Identification of Crocin as a New hIAPP Amyloid Inhibitor via a Simple Yet Highly Biospecific Screening System.

Amylin (hIAPP) amyloid formation plays an important role in the pathogenesis of type 2 diabetes (T2D), which makes it a promising therapeutic target for T2D. In this study, we established a screening tool for identifying chemicals affecting hIAPP amyloid formation based on a reported genetic tool, which constantly tracks protein aggregates in Saccharomyces cerevisiae. In order to obtain the hIAPP with better aggregation ability, the gene of hIAPP was tandemly ligated to create 1×, 2×, 4× or 6×-hIAPP expressing strains. By measuring the cell density and fluorescence intensity of green fluorescent protein (GFP) regulated by the aggregation status of hIAPP, it was found that four intramolecular ligated hIAPP (4×hIAPP) could form obvious amyloids with mild toxicity. The validity and reliability of the screening tool were verified by testing six reported hIAPP inhibitors, including curcumin, epigallocatechin gallate and so on. Combined with surface plasmon resonance (SPR) and the screening tool, which could be a screening system for hIAPP inhibitors, we found that crocin specifically binds to hIAPP and acts inhibit amyloid formation of hIAPP. The effect of crocin was further confirmed by Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) analysis. Thus, a screening system for hIAPP amyloid inhibitors and a new mechanism of crocin on anti-T2D were obtained as a result of this study.

Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity.

Washingtonia filifera seeds have revealed to possess antioxidant properties, butyrylcholinesterase and xanthine oxidase inhibition activities. The literature has indicated a relationship between Alzheimer's disease (AD) and type-2 diabetes (T2D). Keeping this in mind, we have now evaluated the inhibitory properties of W. filifera seed extracts on α-amylase, α-glucosidase enzyme activity and the Islet Amyloid Polypeptide (IAPP) fibrils formation. Three extracts from seeds of W. filifera were evaluated for their enzyme inhibitory effect and IC50 values were calculated for all the extracts. The inhibition mode was investigated by Lineweaver-Burk plot analysis and the inhibition of IAPP aggregate formation was monitored. W. filifera methanol seed extract appears as the most potent inhibitor of α-amylase, α-glucosidase, and for the IAPP fibril formation. Current findings indicate new potential of this extract that could be used for the identification or development of novel potential agents for T2D and AD.

Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils.

Human islet amyloid polypeptide (hIAPP) functions as a glucose-regulating hormone but deposits as amyloid fibrils in more than 90% of patients with type II diabetes (T2D). Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically related protofilaments with ordered residues 14-37. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20-29 constitute the core of the hIAPP amyloid; (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G; (iii) explains why the six residue substitutions in rodent IAPP prevent aggregation; and (iv) suggests regions responsible for the observed hIAPP cross-seeding with ß-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors. Four of the designed peptides delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof of concept that the capping strategy can be used on full-length cryo-EM fibril structures.

Inhibitory activities of phenylpropanoids from Lycopus lucidus on amyloid aggregation related to Alzheimer's disease and type 2 diabetes.

The number of patients with Alzheimer's disease (AD) and type 2 diabetes (T2D) is increasing rapidly, and thus more research has been focused on the relationship between these two age-related chronic diseases. According to the amyloid hypothesis, prevention of the aggregation of amyloid ß (Aß) and human islet amyloid polypeptide (hIAPP) is a promising strategy for AD and T2D. In this study, thioflavin-T assay and transmission electron microscopy were performed to evaluate the inhibitory effect of three phenylpropanoids isolated from Lycopus lucidus-schizotenuin A and lycopic acids A and B-on both Aß and hIAPP fibrillization. All tested compounds exhibited similarly strong inhibitory activity toward amyloid aggregation. These results suggested that catechol moieties play important roles in the inhibition of amyloid plaque formation.

Cichoric acid from witloof inhibit misfolding aggregation and fibrillation of hIAPP.

The misfolding, aggregation and fibrillation of human islet amyloid polypeptide (hIAPP) has been acknowledged as a hallmark event in type-II diabetes. Hence, inhibiting the misfolding, aggregation and fibrillation of hIAPP have been accepted as a vital factor to treat the disease. Here cichoric acid was extracted from witloof to explore its inhibition effects on misfolding, aggregation and fibrillation of hIAPP. Thioflavin-T (ThT) fluorescence assay, dynamic light scattering (DLS) and atomic force microscopy (AFM) images showed that cichoric acid inhibited the aggregation and fibrillation of hIAPP in a dosage-dependent manner. Circular dichroism (CD) spectra showed that cichoric acid inhibited the misfolding of hIAPP from unfolded to ß-sheet. Molecular docking and further experiments revealed interactions between hIAPP and cichoric acid. Cichoric acid could bind to K1 and R11 of hIAPP via electrostatic interaction. In addition, cichoric acid could form π-π stacking with hIAPP residues F15 and F23. These interactions inhibited the misfolding, aggregation and fibrillation of hIAPP. These results, together with cichoric acid's good cytocompatibility and significant protective effects in hIAPP lesioned cell models, not only showed that cichoric acid could be used to fight against amyloidosis, but also brought a new perspective for Chinese herbal medicine as natural compound's medical potential.

Amylin Amyloid Inhibition by Flavonoid Baicalein: Key Roles of Its Vicinal Dihydroxyl Groups of the Catechol Moiety.

