Diabetes mellitus drug discovery: insights into targeting feline and human amylin with small molecules.

BACKGROUND: Type 2 diabetes (T2D) is a health concern for both humans and cats, with cases rising over the past decade. Around 70% of patients from either species exhibit pancreatic aggregates of islet amyloid polypeptide (IAPP), a protein that proves toxic upon misfolding. These misfolded protein aggregates congregate in the islets of Langerhans of the pancreas, diminishing the capability of ß-cells to produce insulin and further perpetuating disease. OBJECTIVE: Our team's drug discovery program is investigating newly synthesized compounds that could diminish aggregates of both human and feline IAPP, potentially disrupting the progression of T2D. MATERIAL AND METHODS: We prepared 24 compounds derived from diaryl urea, as ureas have previously demonstrated great potential at reducing accumulations of misfolded proteins. Biophysical methods were employed to analyze the anti-aggregation activity of these compounds at inhibiting and/or disrupting IAPP fibril formation in vitro. RESULTS: The results demonstrate that compounds 12 and 24 were most effective at reducing the fibrillization and aggregation of both human and feline IAPP. When compared with the control for each experiment, samples treated with either compound 12 or 24 exhibited fewer accumulations of amyloid-like fibrils. CONCLUSION: Urea-based compounds, such as compounds 12 and 24, may prove crucial in future pre-clinical studies in the search for therapeutics for T2D.

Molecular Mechanisms of Amylin Turnover, Misfolding and Toxicity in the Pancreas.

Amyloidosis is a common pathological event in which proteins self-assemble into misfolded soluble and insoluble molecular forms, oligomers and fibrils that are often toxic to cells. Notably, aggregation-prone human islet amyloid polypeptide (hIAPP), or amylin, is a pancreatic hormone linked to islet ß-cells demise in diabetics. The unifying mechanism by which amyloid proteins, including hIAPP, aggregate and kill cells is still matter of debate. The pathology of type-2 diabetes mellitus (T2DM) is characterized by extracellular and intracellular accumulation of toxic hIAPP species, soluble oligomers and insoluble fibrils in pancreatic human islets, eventually leading to loss of ß-cell mass. This review focuses on molecular, biochemical and cell-biology studies exploring molecular mechanisms of hIAPP synthesis, trafficking and degradation in the pancreas. In addition to hIAPP turnover, the dynamics and the mechanisms of IAPP-membrane interactions; hIAPP aggregation and toxicity in vitro and in situ; and the regulatory role of diabetic factors, such as lipids and cholesterol, in these processes are also discussed.

Novel Human Insulin Isoforms and Cα-Peptide Product in Islets of Langerhans and Choroid Plexus.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells, where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains ß-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP, and its amyloid formation was inhibited in vitro more efficiently by Cα-peptide than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase-processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus autopsy donors. Intriguingly, 100 years after the discovery of insulin, we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

Simultaneous Detection of Tyrosine and Structure-Specific Intrinsic Fluorescence in the Fibrillation of Alzheimer's Associated Peptides.

Understanding of the structural changes during their aggregation and interaction is a prerequisite for establishing the precise clinical relevance of human islet amyloid polypeptide (hIAPP) (involved in Type-II Diabetes Mellitus) in the treatment of Alzheimer's disease stemmed from beta-amyloid (Aß). Herein, we show that the steady-state emission spectra obtained from photoluminescence (PL) simultaneously capture both the tyrosine derivative (tyrosinate) and the structure-specific intrinsic fluorescence during the aggregation of Aß and hIAPP. We observe multiple peaks in the emission spectra which exist for structure-specific intrinsic fluorescence, and use the second derivative UV-Vis spectra and the shift in the tyrosine peak as a quantitative measure of the dissimilitude in the electronic states and the fibril growth. We further applied these techniques to detect the static electric field (0, 40, 120, 200 V/cm) induced promotion and inhibition of fibrillation in Aß, hIAPP and their electric field dependent role in the fibrillation of Aß : hIAPP(1 : 1). The results were corroborated by field-emission scanning electron microscopy (FESEM), and the determinations of secondary structures by Fourier transform infrared spectroscopy (FTIR). The results indicate that the emission spectrum can be used as a sensor to detect the presence of fibrils; hence for screening potential inhibitors of amyloid fibrillation.

Membrane affinity of individual toxic protein oligomers determined at the single-molecule level.

