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Currently, there are more than 10 million patients with diabetes mellitus in Japan. Therefore, the need to explore the pathogenesis of diabetes and the complications leading to its cure is becoming increasingly urgent. Pathological examination of pancreatic tissues from patients with type 2 diabetes reveals a decrease in the volume of beta cells because of a combination of various stresses. In human type 2 diabetes, islet amyloid deposition is a unique pathological change characterized by proinflammatory macrophage (M1) infiltration into the islets. The pathological changes in the pancreas with islet amyloid were different according to clinical factors, which suggests that type 2 diabetes can be further subclassified based on islet pathology. On the other hand, diabetic peripheral neuropathy is the most frequent diabetic complication. In early diabetic peripheral neuropathy, M1 infiltration in the sciatic nerve evokes oxidative stress or attenuates retrograde axonal transport, as clearly demonstrated by in vitro live imaging. Furthermore, islet parasympathetic nerve density and beta cell volume were inversely correlated in type 2 diabetic Goto‐Kakizaki rats, suggesting that diabetic peripheral neuropathy itself may contribute to the decrease in beta cell volume. These findings suggest that the pathogenesis of diabetes mellitus and diabetic peripheral neuropathy may be interrelated. [ABSTRACT FROM AUTHOR]

The deposition of insoluble proteinaceous aggregates in the form of amyloid fibrils within the extracellular space of tissues is associated with numerous diseases. The development of molecular approaches to arrest amyloid formation and prevent cellular degeneration remains very challenging due to the complexity of the process of protein aggregation, which encompasses an infinite array of conformations and quaternary structures. Polyanionic biopolymers, such as glycosaminoglycans and RNAs, have been shown to modulate the self‐assembly of amyloidogenic polypeptides and to reduce the toxicity induced by the formation of oligomeric and/or pre‐fibrillar proteospecies. This study evaluates the effects of double‐stranded DNA (dsDNA) nanostructures (1D, 2D, and 3D) on amyloid self‐assembly, fibril disaggregation, and the cytotoxicity associated with amyloidogenesis. Using the islet amyloid polypeptide (IAPP) whose pancreatic accumulation is the hallmark of type 2 diabetes, it was observed that dsDNA nanostructures inhibit amyloid formation by inducing the formation of spherical complexes in which the peptide adopts a random coil conformation. Interestingly, the DNA nanostructures showed a persistent ability to disassemble enzymatically and thermodynamically stable amyloid fibrils into nanoscale DNA/IAPP entities that are fully compatible with β‐pancreatic cells and are biodegradable by proteolysis. Notably, dsDNA nanostructures avidly trapped highly toxic soluble oligomeric species in complete cell culture media and converted them into non‐toxic binary complexes. Overall, these results expose the potent modulatory effects of dsDNA on amyloidogenic pathways, and these DNA nanoscaffolds could be used as a source of inspiration for the design of molecules to fight amyloid‐related disorders. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) results from insulin secretory dysfunction arising in part from the loss of pancreatic islet β‐cells. Several factors contribute to β‐cell loss, including islet amyloid formation, which is observed in over 90% of individuals with T2D. The amyloid is comprised of human islet amyloid polypeptide (hIAPP). Here we provide evidence that early in aggregation, hIAPP forms toxic oligomers prior to formation of amyloid fibrils. The toxic oligomers contain α‐sheet secondary structure, a nonstandard secondary structure associated with toxic oligomers in other amyloid diseases. De novo, synthetic α‐sheet compounds designed to be nontoxic and complementary to the α‐sheet structure in the toxic oligomers inhibit hIAPP aggregation and neutralize oligomer‐mediated cytotoxicity in cell‐based assays. In vivo administration of an α‐sheet design to mice for 4 weeks revealed no evidence of toxicity nor did it elicit an immune response. Furthermore, the α‐sheet designs reduced endogenous islet amyloid formation and mitigation of amyloid‐associated β‐cell loss in cultured islets isolated from an hIAPP transgenic mouse model of islet amyloidosis. Characterization of the involvement of α‐sheet in early aggregation of hIAPP and oligomer toxicity contributes to elucidation of the molecular mechanisms underlying amyloid‐associated β‐cell loss. [ABSTRACT FROM AUTHOR]

Aims: This study aims to study the association between pancreatic islet amyloid polypeptide (IAPP) and Alzheimer's disease neuropathological change (ADNC) in brain biopsies obtained from subjects with idiopathic normal pressure hydrocephalus (iNPH) and in post‐mortem (PM) brain samples obtained from aged individuals. Methods: For the immunohistochemical (IHC) analyses, two IAPP antibodies (Abs), monoclonal and polyclonal, and Abs directed towards ADNC were applied. Results: The iNPH cohort included 113 subjects. Amyloid‐β (Aβ) was detected in 50% and hyperphosphorylated τ (HPτ) in 47% of the cases. Concomitant pathology was seen in 32%. The PM cohort included 77 subjects. Aβ was detected in 69% and HPτ in 91% of the cases. Combined Aβ/HPτ pathology was seen in 62%. Reactivity for the monoclonal IAPP was not detected in the brain tissue in either of the cohorts. Reactivity for the polyclonal IAPP was observed in all 77 PM brain samples. Conclusions: There was no specific expression of IAPP in human brain tissue; hence, an association between IAPP and ADNC is not assessable. Of note, the observed reactivity of the polyclonal IAPP Ab was not reproduced with a specific monoclonal Ab; thus, we considered the observed staining with the polyclonal Ab to be unreliable. When using IHC, several pitfalls, especially the choice of an Ab, always need to be considered. Polyclonal Abs cross‐react with other epitopes and proteins, thus leading to false‐positive results. This seems to be the case for the polyclonal IAPP Abs in the human brain. [ABSTRACT FROM AUTHOR]

Human islet amyloid polypeptide (hIAPP) is a naturally occurring, intrinsically disordered protein (IDP) whose abnormal aggregation into toxic soluble oligomers and insoluble amyloid fibrils is a pathological feature in type‐2 diabetes. Rat IAPP (rIAPP) differs from hIAPP by only six amino acids yet has a reduced tendency to aggregate or form fibrils. The structures of the monomeric forms of IAPP are difficult to characterize due to their intrinsically disordered nature. Molecular dynamics simulations can provide a detailed characterization of the monomeric forms of rIAPP and hIAPP in near‐physiological conditions. In this work, the conformational landscapes of rIAPP and hIAPP as a function of secondary structure content were predicted using well‐tempered bias exchange metadynamics simulations. Several combinations of commonly used biomolecular force fields and water models were tested. The predicted conformational preferences of both rIAPP and hIAPP are typical of IDPs, exhibiting dominant random coil structures but showing a low propensity for transient α‐helical conformations. Predicted nuclear magnetic resonance Cα chemical shifts reveal different preferences with each force field towards certain conformations, with AMBERff99SBnmr2/TIP4Pd showing the best agreement with the experiment. Comparisons of secondary structure content demonstrate residue‐specific differences between hIAPP and rIAPP that may reflect their different aggregation propensities. [ABSTRACT FROM AUTHOR]

