Activation of TRPV4 channel in pancreatic INS-1E beta cells enhances glucose-stimulated insulin secretion via calcium-dependent mechanisms

Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca²⁺- and Mg²⁺-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca²⁺ and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca²⁺ and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca²⁺]_i and insulin secretion in INS-1E cells.     

A hIAPP-derived all-d-amino-acid inhibits hIAPP fibrillation efficiently at membrane surface by targeting α-helical oligomeric intermediates

A variety of peptides and peptide derivatives have been constructed using the “β-sheet core segment” of amyloid proteins as inhibitors of amyloidogenic fibrillation. A novel all-d-amino-acid from hIAPP β-sheet core segment (hIAPP 22–27) is demonstrated to inhibit hIAPP fibril formation efficiently both at the phospholipid membrane and in bulk solution. The inhibitor terminates hIAPP aggregation to the α-helical oligomeric intermediates at the membrane surface, whereas it stops the aggregation at the stage of β-sheet oligomeric intermediates in bulk solution. This is the first evidence that the inhibition mechanism of the inhibitor at membrane surface is significantly different from that in bulk solution.     

Separate Molecular Determinants in Amyloidogenic and Antimicrobial Peptides

Several amyloid-forming and antimicrobial peptides (AMYs and AMPs) have the ability to bind to and damage cell membranes. In addition, some AMYs possess antimicrobial activity and some AMPs form amyloid-like fibrils, relating the two peptide types and their properties. However, a comparison of their sequence characteristics reveals important differences. The high β-strand and aggregation propensities typical of AMYs are largely absent in α-helix-forming AMPs, which are instead marked by a strong amphipathic moment not generally found in AMYs. Although a few peptides, for example, islet amyloid polypeptide and dermaseptin S9, combine some determinants of both groups, the structural distinctions suggest that antimicrobial activity and amyloid formation are separate features not generally associated.     

The association of lipids with amyloid fibrils

Amyloid formation continues to be a widely studied area because of its association with numerous diseases, such as Alzheimer’s and Parkinson’s diseases. Despite a large body of work on protein aggregation and fibril formation, there are still significant gaps in our understanding of the factors that differentiate toxic amyloid formation in vivo from alternative misfolding pathways. In addition to proteins, amyloid fibrils are often associated in their cellular context with several types of molecule, including carbohydrates, polyanions, and lipids. This review focuses in particular on evidence for the presence of lipids in amyloid fibrils and the routes by which those lipids may become incorporated. Chemical analyses of fibril composition, combined with studies to probe the lipid distribution around fibrils, provide evidence that in some cases, lipids have a strong association with fibrils. In addition, amyloid fibrils formed in the presence of lipids have distinct morphologies and material properties. It is argued that lipids are an integral part of many amyloid deposits in vivo, where their presence has the potential to influence the nucleation, morphology, and mechanical properties of fibrils. The role of lipids in these structures is therefore worthy of further study.     

Stabilities and structures of islet amyloid polypeptide (IAPP22–28) oligomers: From dimer to 16-mer

Background

The formation of amyloid fibrils is associated with many age-related degenerative diseases. Nevertheless, the molecular mechanism that directs the nucleation of these fibrils is not fully understood.

Methods

Here, we performed MD simulations for the NFGAILS motif of hIAPP associated with the type II diabetes to estimate the stabilities of hIAPP_22–28 protofibrils with different sizes: from 2 to 16 chains. In addition, to study the initial self-assembly stage, 4 and 8 IAPP_22–28 chains in explicit solvent were also simulated.

Results

Our results indicate that the ordered protofibrils with no more than 16 hIAPP_22–28 chains will be structurally stable in two layers, while one-layer or three-layer models are not stable as expected. Furthermore, the oligomerization simulations show that the initial coil structures of peptides can quickly aggregate and convert to partially ordered β-sheet-rich oligomers.

Conclusions

Based on the obtained results, we found that the stability of an IAPP_22–28 oligomer was not only related with its size but also with its morphology. The driving forces to form and stabilize an oligomer are the hydrophobic effects and backbone H-bond interaction. Our simulations also indicate that IAPP_22–28 peptides tend to form an antiparallel strand orientation within the sheet.

General significance

Our finding can not only enhance the understanding about potential mechanisms of hIAPP nuclei formation and the extensive structural polymorphisms of oligomers, but also provide valuable information to develop potential β-sheet formation inhibitors against type II diabetes.     

The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet β-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP_1-19, plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP_1-19 in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP_1-19 is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP_1-19 is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo.     