Oligomers are the key suspects in protein aggregation-linked diseases, such as Alzheimer's and Type II diabetes, and most likely exert their toxicity by interacting with lipid membranes. However, the "which oligomer" question remains an obstacle in understanding the disease mechanism, as the exact identity of the toxic oligomer(s) is not yet known. Oligomers exist as a mixture of species of different sizes (i.e. as different 'n-mers') in a physiological solution, making it difficult to determine the properties of individual species. Here we demonstrate a method based on single-molecule photo-bleaching (smPB) which can provide an answer to the "which oligomer" question, at least as far as membrane affinity is concerned. We calculate the ratio of the oligomer size distribution of human Islet Amyloid Polypeptide (IAPP) in the aqueous phase and that on a coexisting artificial lipid bilayer, and this measures the relative membrane affinity of individual oligomeric species. A problem with smPB measurements is that they can be very sensitive to pre-measurement bleaching. Here we correct for pre-bleaching using a covalently linked multimeric peptide as a bleaching standard. We find that the order of membrane affinity for IAPP n-mers is trimer > dimer > tetramer ≫ monomer. Our results agree well with the average membrane affinity values of oligomeric and monomeric solutions previously measured with Fluorescence Correlation Spectroscopy. The "which oligomer" question, in the context of membrane affinity, can therefore, be solved quantitatively for any membrane-active toxic protein aggregate.

In Vivo and In Vitro Monitoring of Amyloid Aggregation via BSA@FGQDs Multimodal Probe.

Early detection of peptide aggregate intermediates is quite challenging because of their variable and complex nature as well as due to lack of reliable sensors for diagnosis. Herein, we report the detection of monomers and oligomers using specified fluorescence and a magnetic resonance imaging (MRI) multimodal probe based on bovine-serum-albumin-capped fluorine functionalized graphene quantum dots (BSA@FGQDs). This probe enables in vitro fluorescence-based monitoring of human islet amyloid polypeptide (hIAPP), insulin, and amyloid ß(1-42) (Aß42) monomers and oligomers during the fibrillogenesis dynamic. Up to 90% fluorescence quenching of BSA@FGQDs probe upon addition of amyloid monomers/oligomers was observed due to static quenching and nonradiative energy transfer. Moreover, the BSA@FGQDs probe shows 10 times higher signals in detecting amyloid intermediates and fibrils than that of conventional thioflavin dye. A negative Δ G° value (-36.21 kJ/mol) indicates spontaneous interaction of probe with the peptide. These interactions are hydrogen bonding and hydrophobic as proved by thermodynamic parameters. Visual binding clues of BSA@FGQDs with different morphological states of amyloid protein was achieved through electron microscopy. Furthermore, intravenous and intracranial injection of BSA@FGQDs probe in Alzheimer model mice brain enabled in vivo detection of amyloid plaques in live mice brain by 19F MRI through contrast enhancement. Our proposed probe not only effectively monitors in vitro fibrillation kinetics of number of amyloid proteins with higher sensitivity and specificity than thioflavin dye, but also, the presence of a 19F center makes BSA@FGQDs an effective probe as a noninvasive and nonradiative in vivo detection probe for amyloid plaques.

Amperometric immunoassay for the obesity biomarker amylin using a screen printed carbon electrode functionalized with an electropolymerized carboxylated polypyrrole.

Amylin (the islet amyloid polypeptide) is a hormone related to adiposity, hunger and satiety. It is co-secreted with insulin from pancreatic B-cells. An amperometric immunosensor is presented here for the determination of amylin. It is making use of a screen printed carbon electrode (SPCE) functionalized with electropolymerized poly(pyrrole propionic acid) (pPPA) with abundant carboxyl groups that facilitate covalent binding of antibody against amylin. A competitive immunoassay was implemented using biotinylated amylin and streptavidin labeled with horse radish peroxidase (HRP-Strept) as the enzymatic tracer. The amperometric detection of H2O2 mediated by hydroquinone was employed as an electrochemical probe to monitor the affinity reaction. The variables involved in the preparation and function of the immunosensor were optimized and the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The calibration graph for amylin, obtained by amperometry at -200 mV vs Ag pseudo-reference electrode, showed a range of linearity extending from 1.0 fg∙mL-1 to 50 pg∙mL-1, with a detection limit of 0.92 fg∙mL-1. This is approximately 7000 times lower than the minimum detectable concentration reported for the ELISA immunoassays available for amylin. The assay has excellent reproducibility and good selectivity over potential interferents. Graphical abstract Schematic of an amperometric competitive immunoassay for the obesity biomarker amylin using a poly(pyrrole propionic acid)-modified screen-printed electrode. The detection limit is 0.92 fg∙mL-1 amylin. The method provides excellent reproducibility for the measurements, good selectivity and successful applicability to human urine and serum samples.