In humans with type 2 diabetes, at least 70% of patients exhibit islet amyloid plaques formed by misfolding islet amyloid polypeptides (IAPP). The oligomeric conformation and accumulation of the IAPP plaques lead to a panoply of cytotoxic effects on the islet β‐cells. Currently, no marketed therapies for the prevention or elimination of these amyloid deposits exist, and therefore significant efforts are required to address this gap. To date, most of the experimental treatments are limited to only in vitro stages of testing. In general, the proposed therapeutics use various targeting strategies, such as binding to the N‐terminal region of islet amyloid polypeptide on residues 1–19 or the hydrophobic region of IAPP. Other strategies include targeting the peptide self‐assembly through π‐stacking. These methods are realized by using several different families of compounds, four of which are highlighted in this review: naturally occurring products, small molecules, organometallic compounds, and nanoparticles. Each of these categories holds immense potential to optimize and develop inhibitor(s) of pancreatic amyloidosis in the near future. [ABSTRACT FROM AUTHOR]

Amylin is a 37‐residue peptide hormone, which is co‐secreted with insulin and prevents postprandial spikes in blood glycemia by slowing gastric emptying and promoting satiety. Amylin is prone to aggregate into oligomers and amyloid fibrils, which is related to the onset of type 2 diabetes, and hampers its use as a biopharmaceutical. To overcome the instability and extend its in vivo half‐life it has been proposed the conjugation of amylin with polyethylene glycol (PEG) at the HNζ or the HNα amines of Lys1. Here we used two‐dimensional nuclear magnetic resonance spectra aiming the unambiguous identification of the site of covalent modification on amylin. The coupling of PEG causes both a substantial decrease in the chemical exchange of their HN and alterations in the chemical shifts at both the HN and the neighborhood hydrocarbon groups including CHα, CHδ and CHε of Lys1. Additional analysis of chemical shifts indicates alteration in the HNα solvent accessibility of residues Cys2, Asn3, Ala5, Cys7, and Gln10, and confirmed the presence of oxidized Cys2 and Cys7. We believe that the methodology described here is a reference for the characterization of chemical coupling of a number of biopharmaceuticals. [ABSTRACT FROM AUTHOR]

Islet amyloid polypeptide (IAPP or amylin) is a hormone co‐secreted with insulin by pancreatic β‐cells and is the major component of islet amyloid. Islet amyloid is found in the pancreas of patients with type 2 diabetes (T2D) and may be involved in β‐cell dysfunction and death, observed in this disease. Thus, investigating the aspects related to amyloid formation is relevant to the development of strategies towards β‐cell protection. In this sense, IAPP misprocessing, IAPP overproduction, and disturbances in intra‐ and extracellular environments seem to be decisive for IAPP to form islet amyloid. Islet amyloid toxicity in β‐cells may be triggered in intra‐ and/or extracellular sites by membrane damage, endoplasmic reticulum stress, autophagy disruption, mitochondrial dysfunction, inflammation, and apoptosis. Importantly, different approaches have been suggested to prevent islet amyloid cytotoxicity, from inhibition of IAPP aggregation to attenuation of cell death mechanisms. Such approaches have improved β‐cell function and prevented the development of hyperglycemia in animals. Therefore, counteracting islet amyloid may be a promising therapy for T2D treatment. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) are long and unbranched anionic heteropolysaccharides that have been associated with virtually all amyloid deposits. Soluble sulfated GAGs are known for their propensity to promote the self‐assembly of numerous amyloidogenic proteins and to modulate their cytotoxicity. Nonetheless, although GAGs are prevalent on the outer leaflet of eukaryotic cell plasma membrane as part of proteoglycans, their contributions in the perturbation of lipid bilayer induced by amyloid polypeptides remain unknown. Herein, we investigate the roles of GAGs in the cytotoxicity and plasma membrane perturbation induced by the islet amyloid polypeptide (IAPP), whose deposition in the pancreatic islets is associated with type II diabetes. Cellular assays using GAG‐deficient cells reveal that GAGs exacerbate IAPP‐induced cytotoxicity and permeabilization of the plasma membrane. Confocal microscopy and flow cytometry analyses show that IAPP sequestration at the cell surface is dependent of GAGs and of the aggregation propensity of the peptide. Using giant plasma membrane vesicles (GPMVs) prepared from GAG‐deficient cells, we investigate the direct contributions of membrane‐embedded proteoglycans in IAPP‐induced membrane disassembly. In sharp contrast to soluble sulfated GAGs, kinetics of amyloid self‐assembly expose that the presence of GAGs on GPMVs does not significantly modulate in vitro amyloid formation. Overall, this study indicates that cell surface GAGs increase the local concentration of IAPP in the vicinity of the plasma membrane, promoting lipid bilayer perturbation and cell death. [ABSTRACT FROM AUTHOR]

Introduction: This study assessed the hypothesis that circulating human amylin (amyloid-forming) cross-seeds with amyloid beta (Aβ) in early Alzheimer’s disease (AD). Methods: Evidence of amylin-AD pathology interaction was tested in brains of 31 familial AD mutation carriers and 20 cognitively unaffected individuals, in cerebrospinal fluid (CSF) (98 diseased and 117 control samples) and in genetic databases. For functional testing, we genetically manipulated amylin secretion in APP/PS1 and non-APP/PS1 rats. Results: Amylin-Aβ cross-seeding was identified in AD brains. High CSF amylin levels were associated with decreased CSF Aβ42 concentrations. AD risk and amylin gene are not correlated. Suppressed amylin secretion protected APP/PS1 rats against AD-associated effects. In contrast, hypersecretion or intravenous injection of human amylin in APP/PS1 rats exacerbated AD-like pathology through disruption of CSF-brain Aβ exchange and amylin-Aβ cross-seeding. Discussion: These findings strengthened the hypothesis of circulating amylin-AD interaction and suggest that modulation of blood amylin levels may alter Aβ-related pathology/symptoms. [ABSTRACT FROM AUTHOR]

Amyloid self‐assembly is linked to the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes (T2D), but so far, no anti‐amyloid compound has reached the clinic. Macrocyclic peptides belong to the most attractive drug candidates. Herein we present macrocyclic peptides (MCIPs) designed using minimal IAPP‐derived recognition elements as a novel class of nanomolar amyloid inhibitors of both Aβ40(42) and IAPP or Aβ40(42) alone and show that chirality controls inhibitor selectivity. Sequence optimization led to the discovery of an Aβ40(42)‐selective MCIP exhibiting high proteolytic stability in human plasma and human blood–brain barrier (BBB) crossing ability in a cell model, two highly desirable properties for anti‐amyloid AD drugs. Owing to their favorable properties, MCIPs should serve as leads for macrocyclic peptide‐based anti‐amyloid drugs and scaffolds for the design of small‐molecule peptidomimetics for targeting amyloidogenesis in AD or in both AD and T2D. Taking the lead: Amyloid self‐assembly is linked to Alzheimer's disease (AD) and type 2 diabetes (T2D). In a minimalistic approach, macrocyclic peptides (MCIPs) were designed as potent amyloid inhibitors of Aβ40(42) and IAPP and a selective MCIP (see structure) for Aβ40(42) with high proteolytic stability in human plasma and blood–brain‐barrier‐crossing ability in a cell model was discovered. MCIPs are promising leads for amyloid targeting in AD or in both AD and T2D. [ABSTRACT FROM AUTHOR]