The two-fold aspect of the interplay of amyloidogenic proteins with lipid membranes

Investigating the pathways leading to the formation of amyloid protein aggregates and the mechanism of their cytotoxicity is fundamental for a deeper understanding of a broad range of human diseases. Increasing evidence indicates that early aggregates are responsible for the cytotoxic effects. This paper addresses the catalytic role of lipid surfaces in promoting aggregation of amyloid proteins and the permeability changes that these aggregates induce on lipid membranes. Effects of amyloid aggregates on model systems such as monolayers, vesicles, liposomes and supported lipid bilayers are reviewed. In particular, the relevance of atomic force microscopy in detecting both kinetics of amyloid formation and amyloid-membrane interactions is emphasized.     

Glucose- and time-dependence of islet amyloid formation in vitro

Islet amyloid contributes to the loss of beta-cell mass in type 2 diabetes. To examine the roles of glucose and time on amyloid formation, we developed a rapid in vitro model using isolated islets from human islet amyloid polypeptide (hIAPP) transgenic mice. Islets from hIAPP transgenic and non-transgenic mice were cultured for up to 7 days with either 5.5, 11.1, 16.7 or 33.3mmol/l glucose. At various time-points throughout the culture period, islets were harvested for determination of amyloid and beta-cell areas, and for measures of cell viability, insulin content, and secretion. Following culture of hIAPP transgenic islets in 16.7 or 33.3mmol/l glucose, amyloid formation was significantly increased compared to 5.5 or 11.1mmol/l glucose culture. Amyloid was detected as early as day 2 and increased in a time-dependent manner so that by day 7, a decrease in the proportion of beta-cell area in hIAPP transgenic islets was evident. When compared to non-transgenic islets after 7-day culture in 16.7mmol/l glucose, hIAPP transgenic islets were 24% less viable, had decreased beta-cell area and insulin content, but displayed no change in insulin secretion. Thus, we have developed a rapid in vitro model of light microscopy-visible islet amyloid formation that is both glucose- and time-dependent. Formation of amyloid in this model is associated with reduced cell viability and beta-cell loss but adequate functional adaptation. It thus enables studies investigating the mechanism(s) underlying the amyloid-associated loss of beta-cell mass in type 2 diabetes.     

Cloning and expression of human islet amyloid polypeptide in cultured cells

Efforts to clone amyloidogenic proteins in the cells often have resulted in cell death. We report successful cloning and expression of recombinant human islet amyloid polypeptide (hIAPP) in cultured mammalian cells. Amylin gets secreted, forms fibrils that are toxic to target cells like beta cells of rat and human. The study involves cloning of full-length amylin in fluorescent protein vector followed by transfection into mammalian cells. The transfected cells with recombinant human amylin, secrete the translated protein corresponding to 37-amino acid native mature IAPP. The mature IAPP secreted out of the cell is purified and characterized by MALDI-TOF/TOF-MS and Western blotting. Purified IAPP forms fibrils as seen by Thioflavin-T fluorescence and AFM, and these fibrils were cytotoxic towards pancreatic cell line RIN5mf cells.     

Comparing the structural properties of human and rat islet amyloid polypeptide by MD computer simulations

Conformational properties of the full-length human and rat islet amyloid polypeptide 1-37 (amyloidogenic hIAPP and non-amyloidogenic rIAPP, respectively) were studied at 310 and 330 K by MD simulations both for the cysteine (reduced IAPP) and cystine (oxidized IAPP) moieties. At all temperatures studied, IAPP does not adopt a well-defined conformation and is essentially random coil in solution, although transient helices appear forming along the peptide between residues 8 and 22, particularly in the reduced form. Above the water percolation transition (at 320 K), the reduced hIAPP moiety presents a considerably diminished helical content remaining unstructured, while the natural cystine moiety reaches a rather compact state, presenting a radius of gyration that is almost 10% smaller and characterized by intrapeptide H-bonds that form many β-bridges in the C-terminal region. This compact conformation presents a short end-to-end distance and seems to form through the formation of β-sheet conformations in the C-terminal region with a minimization of the Y/F distances in a two-step mechanism: the first step taking place when the Y37/F23 distance is ~ 1.1 nm, and subsequently Y37/F15 reaches its minimum of ~ 0.86 nm. rIAPP, which does not aggregate, also presents transient helical conformations. A particularly stable helix is located in proximity of the C-terminal region, starting from residues L27 and P28. Our MD simulations show that P28 in rIAPP influences the secondary structure of IAPP by stabilizing the peptide in helical conformations. When this helix is not present, the peptide presents bends or H-bonded turns at P28 that seem to inhibit the formation of the β-bridges seen in hIAPP. Conversely, hIAPP is highly disordered in the C-terminal region, presenting transient isolated β-strand conformations, particularly at higher temperatures and when the natural disulfide bond is present. Such conformational differences found in our simulations could be responsible for the different aggregational propensities of the two different homologues. In fact, the fragment 30-37, which is identical in both homologues, is known to aggregate in vitro, hence the overall sequence must be responsible for the amyloidogenicity of hIAPP. The increased helicity in rIAPP induced by the serine-to-proline variation at residue 28 seems to be a plausible inhibitor of its aggregation.