Study of forced degradation behavior of pramlintide acetate by HPLC and LC-MS.

Pramlintide acetate (Symlin®), a synthetic analogue of the human hormone amylin. It was approved in March 2005 as a subcutaneous injection for the adjunctive treatment of patients who have type 1 or 2 diabetes mellitus. The objective of current investigation was to study the degradation behavior of pramlintide acetate under different ICH recommended stress conditions by HPLC and LC-MS. Pramlintide acetate was subjected to stress conditions of hydrolysis (acidic or alkaline), oxidation, photolysis and thermal decomposition. Extensive degradation products were observed under the hydrolysis, oxidation or thermal stress conditions, while minimal degradation was found in the photolytic conditions. Successful separation of drug from the degradation products was achieved by the validated chromatography (RP-HPLC and SCX-HPLC) methods. Subsequent to isolation, the molecular weight of each component was determined by LC-MS. The LC-MS m/z values and fragmentation patterns of 4 impurities matched with the predicted degradation products of pramlintide acetate.

Advances in Diabetes Pharmacotherapy: An Update for the Emergency Provider.

BACKGROUND: Diabetes mellitus is a disease that affects millions of Americans, and its prevalence is only anticipated to increase in coming years. It is estimated that diabetes-related visits account for 1% of all emergency department (ED) encounters. In recent years, there have been several new categories of medications approved for the treatment of diabetes, including new insulins, glucagon-like peptide-1 receptor agonists, dipeptidyl peptidase-4 inhibitors, an amylin analogue, and sodium-glucose cotransporter-2 inhibitors. OBJECTIVE OF THE REVIEW: This review presents recently approved agents to treat diabetes, with a focus on basic mechanism, place in therapy, and toxicities the ED provider may encounter. DISCUSSION: Many of these new therapies have been incorporated as first- and second-line agents for the management of diabetes. Recently approved diabetes medications often have different mechanisms of action and adverse effect and overdose profiles compared to traditional agents, such as sulfonylureas and metformin. CONCLUSIONS: Emergency providers will encounter patients taking these newly approved medications, as well as treat those presenting with adverse effects and overdoses from them. As such, emergency providers must have a basic understanding of these new therapies so that they can optimally care for diabetic patients.

Thioflavin T fluorescence to analyse amyloid formation kinetics: Measurement frequency as a factor explaining irreproducibility.

The most frequent method to monitor amyloid formation relies on the fluorescence of thioflavin T (ThT). The present study reports a novel factor of irreproducibility in ThT kinetic assays performed in microplate. Discrepancies among kinetics of amyloid assembly, performed under quiescent conditions, were associated with the frequency of fluorescence measurement. Evaluating self-assembly of the islet amyloid polypeptide at short intervals hastened its fibrillization. This observation was confirmed by transmission electron microscopy, circular dichroism spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence. This effect, attributed to agitation during microplate displacements between fluorescence measurements, reinforces the importance of a better standardization in amyloid formation assays.

Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting.

AIM: Gadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses. MATERIALS & METHODS: The ability of the functionalized nanoparticles to target amyloid fibrils made of ß-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy. RESULTS: Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with KD values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues. CONCLUSION: Such functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimer's disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy.

Potentiometric and spectroscopic studies on the copper(II) complexes of rat amylin fragments. The anchoring ability of specific non-coordinating side chains.

Copper(ii) complexes of peptides modelling the sequence of the 17-22 residues of rat amylin have been studied by potentiometric, UV-Vis, CD and ESR spectroscopic methods. The peptides were synthesized in N-terminally free forms, NH2-VRSSNN-NH2, NH2-VRSSAA-NH2, NH2-VRAANN-NH2, NH2-VRSS-NH2, NH2-SSNN-NH2, NH2-SSNA-NH2 and NH2-AANN-NH2, providing a possibility for the comparison of the metal binding abilities of the amino terminus and the -SSNN- domain. The amino terminus was the primary ligating site in all cases and the formation of only mononuclear complexes was obtained for the tetrapeptides. The thermodynamic stability of the (NH2, N(-), N(-)) coordinated complexes was, however, enhanced by the asparaginyl moiety in the case of NH2-SSNN-NH2, NH2-SSNA-NH2 and NH2-AANN-NH2. Among the hexapeptides the formation of dinuclear complexes was characteristic for NH2-VRSSNN-NH2 demonstrating the anchoring ability of the -SSNN- (SerSerAsnAsn) domain. The complexes of the heptapeptide NH2-GGHSSNN-NH2 were also studied and the data supported the above mentioned anchoring ability of the -SSNN- site.

