Pancreatic Β-cell Model Research Articles

Pharmacological and phytochemical insights on the pancreatic β-cell modulation by Angelica L. roots

Ethnopharmacological relevanceAngelica roots are a significant source of traditional medicines for various cultures around the northern hemisphere, from indigenous communities in North America to Japan. Among its many applications, the roots are used to treat type 2 diabetes mellitus; however, this application is not mentioned often. Ethnopharmacological studies have reported the use of A. japonica var. hirsutiflora, A. furcijuga, A. shikokiana, and A. keiskei to treat diabetes symptoms, and further reports have demonstrated the three angelica roots, i.e., A. japonica var. hirsutiflora, A. reflexa, and A. dahurica, exhibit insulin secretagogue activity. Aim of the studyThis study aimed to phytochemically characterize and compare angelica roots monographed in the European Pharmacopeia 11th, isolate major plant metabolites, and assess extracts and isolates' capability to modulate pancreatic β-cell function. Materials and methodsRoot extracts of Angelica archangelica, Angelica dahurica, Angelica biserrata, and Angelica sinensis were phytochemically profiled using liquid chromatography method coupled with mass spectrometry. Based on this analysis, simple and furanocoumarins were isolated using chromatography techniques. Extracts (1.6–50 μg/mL) and isolated compounds (5–40 μmol/L) were studied for their ability to modulate insulin secretion in the rat insulinoma INS-1 pancreatic β-cell model. Insulin was quantified by the homogeneous time-resolved fluorescence method. ResultsForty-one secondary metabolites, mostly coumarins, were identified in angelica root extracts. A. archangelica, A. dahurica, and A. biserrata root extracts at concentration of 12.5–50 μg/mL potentiated glucose-induced insulin secretion, which correlated with their high coumarin content. Subsequently, 23 coumarins were isolated from these roots and screened using the same protocol. Coumarins substituted with the isoprenyl group were found to be responsible for the extracts' insulinotropic effect. ConclusionsInsulinotropic effects of three pharmacopeial angelica roots were found, the metabolite profiles and pharmacological activities of the roots were correlated, and key structures responsible for the modulation of pancreatic β-cell function were identified. These findings may have implications for the traditional use of angelica roots in treating diabetes. Active plant metabolites may also become lead structures in the search for new antidiabetic treatments.

ODP254 WFS1 Loss of Function Causes ER Stress-Mediated Inflammation in Pancreatic β-Cells

Abstract Wolfram syndrome is a rare genetic disorder characterized by juvenile-onset diabetes mellitus, optic nerve atrophy, hearing loss, diabetes insipidus, and progressive neurodegeneration. Pathogenic variants in the WFS1 gene are the main causes of Wolfram syndrome. WFS1 encodes a transmembrane protein localized to the endoplasmic reticulum (ER) and regulates the unfolded protein response (UPR). Loss of function of WFS1 leads to dysregulation of insulin production and secretion, ER calcium depletion, and cytosolic calpains activation, resulting in the activation of apoptotic cascades. Although the terminal UPR is well known to yield an inflammatory response called "sterilized inflammation" that accelerates β-cell dysfunction and death in diabetes, the contribution of β-cell inflammation to the development of diabetes in Wolfram syndrome has not been fully understood. Here we show that WFS1-deficiency enhances the gene expression of pro-inflammatory cytokines and chemokines, leading to cytokine-induced ER-stress and cell death in pancreatic β-cells. Under thechronic[BL1] high-glucose condition, CRISPR/Cas9-mediated Wfs1-knockout (KO) INS-1 832/13 cells, the pancreatic β-cell model of Wolfram syndrome, showed enhanced gene expression levels of ER stress markers (Chop, sXbp1, Bip, and Txnip). Moreover, the gene expression levels of pro-inflammatory cytokines (Il-1β, Il-6), chemokine (Ccl2), and vascular endothelial growth factor A (VegfA) were higher in Wfs1- KO INS-1 832/13 cells when compared to Wfs1-wild type INS-1 832/13 cells. [BL2] The high glucose-induced pro-inflammatory cytokine genes in Wfs1-KO INS-1 832/13 cells were mediated by the PKR-like ER-resident kinase (PERK) and inositol-requiring enzyme 1α (IRE1α)pathways. Furthermore, the IFN-γ and IL-1β treatment enhanced ER stress-mediated cell death and upregulated the pro-inflammatory cytokine gene expression in Wfs1-KO INS-1 832/13 cells. To confirm these findings, we investigated whether inflammation occurs in a mouse model of Wolfram syndrome. Compared to wild-type mice, 10-month-old whole body Wfs1-KO mouse islets showed a significantly higher density of Iba1, which is a marker for macrophage expression. The islet-infiltrated macrophages in these Wfs1-KO mice expressed the M1-macrophage marker (CD68). There was also strong fibrosis and a high density of endothelial cell marker (CD31) in the Wfs1-KO mouse islets. These results demonstrate that WFS1 loss-of-function enhances the inflammatory responses in pancreatic β-cells. The results in our study identified sterile inflammation as a new pathological mechanism in the development of diabetes in Wolfram syndrome. In Wolfram syndrome model mice, islets are highly infiltrated with macrophages, suggesting that the vicious cycle of ER stress and sterile inflammation accelerates the progression of diabetes in a cell-autonomous and cell-nonautonomous manner. A deeper understanding of the pathophysiology of Wolfram syndrome is essential for developing novel therapeutic targets for the disease. Presentation: No date and time listed