Amyloid formation of the 37-residue amylin is involved in the pathogenesis of type 2 diabetes and, potentially, diabetes-induced neurological deficits. Numerous flavonoids exhibit inhibitory effects against amylin amyloidosis, but the mechanisms of inhibition remain unclear. Screening a library of natural compounds uncovered a potent lead compound, the flavone baicalein. Baicalein inhibits amylin amyloid formation and reduces amylin-induced cytotoxicity. Analogue analyses demonstrated, for the first time, key roles of the vicinal hydroxyl groups on the A-ring. We provided mass spectrometric evidence that incubating baicalein and amylin leads to their conjugation, consistent with a Schiff base mechanism.

Hypothalamic Amylin Acts in Concert with Leptin to Regulate Food Intake.

In this report we evaluated the functions of hypothalamic amylin in vivo and in vitro. Profiling of hypothalamic neurons revealed that islet amyloid polypeptide (Iapp, precursor to amylin) is expressed in neurons in the lateral hypothalamus, arcuate nucleus, medial preoptic area, and elsewhere. Hypothalamic expression of lapp is markedly decreased in ob/ob mice and normalized by exogenous leptin. In slices, amylin and leptin had similar electrophysiologic effects on lateral hypothalamic leptin receptor ObRb-expressing neurons, while the amylin antagonist AC187 inhibited their activity and blunted the effect of leptin. Finally, i.c.v. infusion of AC187 acutely reduced the anorectic effects of leptin. These data show that hypothalamic amylin is transcriptionally regulated by leptin, that it can act directly on ObRb neurons in concert with leptin, and that it regulates feeding. These findings provide a potential mechanism for the increased efficacy of a metreleptin/pramlintide combination therapy for obesity.

Isoliquiritigenin is a potent inhibitor of NLRP3 inflammasome activation and diet-induced adipose tissue inflammation.

Inflammasome activation initiates the development of many inflammatory diseases, including obesity and type 2 diabetes. Therefore, agents that target discrete activation steps could represent very important drugs. We reported previously that ILG, a chalcone from Glycyrrhiza uralensis, inhibits LPS-induced NF-κB activation. Here, we show that ILG potently inhibits the activation of NLRP3 inflammasome, and the effect is independent of its inhibitory potency on TLR4. The inhibitory effect of ILG was stronger than that of parthenolide, a known inhibitor of the NLRP3 inflammasome. GL, a triterpenoid from G. uralensis, had similar inhibitory effects on NLRP3 activity, but high concentrations of GL were required. In contrast, activation of the AIM2 inflammasome was inhibited by GL but not by ILG. Moreover, GL inhibited NLRP3- and AIM2-activated ASC oligomerization, whereas ILG inhibited NLRP3-activated ASC oligomerization. Low concentrations of ILG were highly effective in IAPP-induced IL-1ß production compared with the sulfonylurea drug glyburide. In vivo analyses revealed that ILG potently attenuated HFD-induced obesity, hypercholesterolemia, and insulin resistance. Furthermore, ILG treatment improved HFD-induced macrovesicular steatosis in the liver. Finally, ILG markedly inhibited diet-induced adipose tissue inflammation and IL-1ß and caspase-1 production in white adipose tissue in ex vivo culture. These results suggest that ILG is a potential drug target for treatment of NLRP3 inflammasome-associated inflammatory diseases.

Proanthocyanidins are the major anti-diabetic components of cinnamon water extract.

Cinnamon consumption has been found to associate with the attenuation of diabetes mellitus. The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as a causative factor of type 2 diabetes mellitus (T2DM). Here, we investigated whether cinnamon has any beneficial effect on the toxic aggregation of hIAPP. We found that cinnamon water extract (CWE) inhibited the amyloid formation of hIAPP in a dose-dependent manner, and identified proanthocyanidins as the major anti-amyloidogenic compounds of CWE. Proanthocyanidins affected the secondary structures of hIAPP and delayed the structural transition from unstructured coils to ß-sheet-rich structures. Further studies showed that proanthocyanidins not only inhibited the formation of hIAPP oligomers, but also significantly attenuated the membrane damaging and cytotoxic effects caused by the hIAPP aggregation. Together, these results suggest a possible way by which cinnamon shows beneficial effects on T2DM, and indicate a potential pharmacological usage of proanthocyanidins as an anti-diabetic drug candidate.

Salvianolic acid B inhibits the amyloid formation of human islet amyloid polypeptide and protects pancreatic beta-cells against cytotoxicity.

The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of type 2 diabetes mellitus (T2DM). Salvia miltiorrhiza (Danshen), one of the most commonly used of traditional Chinese medicines, is often used in Compound Recipes for treating diabetes, however with unclear mechanisms. Since salvianolic acid B (SalB) is the most abundant bioactive ingredient of salvia miltiorrhiza water-extract. In this study, we tested whether SalB has any effect on the amyloidogenicity of hIAPP. Our results clearly suggest that SalB can significantly inhibit the formation of hIAPP amyloid and disaggregate hIAPP fibrils. Furthermore, photo-crosslinking based oligomerization studies suggest SalB significantly suppresses the toxic oligomerization of hIAPP monomers. Cytotoxicity protection effects on pancreatic INS-1 cells by SalB were also observed using MTT-based assays, potentially due to the inhibition on the membrane disruption effects and attenuated mitochondria impairment induced by hIAPP. These results provide evidence that SalB may further be studied on the possible pharmacological treatment for T2DM.