in its monomeric state but forms islet amyloid in T2DM. In common with other misfolded and aggregated proteins, amyloid formation involves aggregation of monomers of hIAPP into oligomers, fibrils, and ultimately mature amyloid deposits. hIAPP is coproduced and stored with insulin by the pancreatic islet β-cells and is released in response to the stimuli that lead to insulin secretion. Accumulating evidence suggests that hIAPP amyloid deposits that accompany T2DM are not just an insignificant phenomenon derived from the disease progression but that hIAPP aggregation induces processes that impair the functionality and the viability of β-cells. In this review, we particularly focus on hIAPP structure, hIAPP aggregation, and hIAPP-membrane interactions. We will also discuss recent findings on the mechanism of hIAPP-membrane damage and on hIAPP-induced cell death. Finally, the development of successful antiamyloidogenic agents that prevent hIAPP fibril formation will be examined.

The effects of organic solvents on the membrane-induced fibrillation of human islet amyloid polypeptide and on the inhibition of the fibrillation

The organic solvent dimethylsulphoxide (DMSO) and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) have been widely used as a pre-treating agent of amyloid peptides and as a vehicle for water-insoluble inhibitors. These solvents are left in many cases as a trace quantity in bulk and membrane environments with treated amyloid peptides or inhibitors. In the present work, we studied the effects of the two organic solvents on the aggregation behaviors of human islet amyloid polypeptide (hIAPP) and the performances of an all-D-amino-acid inhibitor D-NFGAIL in preventing hIAPP fibrillation both in bulk solution and at phospholipid membrane. We showed that the presence of 1% v/v DMSO or HFIP decreases the rate of fibril formation of hIAPP at the lipid membrane rather than accelerates the fibril formation as what happened in bulk solution. We also showed that the presence of 1% v/v DMSO or HFIP impairs the activity of the inhibitor at the lipid membrane surface dramatically, while it affects the efficiency of the inhibitor in bulk solution slightly. We found that the inhibitor inserts into the lipid membrane more deeply or with more proportion in the presence of the organic solvents than it does in the absence of the organic solvents, which may hinder the binding of the inhibitor to hIAPP at the lipid membrane. Our results suggest that the organic solvents should be used with caution in studying membrane-induced fibrillogenesis of amyloid peptides and in testing amyloid inhibitors under membrane environments to avoid incorrect evaluation to the fibrillation process of amyloid peptides and the activity of inhibitors.

Defining the molecular basis of amyloid inhibitors: human islet amyloid polypeptide-insulin interactions.

Human islet amyloid polypeptide (hIAPP or Amylin) is a 37 residue hormone that is cosecreted with insulin from the pancreatic islets. The aggregation of hIAPP plays a role in the progression of type 2 diabetes and contributes to the failure of islet cell grafts. Despite considerable effort, little is known about the mode of action of IAPP amyloid inhibitors, and this has limited rational drug design. Insulin is one of the most potent inhibitors of hIAPP fibril formation, but its inhibition mechanism is not understood. In this study, the aggregation of mixtures of hIAPP with insulin, as well as with the separate A and B chains of insulin, were characterized using ion mobility spectrometry-based mass spectrometry and atomic force microscopy. Insulin and the insulin B chain target the hIAPP monomer in its compact isoform and shift the equilibrium away from its extended isoform, an aggregation-prone conformation, and thus inhibit hIAPP from forming β-sheets and subsequently amyloid fibrils. All-atom molecular modeling supports these conclusions.