Abstract: Proteotoxicity plays a key role in many devastating human disorders, including Alzheimer's, Huntington's and Parkinson's diseases; type 2 diabetes; systemic amyloidosis; and cardiac dysfunction, to name a few. The cellular mechanisms of proteotoxicity in these disorders have been the focus of considerable research, but their role in prevalent and morbid disorders, such as diabetes, is less appreciated. There is a large body of literature on the impact of glucotoxicity and lipotoxicity on insulin‐producing pancreatic β‐cells, and there is increasing recognition that proteotoxicty plays a key role. Pancreatic islet amyloidosis by the hormone IAPP, the production of advanced glycation endproducts (AGE), and insulin misprocessing into cytotoxic aggregates are all sources of β‐cell proteotoxicity in diabetes. AGE, produced by the reaction of reducing sugars with proteins and lipids are ligands for the receptor for AGE (RAGE), as are the toxic pre‐fibrillar aggregates of IAPP produced during amyloid formation. The mechanisms of amyloid formation by IAPP in vivo or in vitro are not well understood, and the cellular mechanisms of IAPP‐induced β‐cell death are not fully defined. Here, we review recent findings that illuminate the factors and mechanisms involved in β‐cell proteotoxicity in diabetes. Together, these new insights have far‐reaching implications for the establishment of unifying mechanisms by which pathological amyloidoses imbue their injurious effects in vivo. [ABSTRACT FROM AUTHOR]

Summary: Immunohistochemical techniques were employed to investigate the distribution of amylin‐like immunoreactive cells in the pancreas of gecko Homopholis fasciata. Four types of endocrine cells were distinguished: insulin immunoreactive (B cells), pancreatic polypeptide immunoreactive (PP cells), glucagon and pancreatic polypeptide immunoreactive (A/PP cells) and somatostatin immunoreactive cells (D cells). Pancreatic islets contained B, A/PP and D cells, whereas extrainsular regions contained B, D and PP cells. In the pancreatic islets, amylin‐like immunoreactive cells corresponded to B cells, but not to A/PP or D cells. In the extrainsular regions, amylin‐like immunoreactive cells corresponded to either B or PP cells. Amylin secreted from intrainsular B cells may regulate pancreatic hormone secretion in an autocrine and/or a paracrine fashion. On the other hand, amylin secreted from extrainsular PP and B cells, and/or intrainsular B cells may participate in the modulation of calcium homoeostasis in an endocrine fashion. [ABSTRACT FROM AUTHOR]

Type 2 diabetes mellitus, a kind of conformational disease, has become an epidemic disease, which seriously endangers the quality of life and health of human beings. The deposition of human islet amyloid polypeptide (hIAPP) has been considered as one of the major pathological features of type 2 diabetes mellitus. As lipopeptides have some hydrophobic groups, which are similar to the reported aggregation inhibitors, and some lipopeptides could prevent cells from depositing of amyloid fibrils, several potential lipopeptide inhibitors have been engineered and synthesized, which have been assessed for their inhibitory effect in preventing amyloid fibrils formation of hIAPP11‐20 by using the conventional thioflavin‐T fluorescence assay and new technique microscale thermophoresis (MST). The final amyloid fibrils of hIAPP11‐20 were characterized by transmission electron microscopy. Results suggested that with the increasing length of alkyl chain, the antiaggregation efficiency of lipopeptide inhibitors towards hIAPP11‐20 increased gradually. Meanwhile, the amount of arginines, which represent the head groups of lipopeptides, may also have some influence. The binding events also showed that the inhibitory efficiency of these lipopeptide inhibitors was enhanced with the increase of affinities between lipopeptides and hIAPP11‐20, which were obtained from MST. This study demonstrated the efficiency of lipopeptides in inhibiting the aggregation of hIAPP11‐20 and proved that MST could be regarded as an appropriate and rapid method to screen potential inhibitors of hIAPP11‐20 or other amyloid proteins. This study also broadens the types of inhibitors on inhibiting amyloid formation of hIAPP. [ABSTRACT FROM AUTHOR]

Much of our knowledge of diabetes is derived from studies of rodent models. An alternative approach explores evolutionary solutions to physiological stress by studying organisms that face challenging metabolic environments. Polar bears eat an enormously lipid-rich diet without deleterious metabolic consequences. In contrast, transgenic rodents expressing the human neuropancreatic polypeptide hormone amylin develop hyperglycemia and extensive pancreatic islet amyloid when fed a high-fat diet. The process of islet amyloid formation by human amylin contributes to β-cell dysfunction and loss of β-cell mass in type 2 diabetes. We show that ursine amylin is considerably less amyloidogenic and less toxic to β-cells than human amylin, consistent with the hypothesis that part of the adaptation of bears to metabolic challenges might include protection from islet amyloidosis-induced β-cell toxicity. Ursine and human amylin differ at four locations: H18R, S20G, F23L, and S29P. These are interesting from a biophysical perspective, since the S20G mutation accelerates amyloid formation, but the H18R slows it. An H18RS20G double mutant of human amylin behaves similarly to the H18R mutant, indicating that the substitution at position 18 dominates the S20G replacement. These data suggest one possible mechanism underpinning the protection of bears against metabolic challenges and provide insight into the design of soluble analogs of human amylin. [ABSTRACT FROM AUTHOR]

Aggregation of islet amyloid polypeptide ( IAPP) is implicated in the development of type 2 diabetes. The modified NFGAIL with double N-methylated at Gly24 and Ile26 has the property of soluble, non-amyloidogenic, non-cytotoxic, and the ability of inhibiting amyloid formation and cytotoxicity of IAPP. To discover the inhibition mechanism of this peptide inhibitor and provide useful information to design more potential peptide inhibitors, molecular dynamics simulations in explicit solvent were performed. The simulation results reveal that Gly24 and Ile26 are of importance in IAPP aggregation, and N-methylation at these two key residues will disrupt the stability of formed oligomer and prevent the conformation transition of free monomer near the oligomer template. The origin of the N-methylated peptide inhibitor inhibiting IAPP aggregation is that it can keep good binding with IAPP template by stable hydrogen bonding interaction. Furthermore, it cannot induce the conformational transition of free monomer by preventing the hydrogen bond interaction between free monomer and boundary peptide. The structural environment can largely affect the stacking of free monomers to the template. Our study sheds light on the inhibition mechanism of peptide inhibitor at molecular level and may provide guidance for the future design and discovery of new peptide inhibitors. [ABSTRACT FROM AUTHOR]