Determination of Optimal Sample Size for Quantification of ß-Cell Area, Amyloid Area and ß-Cell Apoptosis in Isolated Islets.

Culture of isolated rodent islets is widely used in diabetes research to assess different endpoints, including outcomes requiring histochemical staining. As islet yields during isolation are limited, we determined the number of islets required to obtain reliable data by histology. We found that mean values for insulin-positive ß-cell area/islet area, thioflavin S-positive amyloid area/islet area and ß-cell apoptosis do not vary markedly when more than 30 islets are examined. Measurement variability declines as more islets are quantified, so that the variability of the coefficient of variation (CV) in human islet amyloid polypeptide (hIAPP) transgenic islets for ß-cell area/islet area, amyloid area/islet area and ß-cell apoptosis are 13.20% ± 1.52%, 10.03% ± 1.76% and 6.78% ± 1.53%, respectively (non-transgenic: 7.65% ± 1.17% ß-cell area/islet area and 8.93% ± 1.56% ß-cell apoptosis). Increasing the number of islets beyond 30 had marginal effects on the CV. Using 30 islets, 6 hIAPP-transgenic preparations are required to detect treatment effects of 14% for ß-cell area/islet area, 30% for amyloid area/islet area and 23% for ß-cell apoptosis (non-transgenic: 9% for ß-cell area/islet area and 45% for ß-cell apoptosis). This information will be of value in the design of studies using isolated islets to examine ß cells and islet amyloid.

A Comparison of Three Fluorophores for the Detection of Amyloid Fibers and Prefibrillar Oligomeric Assemblies. ThT (Thioflavin T); ANS (1-Anilinonaphthalene-8-sulfonic Acid); and bisANS (4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic Acid).

Amyloid fiber formation is a key event in many misfolding disorders. The ability to monitor the kinetics of fiber formation and other prefibrillar assemblies is therefore crucial for understanding these diseases. Here we compare three fluorescent probes for their ability to monitor fiber formation, ANS (1-anilinonaphthalene-8-sulfonic acid) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid) along with the more widely used thioflavin T (ThT). For this, we have used two highly amyloidogenic peptides: amyloid-ß (Aß) from Alzheimer's disease and islet amyloid polypeptide (IAPP) associated with type II diabetes. Using a well-plate reader, we show all three fluorophores can report the kinetics of fiber formation. Indeed, bis-ANS is markedly more sensitive to fiber detection than ThT and has a submicromolar affinity for Aß fibers. Furthermore, we show that fluorescence detection is very sensitive to the presence of excess fluorophore. In particular, beyond a 1:1 stoichiometry these probes demonstrate marked fluorescence quenching, for both Aß and IAPP. Indeed, the fiber-associated fluorescence signal is almost completely quenched in the presence of excess ThT. There is also intense interest in the detection of prefibrillar amyloid assemblies, as oligomers and protofibrils are believed to be highly cytotoxic. We generate stable, fiber-free, prefibrillar assemblies of Aß and survey their fluorescence with ANS and bis-ANS. Fluorescence from ANS has often been used as a marker for oligomers; however, we show ANS can fluoresce more strongly in the presence of fibers and should therefore be used as a probe for oligomers with caution.

[Amylin under examination. Fibrillation - cytotoxic pancreatic polypeptide aggregation]. / Amylina w badaniach eksperymentalnych.Fibrylacja ­ cytotoksyczna agregacja polipeptydutrzustki.

In patients or animals affected by type 2 diabetes mellitus (DM2, non-insulin dependent diabetes mellitus [NIDDM]), some pathological deposits, called amyloid, are observed among cells of islets of Langerhans. Among other constituents, the deposits consist of an insoluble, fibrillar form of polypeptide neurohormone called amylin, produced by pancreatic beta cells. It is thought that formation of fibrillar deposits of misfolded and aggregated polypeptide is highly toxic to beta cells and leads to cell dysfunction, cell loss, pancreas destruction and progress of the disease. Due to the extreme insolubility of this polypeptide and its instant fibrillation, amylin constitutes a methodological problem, and there is a need for a special methodology in experiments. Some mechanisms and factors that govern amylin fibrillization are rather poorly understood. This article presents amylin as a fibrillating molecule and some methods and methodological aspects and problems that emerge at successive steps during the fibrillation process, including hypothesized cytotoxicity mechanisms of this polypeptide.

