6 research outputs found
Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation
No full textBackground: Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death. Methodology/Principal Findings: We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elavC(155,Gal4) showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures. Conclusions/Significance: These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology.Original Publication: Sebastian Schultz, Peter Nilsson and Gunilla Torstensdotter Westermark, Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation, 2011, PLoS ONE, (6), 6. http://dx.doi.org/10.1371/journal.pone.0020221 Copyright: Public Library of Science (PLoS) http://www.plos.org/</p
A new biological prospective for the 2-phenylbenzofurans as inhibitors of α-glucosidase and of the islet amyloid polypeptide formation
No full textIn this study, we have investigated a series of hydroxylated 2-phenylbenzofurans compounds for their inhibitory activity against α-amylase and α-glucosidase activity.
Inhibitors of carbohydrate degrading enzymes seem to have an important role as antidiabetic drugs.
Diabetes mellitus is a wide-spread metabolic disease characterized by elevated levels of blood glucose. The most common is type 2 diabetes, which can lead to severe complications. Since the aggregates of islet amyloid polypeptide (IAPP) are common in diabetic patients, the effect of compounds to inhibit amyloid fibril formation was also determined.
All the compounds assayed showed to be more active against α-glucosidase. Compound 16 showed the lowest IC50 value of the series, and it is found to be 167 times more active than acarbose, the reference compound. The enzymatic activity assays showed that compound 16 acts as a mixed-type inhibitor of α-glucosidase. Furthermore, compound 16 displayed effective inhibition of IAPP aggregation and it manifested no significant cytotoxicity.
To predict the binding of compound 16 to IAPP and α-glucosidase protein complexes, molecular docking studies were performed.
Altogether, our results support that the 2-phenylbenzofuran derivatives could represent a promising candidate for developing molecules able to modulate multiple targets involved in diabetes mellitus disorder
Amyloid Deposition in Transplanted Human Pancreatic Islets : A Conceivable Cause of Their Long-Term Failure
No full textFollowing the encouraging report of the Edmonton group, there was a rejuvenation of the islet transplantation field. After that, more pessimistic views spread when long-term results of the clinical outcome were published. A progressive loss of the beta-cell function meant that almost all patients were back on insulin therapy after 5 years. More than 10 years ago, we demonstrated that amyloid deposits rapidly formed in human islets and in mouse islets transgenic for human IAPP when grafted into nude mice. It is, therefore, conceivable to consider amyloid formation as one potential candidate for the long-term failure. The present paper reviews attempts in our laboratories to elucidate the dynamics of and mechanisms behind the formation of amyloid in transplanted islets with special emphasis on the impact of long-term hyperglycemia.Original Publication:Arne Andersson, Sara Bohman, L A Hakan Borg, Johan Paulsson, Sebastian Schultz, Gunilla Westermark and Per Westermark, Amyloid Deposition in Transplanted Human Pancreatic Islets: A Conceivable Cause of Their Long-Term Failure, 2008, EXPERIMENTAL DIABETES RESEARCH, (2008), 562985.http://dx.doi.org/10.1155/2008/562985Copyright: Author
Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation
No full textAmyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP). Peptides were added in solubilized from (50 mu M) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occurein cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.Original Publication:Johan F Paulsson, Sebastian Schultz, Martin Kohler, Ingo Leibiger, Per-Olof Berggren and Gunilla Westermark, Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation, 2008, EXPERIMENTAL DIABETES RESEARCH, (2008), 865850.http://dx.doi.org/10.1155/2008/865850Copyright: Author
Studies on Islet Amyloid Polypeptide Aggregation [Elektronisk resurs] : From Model Organism to Molecular Mechanisms
No full textThe proper folding of a protein into its defined three--‐dimensional structure is one of the many fundamental challenges a cell encounters. A number of tightly controlled pathways have evolved to assist in the proper folding of a protein, but also to aid in the removal of misfolded proteins. Despite the presence of these pathways accumulation of misfolded proteins can still occur. Amyloid deposits consist of misfolded proteins with a characteristic highly ordered fibrillar structure that will exert affinity for the amyloid dye Congo red and has a unique X-ray diffraction pattern. Currently 27 different proteins have been identified as amyloid forming proteins in human, however the exact role of amyloid in the pathogenesis of the connected disease is most often unclear.Islet amyloid is made up of the beta cell derived hormone islet amyloid polypeptide (IAPP) and is associated with the development of type 2 diabetes. Propagation of IAPP-fibrils is believed to be one important cause of the pancreatic beta cell death detected in patients with type 2 diabetes. IAPP is a naturally occurring polypeptide hormone stored and secreted together with insulin. IAPP and insulin arise from posttranslational processing of their biological inactive precursors proIAPP and proinsulin. In addition to human, cat and monkey IAPP will form amyloid deposits in conditions resembling human type 2 diabetes. However, IAPP from mouse and rat do not form amyloid as a result of the differences in amino acid sequence.My main research goal was to establish a unique model system suitable to study the effects of proIAPP and IAPP aggregation. I selected Drosophila melanogaster due to its many suitable characteristics as a model organism and its superior genetic toolbox. I have demonstrated that over--‐expression of hproIAPP and hIAPP in the central nervous system (CNS) results in aggregate formation in the brain and neighbouring fat body. Consistent with previous studies, expression of mIAPP does not result in the formation of aggregates. To investigate the intracellular effects of hproIAPP and hIAPP aggregation on a specific population of neurons, we targeted the expression of these peptides specifically to 16 neurons in the brain, the pdf- neurons. These pdf-neurons are divided into 2 clusters of 8 cells per brain hemisphere. First I showed that expression of aggregation prone hIAPP and hproIAPP resulted in significant death of the 8 cells, whereas expression of mIAPP had no such effect. In efforts to pinpoint the mechanisms behind the observed cell death I demonstrated that hproIAPP and hIAPP both pass the ERs quality control for protein folding and that the initiated cell death does not occur through classical apoptosis. Instead, selective autophagy is activated by hIAPP and hproIAPP. This activation counteracts the usually neuro-protective effects of autophagy and contributes to cell death. Strikingly, I also showed that Aâ, the amyloid protein implicated in Alzheimer’s disease, does not exhibit any intracellular toxicity when expressed in pdf-cells. This supports the existence of separate toxic pathways for different amyloid proteins.</p
Efficient Vascular and Neural Engraftment of Stem Cell–Derived Islets
No full textPluripotent stem cell–derived islets (SC-islets) have emerged as a new source for β-cell replacement therapy. The function of human islet transplants is hampered by excessive cell death posttransplantation; contributing factors include inflammatory reactions, insufficient revascularization, and islet amyloid formation. However, there is a gap in knowledge of the engraftment process of SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at 3 months posttransplantation and observed that cell apoptosis rates were lower but vascular density was similar in SC-islets compared with human islets. Whereas the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. Oxygenation in the SC-islet grafts was twice as high as that in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, reinnervation of the SC-islets was four- to fivefold higher than that of the human islets. Both SC-islets and human islets contained amyloid at 1 and 3 months posttransplantation. We conclude that the vascular and neural engraftment of SC-islets are superior to those of human islets, but grafts of both origins develop amyloid, with potential long-term consequences.</p