Aims Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to β-cell failure in type 2 diabetes, cultured and transplanted islets. We previously showed that biosynthetic hIAPP aggregates induce β-cell Fas upregulation and activation of the Fas apoptotic pathway. We used cultured human and hIAPP-expressing mouse islets to investigate: (1) the role of interleukin-1β ( IL-1β) in amyloid-induced Fas upregulation; and (2) the effects of IL-1β-induced β-cell dysfunction on pro-islet amyloid polypeptide ( proIAPP) processing and amyloid formation. Research Design and Methods Human and h IAPP-expressing mouse islets were cultured to form amyloid without or with the IL-1 receptor antagonist ( IL-1 Ra) anakinra, in the presence or absence of recombinant IL-1β. Human islets in which amyloid formation was prevented (amyloid inhibitor or Ad-proh IAPP-siRNA) were cultured similarly. β-cell function, apoptosis, Fas expression, caspase-8 activation, islet IL-1β, β-cell area, β-/α-cell ratio, amyloid formation, and (pro) IAPP forms were assessed. Results hIAPP aggregates were found to increase IL-1β levels in cultured human islets that correlated with β-cell Fas upregulation, caspase-8 activation and apoptosis, all of which were reduced by IL-1Ra treatment or prevention of amyloid formation. Moreover, IL-1Ra improved culture-induced β-cell dysfunction and restored impaired proIAPP processing, leading to lower amyloid formation. IL-1β treatment potentiated impaired proIAPP processing and increased amyloid formation in cultured human and h IAPP-expressing mouse islets, which were prevented by IL-1Ra. Conclusions IL-1β plays a dual role by: (1) mediating amyloid-induced Fas upregulation and β-cell apoptosis; (2) inducing impaired proIAPP processing thereby potentiating amyloid formation. Blocking IL-1β may provide a new strategy to preserve β cells in conditions associated with islet amyloid formation. [ABSTRACT FROM AUTHOR]

Human islet amyloid polypeptide (hIAPP), a major constituent of islet amyloid deposits, induces pancreatic β-cell apoptosis and eventually contributes to β-cell deficit in patients with type 2 diabetes mellitus (T2DM). In this study, Tat-mediated transduction of biliverdin reductase A (BLVRA) was investigated in INS-1 cells to examine whether exogenous supplementation of BLVRA prevented hIAPP-induced apoptosis and dysfunction in insulin secretion in β-cells. Tat-BLVRA fusion protein was efficiently delivered into INS-1 cells in a time- and dose-dependent manner. Exposure of cells to hIAPP induced apoptotic cell death, which was dose-dependently inhibited by pre-treatment with Tat-BLVRA for 1 h. Transduced Tat-BLVRA reduced hIAPP-evoked generation of reactive oxygen species, a crucial mediator of β-cell destruction. Immunoblot analysis showed that Tat-BLVRA suppressed hIAPP-induced increase in the levels of proteins involved in endoplasmic reticulum (ER) stress and apoptosis signaling. Transduced Tat-BLVRA also recovered hIAPP-induced dysfunction in basal and glucose-stimulated insulin secretions. These results suggested that transduced Tat-BLVRA enhanced the tolerance of β-cells against IAPP-induced cytotoxicity by alleviating oxidative stress and ER stress. Therefore, Tat-mediated transduction of BLVRA may provide a potential tool to ameliorate β-cell deficit in pancreas with T2DM. [ABSTRACT FROM AUTHOR]

Amyloid deposition underlies a broad range of diseases including multiple neurodegenerative diseases, systemic amyloidosis and type-2 diabetes. Amyloid sensitive dyes, particularly thioflavin-T, are widely used to detect ex-vivo amyloid deposits, to monitor amyloid formation in vitro and to follow the kinetics of amyloid self-assembly. We show that the dye SYPRO-orange binds to amyloid fibrils formed by human amylin, the polypeptide responsible for islet amyloid formation in type-2 diabetes. No fluorescence enhancement is observed in the presence of pre-fibrillar species or in the presence of non-amyloidogenic rat amylin. The kinetics of human amylin amyloid formation can be monitored by SYPRO-orange fluorescence and match the time course determined with thioflavin-T assays. Thus, SYPRO-orange offers an alternative to thioflavin-T assays of amylin amyloid formation. The implications for the interpretation of SYPRO-orange-based assays of protein stability and protein-ligand interactions are discussed. [ABSTRACT FROM AUTHOR]

Amyloid deposition is a hallmark of many diseases, such as the Alzheimer's disease. Numerous amyloidogenic proteins, including the islet amyloid polypeptide (IAPP) associated with type II diabetes, are natively unfolded and need to undergo conformational rearrangements allowing the formation of locally ordered structure(s) to initiate self-assembly. Recent studies have indicated that the formation of α-helical intermediates accelerates fibrillization, suggesting that these species are on-pathway to amyloid assembly. By identifying an IAPP derivative with a restricted conformational ensemble that co-assembles with IAPP, we observed that helical species were off-pathway in homogenous environment and in presence of lipid bilayers or glycosaminoglycans. Moreover, preventing helical folding potentiated membrane perturbation and IAPP cytotoxicity, indicating that stabilization of helical motif(s) is a promising strategy to prevent cell degeneration associated with amyloidogenesis. [ABSTRACT FROM AUTHOR]

The design of inhibitors of protein-protein interactions mediating amyloid self-assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self-assembly. Here we present a hot-segment-linking approach to design a series of mimics of the IAPP cross-amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self- and cross-seeded IAPP self-assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer's disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self-assembly and pathogenic interactions of other proteins as well. [ABSTRACT FROM AUTHOR]

Amyloid deposition of human islet amyloid polypeptide (hI APP) within the islets of Langerhans is a pathological feature of type 2 diabetes mellitus. Substantial evidence indicates that the membrane-mediated aggregation and subsequent deposition of hI APP are linked to dysfunction and death of pancreatic β-cells, but the molecular processes of hI APP deposition are poorly understood. In this study, we examined the membrane-mediated aggregation and deposition of hI APP at supported planar lipid bilayers with and without raft components (i.e. cholesterol and sphingomyelin) to provide insight into hI APP-induced membrane dysfunction. The adsorption of hI APP onto the bilayers was studied using a quartz crystal microbalance with dissipation monitoring, which showed enhanced accumulation of the peptide onto the bilayer containing raft components. Microscope observations demonstrated the growth of the aggregates formed from the membrane-adsorbed hI APP. The examination of the membrane interfaces revealed that hI APP aggregates retained the ability to associate with the membranes during the aggregation process, resulting in insertion of the aggregates into the bilayers. We also report the inhibitory effect of insulin on the hI APP deposition. These findings demonstrate the aggregation of hI APP at the membrane interfaces leading to amyloid deposits associated with the membrane and suggest a role for insulin in hI APP deposition. A presumed mechanism regulating hI APP deposition at the membrane interfaces is discussed. [ABSTRACT FROM AUTHOR]