[Amylin under examination. Fibrillogenic polypeptide of pancreatic amyloid]. / Amylina w badaniach eksperymentalnych. Fibrylotwórczy polipeptyd amyloidu trzustkowego

In patients or animals affected by 2 type diabetes mellitus (diabetes mellitus type 2, DM2, non-insulin-dependent diabetes mellitus, NIDDM) or pancreatic tumor disease e.g., insulinoma, some pathological deposits, called amyloid, are observed among cells of islets of Langerhans. Among other constituents, pancreatic deposits consist of an insoluble, fibrillar form of peptide neurohormone termed amylin, produced by pancreatic beta cells. It is thought that formation of fibrillar deposits of misfolded and aggregated peptide is highly toxic to beta cells and leads to cell dysfunction, cell loss, pancreas destruction and progress of the disease. This relatively small, 37-amino acid peptide constitutes a serious scientific, research and to some extent a medical problem. This article presents amylin as a fibrillating molecule which participates in formation of amyloid deposits in human and animal pancreas, Langerhans islets as a microenvironment of pancreatic amyloid formation, occurrence of amylin and amyloid in animals and humans, and physico-chemical requirements to meet to name amylin deposit as amyloid.

Ion mobility spectrometry-mass spectrometry defines the oligomeric intermediates in amylin amyloid formation and the mode of action of inhibitors.

The molecular mechanisms by which different proteins assemble into highly ordered fibrillar deposits and cause disease remain topics of debate. Human amylin (also known as islet amyloid polypeptide/hIAPP) is found in vivo as amyloid deposits in the pancreatic islets of sufferers of type II diabetes mellitus, and its self-aggregation is thought to be a pathogenic factor in disease and to contribute to the failure of islet transplants. Here, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to monitor oligomer formation from IAPP. The detection, identification and characterization of oligomers from both human and rat amylin (rIAPP) are described. Oligomers up to and including hexamers have been detected for both peptides. From ESI-IMS-MS derived collision cross sections (CCS), these species are shown to be elongated in conformation. Collision-induced dissociation (CID-MS/MS) revealed differences in the gas-phase stability of the oligomers formed from hIAPP and rIAPP, which may contribute to their differences in amyloid propensity. Using ESI-IMS-MS, the mode of inhibition of amyloid formation from hIAPP using small molecules or co-incubation with rIAPP was also investigated. We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly. Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition. The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.

Simultaneous monitoring of insulin and islet amyloid polypeptide secretion from islets of Langerhans on a microfluidic device.

A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled changes in the perfusion media within 5 s, allowing rapid exchange of the glucose concentrations delivered to groups of islets. The perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 °C. The immunoassay mixture was injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4-10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis.

Ion-transfer electrochemistry of rat amylin at the water-organogel microinterface array and its selective detection in a protein mixture.

The behavior of proteins and polypeptides at electrified aqueous-organic interfaces is of benefit in label-free detection strategies. In this work, rat amylin (or islet amyloid polypeptide) was studied at the interface formed between aqueous liquid and gelled organic phases. Amylin is a polypeptide that is co-secreted with insulin from islet beta-cells and is implicated in fibril formation. In this study, rat amylin was used, which does not undergo aggregation. The polypeptide underwent an interfacial transfer process, from water to the gelled organic phase, under applied potential stimulation. Cyclic voltammetry revealed steady-state forward and peak-shaped reverse voltammograms, which were consistent with diffusion-controlled water-to-organic transfer and thin-film stripping or desorptive back-transfer. The diffusion-controlled forward current was greater when amylin was present in an acidic aqueous phase than when it was present in an aqueous phase at physiological pH; this reflects the greater charge on the polypeptide under acidic conditions. The amylin transfer current was concentration dependent over the range 2-10 µM, at both acidic and physiological pH. At physiological pH, amylin was selectively detected in the presence of a protein mixture, which illustrated the bioanalytical possibilities for this electrochemical behavior.

A direct fluorescence-based technique for cellular localization of amylin.

Amylin has been implicated in type II diabetes because of its inherent property to misfold into toxic aggregates. Although it has been shown that amylin interacts with cell membranes, no study to date has monitored the association process using a direct approach. The present study uses confocal microscopy to identify the localization of carboxyfluorescein-labeled amylin in RIN-5F cells. In addition, the size of the aggregates that are formed was evaluated using nanoparticle tracking analysis. In support of previous findings, amylin was observed to interact with and remain associated with the cell membrane. The cell membrane-associated aggregates spanned a size range of 130-800 nm.