Amylin or islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone secreted from the pancreatic islets into the blood circulation and is cleared by peptidases in the kidney. IAPP aggregates are strongly associated with β-cell degeneration in type 2 diabetes, as demonstrated by the fact that more than 95% of patients exhibit IAPP amyloid upon autopsy. Recently, it has been reported that metal ions such as copper(II) and zinc(II) are implicated in the aggregation of IAPP as well as able to modulate the proteolytic activity of IAPP degrading enzymes. For this reason, in this work, the role of the latter metal ions in the degradation of IAPP by insulin-degrading enzyme (IDE) has been investigated by a chromatographic and mass spectrometric combined method. The latter experimental approach allowed not only to assess the overall metal ion inhibition of the human and murine IAPP degradation by IDE but also to have information on copper- and zinc-induced changes in IAPP aggregation. In addition, IDE cleavage site preferences in the presence of metal ions are rationalized as metal ion-induced changes in substrate accessibility. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT Glycosaminoglycans (GAGs) are found in association with virtually all extracellular protein deposits related to amyloid diseases. Particularly, GAGs were shown to enhance fibrillogenesis of the islet amyloid polypeptide (IAPP), a peptide hormone whose aggregation is associated with Type-II diabetes pathogenesis. However, the exact molecular mechanism by which GAGs enhance IAPP amyloidogenesis remains unclear as well as the implications of cell surface GAGs in IAPP-mediated cytotoxicity. The aim of this study was to gain conformational and thermodynamics insights about GAGs-IAPP interactions as a function of IAPP protonation state and buffer ionic strength as well as to explore the roles of cell surface GAGs in IAPP cytotoxicity. Isothermal titration calorimetry revealed that protonation of residue His18 increases the binding affinity of IAPP towards heparin and, in turn, strongly stimulates fibrillogenesis. Interaction of IAPP with heparin induces a random coil to helix conformational conversion and the helical intermediates could be on-pathway to amyloid fibrils formation. Using rat beta-cells INS-1 that were enzymatically treated with GAG lyases and a CHO cell line that is deficient in the biosynthesis of GAGs, we observed that the lack of GAGs at the plasma membrane does not prevent IAPP-induced toxicity, whereas the presence of soluble heparin in the cell media inhibits IAPP cytotoxicity. Overall, this study reinforces the postulate that sulfated GAGs are actively implicated in IAPP amyloidogenic process in vivo, where they could play a protective role by interacting with cytotoxic species and converting them into less culprit amyloid fibrils. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 645-655, 2013. [ABSTRACT FROM AUTHOR]

The article discusses research on the potency of n-methylated soluble islet amyloid polypeptide (IAPP) as IAPP receptor agonists and nanomolar inhibitors of cytotoxic self-assembly of IAPP and β-amyloid peptide 40 (Aβ40). It talks about the relationship of IAPP with β-cell degeneration and the pathogenesis of type 2 diabetes (T2D). It also offers information on IAPP as a neuropeptide regulator of glucose homeostasis.

Aims/Introduction Islet amyloid polypeptide ( IAPP) is a main component of islet amyloid in type 2 diabetes and cosecreted from β-cell with insulin. Clinical evidence from the patients with S20 G mutation of the IAPP gene, as well as experimental evidence that insulin could inhibit amyloid formation of IAPP, suggests that a gradual reduction of insulin could be related to the cytotoxicity associated with S20 G- IAPP through long-term deterioration of β-cells in type 2 diabetes. Our objective was to show an effect of human insulin on S20 G- IAPP associated cytotoxicity. Materials and Methods We analyzed the cytotoxicity associated with S20 G- IAPP by controlling human insulin expression using adenovirus vectors with micro ribonucleic acid specifically against human insulin in endocrine At T-20ins cells, which express human insulin permanently. Additionally, we carried out a follow-up study of circulating IAPP and insulin in type 2 diabetic patients. Results S20 G- IAPP expression was associated with a decrease in viability and an increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-positive cells in At T-20ins cells. Furthermore, downregulation of human insulin enhanced the cytotoxicity associated with S20 G- IAPP, and induced the cytotoxicity associated with wild-type ( WT)- IAPP. Reduction of ubiquitin carboxy-terminal hydrolase L1 activity enhanced cytotoxicity under the downregulation of human insulin expression in both S20 G- and WT- IAPP transduced cells. A 5-year follow up of type 2 diabetic patients showed a disproportionate increase of serum fasting IAPP-to-insulin ratio from baseline. Conclusions Human insulin plays a protective role against the cytotoxicity associated with S20 G- IAPP, as well as WT- IAPP. The findings could suggest long-term deterioration of insulin secretion associates with IAPP linked cytotoxicity in type 2 diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Amyloid formation by the neuropancreatic hormone, islet amyloid polypeptide (IAPP or amylin), one of the most amyloidogenic sequences known, leads to islet amyloidosis in type 2 diabetes and to islet transplant failure. Under normal conditions, IAPP plays a role in the maintenance of energy homeostasis by regulating several metabolic parameters, such as satiety, blood glucose levels, adiposity and body weight. The mechanisms of IAPP amyloid formation, the nature of IAPP toxic species and the cellular pathways that lead to pancreatic β-cell toxicity are not well characterized. Several mechanisms of toxicity, including receptor and non-receptor-mediated events, have been proposed. Analogs of IAPP have been approved for the treatment of diabetes and are under investigation for the treatment of obesity. [Copyright &y& Elsevier]

Abstract: Pancreatic islet amyloid is a characteristic feature of type 2 diabetes. The major protein component of islet amyloid is the polypeptide hormone known as islet amyloid polypeptide (IAPP, or amylin). IAPP is stored with insulin in the β-cell secretory granules and is released in response to the stimuli that lead to insulin secretion. IAPP is normally soluble and is natively unfolded in its monomeric state, but forms islet amyloid in type 2 diabetes. Islet amyloid is not the cause of type 2 diabetes, but it leads to β-cell dysfunction and cell death, and contributes to the failure of islet cell transplantation. The mechanism of IAPP amyloid formation is not understood and the mechanisms of cytotoxicity are not fully defined. [Copyright &y& Elsevier]

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self-assembly of human islet amyloid polypeptide (hIAPP)22-29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe-23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe-23 virtually abolish the ability of hIAPP22-29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β-sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the CC ring mode (ca. 1600 cm−1) upon aggregation that has previously been shown to be indicative of π-stacking. While previous work has demonstrated that π-stacking is not an absolute requirement for fibrillization, our findings indicate that Phe-23 also contributes to fibril formation through π-stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self-assembly of hIAPP22-29. © Proteins 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Choline-O-sulfate (2-(trimethylammonio)ethyl sulfate, COS) is a naturally occurring osmolyte that is synthesized by plants, lichens, algae, fungi, and several bacterial species. We examined the inhibitory effects of COS on amyloid formation of the human islet amyloid polypeptide (hIAPP or amylin) using a thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy and transmission electron microscopy. The results showed that COS suppresses a conformational change of hIAPP from a random coil to a β-sheet structure, resulting in the inhibition of amyloid formation. Comparisons with various structural analogs including carnitine, acetylcholine and non-detergent sulfobetaines (NDSBs) using the ThT fluorescence assay showed that COS is the most effective inhibitor of hIAPP amyloid formation, suggesting that the sulfate group, which is unique to COS, significantly contributes to the inhibition. [Copyright &y& Elsevier]

Amyloid accumulation is associated with pathological conditions, including type II diabetes and Alzheimer's disease. Lipids influence amyloidogenesis and are themselves targets for amyloid-mediated cell membrane disruption. Amyloid precursors are surface-active, accumulating at hydrophobic-hydrophilic interfaces (e.g., air-water), where their biophysical and kinetic behaviors differ from those in the bulk solution with significant and underappreciated consequences. Biophysical modeling predicted the probability and rate of β-sheet amyloid dimer formation to be higher and faster at the air-water interface (AWI) than in the bulk (by 14 and ∼1500 times, respectively). Time-course staining experiments with a typical amyloid dye verified our predictions by demonstrating that without AWI, islet amyloid polypeptide (IAPP) fibrilization was abolished or slowed, depending on the conditions. Our controls included undisturbed IAPP reactions, and we ascertained that the AWI removal process (technical or material) did not itself affect the reaction. Furthermore, we showed that the role of membranes in amyloidogenesis has been previously underestimated; in an in vivo-like situation (with no AWI), anionic liposomes (containing dioleoylphosphatidylglycerol) enhanced IAPP fibrilogenesis far more than described previously in conventional assay conditions (in the presence of an AWI). These findings have implications for the protein misfolding field and in assay design to target toxic protein aggregation. [ABSTRACT FROM AUTHOR]

Amylin, or islet amyloid polypeptide, is known as a satiating signal expressed in pancreatic β-cells but not in the brain. In this study, regulations of postpartum mRNA expressions were investigated in the preoptic area of the hypothalamus. mRNA levels of lactating dams and mothers whose pups were removed immediately after delivery were compared in microarray experiments. There were 20 genes identified with significantly increased and 14 with decreased expression 9 days postpartum. Amylin mRNA level demonstrated the largest change, a 25.7 times increase. Quantitative RT-PCR measurements validated the increase in the mRNA level of amylin in the preoptic area of lactating dams while the expression level of other members of the calcitonin gene-related peptide family did not change. In situ hybridization histochemistry for amylin further verified its induction in lactating mothers and demonstrated the distribution of amylin mRNA in the medial preoptic nucleus, parts of the medial preoptic area, and the ventral part of the bed nucleus of the stria terminalis but nowhere else in the rat brain. Immunolabeling verified the postpartum induction of amylin in the preoptic area at the peptide level, as well. The results suggest that amylin may play a part in maternal regulations. [ABSTRACT FROM AUTHOR]

Abstract: Type II diabetes mellitus (T2DM) is a disease characterized by progressive deposition of amyloid in the extracellular matrix of β-cells. We investigated the interaction of the islet amyloid polypeptide (IAPP) with lipid model raft mixtures and INS-1E cells using fluorescence microscopy techniques. Following preferential partitioning of IAPP into the fluid lipid phase, the membrane suffers irreversible damage and predominantly circularly-shaped lipid-containing IAPP amyloid is formed. Interaction studies with the pancreatic β-cell line INS-1E revealed that growing IAPP fibrils also incorporate substantial amounts of cellular membranes in vivo. Additionally, the inhibitory effect of the red wine compound resveratrol on IAPP fibril formation has been studied, alluding to its potential use in developing therapeutic strategies against T2DM. [Copyright &y& Elsevier]

Cross-β amyloid is implicated in over 20 human diseases. Experiments suggest that specific sequence elements within amyloidogenic proteins play a major role in seeding amyloid formation. Identifying these seeding sequences is important for rationalizing the molecular mechanisms of amyloid formation and for elaborating therapeutic strategies that target amyloid. Theoretical techniques play an important role in facilitating the identification and structural characterization of putative seeding sequences; most amyloid species are not amenable to high resolution experimental structure techniques. In this study we have combined a coarse-grained physicochemical protein model with a highly efficient Monte Carlo sampling technique to identify amyloidogenic sequences in four proteins for which respective experimental peptide fragmentation data exist. Peptide sequences were defined as amyloidogenic if the ensemble structure predicted for three interacting peptides described a stable and regular three-stranded β-sheet. For such peptides, free energies were calculated to provide a measure of amyloid propensity. The overall agreement between the experimental and predicted data is good, and we correctly identify several self-recognition motifs proposed to define the cross-β amyloid fibril architectures of two of the proteins. Our results compare very favorably with those obtained using atomistic molecular dynamics methods, though our simulations are 30–40 times faster. © 2008 Wiley Periodicals, Inc. J Comput Chem 2009 [ABSTRACT FROM AUTHOR]

Islet amyloid polypeptide (IAPP, also known as amylin) is the major protein component of pancreatic amyloid fibers in type II diabetes and is normally cosecreted with insulin from the β-cells of the pancreas. IAPP forms amyloid fibrils rapidly at concentrations well below those found in vivo, yet progression of type II diabetes occurs over many years. Insulin, a known inhibitor of IAPP fibrillogenesis, exists as a dense crystalline or near-crystalline core in the secretory vesicle, while IAPP localizes to the region between the crystal and the secretory vesicle membrane. In vitro, IAPP fibrillogenesis is both accelerated by lipid membranes and inhibited by monomeric insulin. In this work, we investigate insulin-IAPP-lipid interactions in vitro under conditions chosen to approximate native secretory vesicle physiology and the amyloid disease state. The effect of insulin on IAPP fibrillogenesis is investigated using fluorescence spectrometry. Additionally, interactions of IAPP and lipids with crystalline insulin are studied using fluorescence microscopy. We find that, while soluble states of insulin and IAPP do not interact significantly, large assemblies of either insulin (crystals) or IAPP (fibers) can lead to stable IAPP-insulin interactions. The results raise the possibility of multiple physiological interactions between these two β-cell hormones. [ABSTRACT FROM AUTHOR]

Amyloidogenesis from soluble protein requires conformational and oligomeric assembly steps. In systems where the precursor protein is natively unfolded, such as islet amyloid polypeptide (IAPP), forces and structural changes relevant to protein unfolding are not thought to participate in the assembly mechanism. Thus, fiber core structure elements should provide the dominant contributions to assembly kinetics. Here we show, however, that residues outside the amyloid core can influence the mechanism of IAPP fiber assembly. IAPP possesses an intramolecular disulfide bond between residues 2 and 7. This short-range disulfide prohibits the N-terminal region from adopting the β-strand structure of an amyloid. We examined the role of this disulfide in fiber formation by generating a truncated construct (IAPP8-37) and a stable reduced form of the full-length protein (IAPPCAM). The fiber structures and assembly kinetics of these variants were assessed via optical and mass spectroscopy. Our data confirm that the disulfide does not contribute to the amyloid fiber core structure. Remarkably, however, it plays a central role in the assembly mechanism. First, loss of the disulfide substantially reduces fiber formation by secondary nucleation, i.e., the ability of pre-existing fibers to participate in the formation of new fibers. Second, the bypass of nucleation by seed addition is a two-step process, termed activation. Loss of the disulfide eliminates this two-step nature of seeded kinetics. [ABSTRACT FROM AUTHOR]

The polypeptide hormone amylin forms amyloid deposits in Type 2 diabetes mellitus and a 10-residue fragment of amylin (amylin20-29) is commonly used as a model system to study this process. Studies of amylin20-29 and several variant peptides revealed that low levels of deamidation can have a significant effect on the secondary structure and aggregation behavior of these molecules. Results obtained with a variant of amylin20-29, which has the primary sequence SNNFPAILSS, are highlighted. This peptide is particularly interesting from a technical standpoint. In the absence of impurities the peptide does not spontaneously aggregate and is not amyloidogenic. This peptide can spontaneously deamidate, and the presence of less than 5% of deamidation impurities leads to the formation of aggregates that have the hallmarks of amyloid. In addition, small amounts of deamidated material can induce amyloid formation by the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for studies of amyloid formation. [ABSTRACT FROM AUTHOR]

A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first-order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems. [ABSTRACT FROM PUBLISHER]

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 preparedAGE-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 β-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. [ABSTRACT FROM AUTHOR]

The role of receptor activity-modifying proteins (RAMPs) in modulating the pharmacological effects of an amylin receptor selective agonist (NN1213) or the dual amylin-calcitonin receptor agonist (DACRA), salmon calcitonin (sCT), was tested in three RAMP KO mouse models, RAMP1, RAMP3 and RAMP1/3 KO. Male wild-type (WT) and knockout (KO) littermate mice were fed a 45% high-fat diet for 20 weeks prior to the 3-week treatment period. A decrease in body weight after NN1213 was observed in all WT mice, whereas sCT had no effect. The absence of RAMP1 had no significant effect on NN1213 efficacy, and sCT was still inactive. However, the absence of RAMP3 impeded NN1213 efficacy but improved sCT efficacy. Similar results were observed in RAMP1/3 KO suggesting that the amylin receptor 3 (AMY3 = CTR + RAMP3) is necessary for NN1213's maximal action on body weight and food intake and that the lack of AMY3 allowed sCT to be active. These results suggest that the chronic use of DACRA such as sCT can have unfavourable effect on body weight loss in mice (which differs from the situation in rats), whereas the use of the amylin receptor selective agonist does not. AMY3 seems to play a crucial role in modulating the action of these two compounds, but in opposite directions. The assessment of a long-term effect of amylin and DACRA in different rodent models is necessary to understand potential physiological beneficial and unfavourable effects on weight loss before its transition to clinical trials., (© 2021 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Aim: To compare the safety, pharmacokinetics and pharmacodynamics of ADO09 with insulin lispro (Lispro) and separate subcutaneous injections of human insulin and pramlintide (Ins&Pram) in 24 subjects with type 1 diabetes., Methods: At three dosing visits, participants received single doses of ADO09, Ins&Pram or Lispro immediately before eating a standardized mixed meal together with 1 g of acetaminophen, which was used as a surrogate marker to evaluate the kinetics of gastric emptying. Premeal blood glucose was adjusted to 126 mg/dL ± 10% by means of insulin and glucose infusions. The insulin dose was 7.5 U and the pramlintide dose was 45 μg. Blood glucose, glucagon and acetaminophen concentrations were assessed as pharmacodynamic endpoints; insulin and pramlintide concentrations were analysed as pharmacokinetic endpoints, and safety and tolerability were assessed., Results: Compared with Lispro, ADO09 reduced postprandial blood glucose (ppBG) excursions by more than 95% in the first hour postmeal (mean ± SD ∆AUC BG 0-1 h: 1.4 ± 9.9 mg*h/dL vs. 43.5 ± 15.3 mg*h/dL; p < .0001). Maximum ppBG was significantly improved with ADO09 (∆BGmax 87.0 ± 35.5 mg/dL) versus both Lispro (109.2 ± 31.1 mg/dL; p = .0133) and Ins&Pram (109.4 ± 44.3 mg/dL; p = .0357). Gastric emptying with ADO09 was similar to Ins&Pram and significantly slower than with Lispro. All treatments were well tolerated and both adverse events and hypoglycaemic events were rare during the meal test procedure., Conclusion: ADO09 was well tolerated and markedly reduced ppBG compared with Lispro. ADO09 formulation was generally similar to the separate administration of insulin and pramlintide, except for a better BG level in the 4-8 h interval postmeal. These positive results warrant further investigations with ADO09., (© 2020 John Wiley & Sons Ltd.)

Aggregation of islet amyloid polypeptide (IAPP) is implicated in the development of type 2 diabetes. The modified NFGAIL with double N-methylated at Gly24 and Ile26 has the property of soluble, non-amyloidogenic, non-cytotoxic, and the ability of inhibiting amyloid formation and cytotoxicity of IAPP. To discover the inhibition mechanism of this peptide inhibitor and provide useful information to design more potential peptide inhibitors, molecular dynamics simulations in explicit solvent were performed. The simulation results reveal that Gly24 and Ile26 are of importance in IAPP aggregation, and N-methylation at these two key residues will disrupt the stability of formed oligomer and prevent the conformation transition of free monomer near the oligomer template. The origin of the N-methylated peptide inhibitor inhibiting IAPP aggregation is that it can keep good binding with IAPP template by stable hydrogen bonding interaction. Furthermore, it cannot induce the conformational transition of free monomer by preventing the hydrogen bond interaction between free monomer and boundary peptide. The structural environment can largely affect the stacking of free monomers to the template. Our study sheds light on the inhibition mechanism of peptide inhibitor at molecular level and may provide guidance for the future design and discovery of new peptide inhibitors., (© 2016 John Wiley & Sons A/S.)

Our knowledge of the complex mechanisms underlying energy homeostasis has expanded enormously in recent years. Food intake and body weight are tightly regulated by the hypothalamus, brainstem and reward circuits, on the basis both of cognitive inputs and of diverse humoral and neuronal signals of nutritional status. Several gut hormones, including cholecystokinin, glucagon-like peptide-1, peptide YY, oxyntomodulin, amylin, pancreatic polypeptide and ghrelin, have been shown to play an important role in regulating short-term food intake. These hormones therefore represent potential targets in the development of novel anti-obesity drugs. This review focuses on the role of gut hormones in short- and long-term regulation of food intake, and on the current state of development of gut hormone-based obesity therapies.

Background: Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type 1 diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation., Methods: ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis., Results: In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. In contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol., Conclusions: A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation.

Background: The production of oxidative stress as a result of postprandial hyperglycaemia is now recognized as an important contributing factor in the development of diabetes complications. The objective of this study was to examine the effects of pramlintide on plasma concentrations of glucose and several markers of oxidative stress in patients with type 2 diabetes following a standardized meal., Methods: This was a randomized, single-blind, placebo-controlled, crossover study conducted at two clinical research centres in the United States. A total of 19 subjects (9 men and 10 women) with type 2 diabetes using mealtime insulin participated in the study. Pramlintide (120 microg), or placebo, and rapid-acting mealtime insulin were administered prior to a standardized meal on two separate study days. Plasma concentrations of glucose, nitrotyrosine (NT), oxidized-LDL cholesterol (OxLDL-C), and total radical trapping parameter (TRAP) were assessed during the 4-h postprandial period., Results: Compared to placebo, pramlintide treatment reduced postprandial excursions of glucose, NT, and OxLDL-C and protected TRAP from consumption. Correlation analysis revealed positive associations between placebo-corrected glucose incremental AUC(0-4 h) and both NT and OxLDL-C and a negative association between placebo-corrected glucose incremental AUC(0-4h) and TRAP., Conclusions: The reduction in postprandial glucose excursions achieved with addition of pramlintide to rapid-acting insulin in type 2 diabetes was associated with a reduction in postprandial markers of oxidative stress., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Uncontrolled diabetes mellitus is associated with both microvascular and macrovascular complications. Despite an array of treatment options available, achievement of euglycemia in most patients with diabetes is still lacking. Pramlintide acetate, a synthetic analog of the human hormone amylin and belonging to a new class of agents, was approved in March 2005 as adjunctive treatment in patients with type 1 or 2 diabetes mellitus. To evaluate the data available on the efficacy and safety of pramlintide, we conducted a search of MEDLINE (January 1966-May 2006) and International Pharmaceutical Abstracts (January 1970-May 2006). Bibliographies of clinical trials were reviewed for additional references. The literature reviewed demonstrated that pramlintide is effective in reducing levels of glycosylated hemoglobin and potentially preventing weight gain. The most commonly reported adverse effects associated with pramlintide were nausea, anorexia, and hypoglycemia. These adverse effects occurred more often during the initiation of therapy and were usually mild to moderate in nature. Whether this therapy is a cost-effective option for patients with type 1 or type 2 diabetes mellitus is yet to be determined.

The hypothalamic regulatory system of body weight which develops in rats during critical periods of early postnatal life seems to express plastic changes depending on nutrition at that time. Adult rats previously exposed to early postnatal overnutrition by raising them in small litters become persistently predisposed to overweight, hyperphagia and hyperleptinaemia. The hypothesis was raised that feeding-related peptides could be involved through altered effects on neuronal activity of the regulatory systems of such rats. This was studied on brain slices of small-litter rats and normal-weight controls between days 60 and 120 of life. Neurons of the medial parvocellular part of the paraventricular nucleus were significantly activated by the adiposity signals leptin, insulin and amylin in controls. This is a kind of negative feedback, because activation of these neurons is known to be followed in vivo by increased energy expenditure. GABAergic mechanisms seem to affect these neuronal responses because the activating effects of insulin and amylin were reduced in the presence of a GABA(A) receptor antagonist. In overweight small-litter rats, however, the neuronal responses to the adiposity signals were significantly changed; activating effects were reduced and inhibitory effects increased. By means of blockade of GABA(A) receptors, significant alterations in the neuronal responses to leptin, insulin and amylin in small-litter rats were prevented. Responses to the peptides were reversed and now resembled those of controls. In conclusion, changes in neuronal wiring with GABAergic interneurons seem to contribute to a persistently reduced negative feedback of adiposity signals in early postnatally overfed rats.

Background: Pramlintide, a human amylin analogue, is a potential new adjunctive therapy to insulin for patients with type 1 diabetes and insulin-using patients with type 2 diabetes. Early clinical trials have shown a transient increased risk of hypoglycaemia in some patients at the time of initiating pramlintide therapy. This may be the result of combining the postprandial glucose, lowering effect of pramlintide with the existing hypoglycaemic potential of insulin without appropriate adjustment of insulin doses. However, the possibility that pramlintide may exert an independent detrimental effect on the physiological responses to insulin-induced hypoglycaemia needs to be excluded., Methods: We conducted three separate randomized, placebo-controlled studies in patients with type 1 diabetes treated with adjunctive pramlintide. These studies utilized pramlintide at high doses (either 0.1-1 mg pramlintide daily or 0.1-0.8 mg pramlintide four times a day for 5 or 6 days) as well as doses closer to those anticipated for therapeutic usage (30, 100 or 300 microg three times daily for 14 days), and examined the hormonal, metabolic and symptomatic responses to an insulin-infusion hypoglycaemic challenge conducted at baseline and after days of therapy., Results and Conclusion: Pramlintide had no effect on the counter-regulatory hormonal, metabolic and symptomatic responses to hypoglycaemia. These findings demonstrated that pramlintide, when used as adjunctive therapy to insulin in patients with type 1 diabetes, has no independent effect on the response to hypoglycaemia.

Background: To assess the postprandial glucose-lowering effect of the human amylin analog pramlintide when given with insulin lispro in subjects with type 2 diabetes, with an emphasis on the optimal dose timing relative to meals., Methods: In this randomized, single-blind, placebo-controlled, five-way crossover study, 19 subjects with type 2 diabetes using insulin lispro underwent five consecutive mixed-meal tests. In randomized order, subjects received subcutaneous injections of placebo at -15 min or 120-microg pramlintide at -15, 0, +15, or +30 min relative to the standardized breakfast after an overnight fast. Insulin lispro was injected at 0 min at doses that were adjusted appropriately for both the content of the standardized meal and the anticipated effects of pramlintide. Plasma glucose concentrations were measured before and during the 4-h postmeal period., Results: When injected at 0 min, pramlintide reduced the postprandial glucose excursion by 81% compared to insulin lispro + placebo (incremental AUC(0-4 h) (mean +/- SE) 2.0 +/- 1.5 vs. 10.4 +/- 2.2 mmol/h/L, P<0.05). When pramlintide was injected at -15, +15, and +30 min, the postprandial incremental glucose AUC(0-4 h) was also significantly reduced (P<0.05), but to a lesser extent (42 to 73%). Pramlintide treatment was well tolerated and no serious adverse events were reported., Conclusions: Administration of pramlintide either at or just prior to a meal caused a greater reduction in postprandial glucose than either administration of placebo or postmeal pramlintide injections in subjects with type 2 diabetes treated with a rapid-acting insulin analog, insulin lispro., (Copyright 2004 John Wiley & Sons, Ltd.)

Aim: Two long-term, randomized, double-blind, placebo-controlled clinical trials in insulin-using patients with type 2 diabetes, spanning a wide range of baseline glycaemic control, have shown that the addition of pramlintide, an analogue of the beta-cell hormone amylin, to pre-existing insulin regimens results in reductions in HbA1c that are accompanied by weight loss., Methods: To assess whether this profile of pramlintide is observed in patients approaching, but not yet reaching, glycaemic targets, we conducted a pooled post hoc analysis of the two trials, including all patients with an entry HbA1c between 7.0 and 8.5%. Within this subset of patients, 80 were treated with placebo + insulin [baseline HbA1c 8.0 +/- 0.3%, weight 87.3 +/- 19.3 kg (mean +/- s.d.)] and 86 with pramlintide (120 micro g bid) + insulin [HbA1c 8.0 +/- 0.4%, weight 92.5 +/- 20.4 kg (mean +/- s.d.)]. Endpoints included changes from baseline to Week 26 in HbA1c, body weight, and the event rate of severe hypoglycaemia., Results: Adjunctive therapy with pramlintide resulted in significant reductions in both HbA1c and body weight from baseline to Week 26 (-0.43% and -2.0 kg differences from placebo, respectively, both p < 0.001). These changes were achieved without a concomitant increase in the overall rate of severe hypoglycaemic events (0.13 pramlintide vs. 0.19 placebo, events/patient year of exposure)., Conclusions: The data from this post hoc analysis indicate that the addition of pramlintide to insulin therapy may help patients with type 2 diabetes who are approaching, but not yet reaching, glycaemic targets to achieve further reductions in HbA1c without concomitant weight gain and increased risk of severe hypoglycaemia.