Front Cover: Cucurbit[7]uril Inhibits Islet Amyloid Polypeptide Aggregation by Targeting N Terminus Hot Segments and Attenuates Cytotoxicity (Chem. Eur. J. 38/2022)

Islet amyloid polypeptide (IAPP) self-assembly is responsible for the decline of pancreatic β-cells, which is implicated in type II diabetes. The macrocyclic host, cucurbit[7]uril (CB[7]), effectively inhibits IAPP aggregation through ion–dipole and hydrophobic interactions with different residues of the monomeric peptide in its primary random-coil conformation. Most importantly, CB[7] rescues rat insulinoma cells (a robust pancreatic β-cell model) from IAPP-assembly-associated cytotoxicity. More information can be found in the Research Article by D. Maity, A. D. Hamilton, and co-workers (DOI: 10.1002/chem.202200456).

Cucurbit[7]uril Inhibits Islet Amyloid Polypeptide Aggregation by Targeting N Terminus Hot Segments and Attenuates Cytotoxicity.

Invited for the cover of this issue is the group of Prof. Hamilton at New York University. The image depicts how cucurbit[7]uril inhibits islet amyloid polypeptide self-assembly that rescues rat insulinoma cells (a pancreatic β-cell model) from assembly-associated cytotoxicity. Read the full text of the article at 10.1002/chem.202200456.

In Vitro Assays to Identify Metabolism-Disrupting Chemicals with Diabetogenic Activity in a Human Pancreatic β-Cell Model.

There is a need to develop identification tests for Metabolism Disrupting Chemicals (MDCs) with diabetogenic activity. Here we used the human EndoC-βH1 β-cell line, the rat β-cell line INS-1E and dispersed mouse islet cells to assess the effects of endocrine disruptors on cell viability and glucose-stimulated insulin secretion (GSIS). We tested six chemicals at concentrations within human exposure (from 0.1 pM to 1 µM). Bisphenol-A (BPA) and tributyltin (TBT) were used as controls while four other chemicals, namely perfluorooctanoic acid (PFOA), triphenylphosphate (TPP), triclosan (TCS) and dichlorodiphenyldichloroethylene (DDE), were used as “unknowns”. Regarding cell viability, BPA and TBT increased cell death as previously observed. Their mode of action involved the activation of estrogen receptors and PPARγ, respectively. ROS production was a consistent key event in BPA-and TBT-treated cells. None of the other MDCs tested modified viability or ROS production. Concerning GSIS, TBT increased insulin secretion while BPA produced no effects. PFOA decreased GSIS, suggesting that this chemical could be a “new” diabetogenic agent. Our results indicate that the EndoC-βH1 cell line is a suitable human β-cell model for testing diabetogenic MDCs. Optimization of the test methods proposed here could be incorporated into a set of protocols for the identification of MDCs.

Selenomethionine modulates insulin secretion in the MIN6-K8 mouse insulinoma cell line.

Selenium is an essential trace element of interest for its potential role in glucose homeostasis. The present study investigated the impact of selenium supplementation as selenomethionine (SeMet) on insulin secretion in MIN6-K8 cells, a pancreatic β-cell model. We found that SeMet enhanced percent glucose-induced insulin secretion, while also increasing tolbutamide- and KCl-induced percent insulin secretion. RNA-sequencing showed that SeMet supplementation altered expression of several selenoproteins, including glutathione peroxidase 3 (Gpx3) and selenoprotein P (SelP). Targeted knockdown of Gpx3 increased both percent and total insulin release, while SelP knockdown increased insulin content and insulin release. Collectively, these studies support a putative role for selenium and selenoproteins in the regulation of insulin secretion, glucose homeostasis, and diabetes risk.

Dimethyl sulfoxide acutely enhances regulated insulin secretion in the MIN6-K8 mouse insulinoma cell line.

Diabetes mellitus is a metabolic disorder projected to afflict 700 million people globally by 2045. Fundamental to the progression of diabetes is an insufficient supply of insulin to meet metabolic demand. The MIN6-K8 cell line is a mouse insulinoma model of pancreatic β-cells frequently used to study the mechanisms of insulin secretion. Here, we evaluated the effects of short-term exposure to dimethyl sulfoxide (DMSO), a polar aprotic solvent commonly used in drug screening, on physiological characteristics of MIN6-K8 cells. Short-term exposure of MIN6-K8 cells to DMSO enhanced glucose-induced and tolbutamide-stimulated insulin secretion without significant effects on basal secretion or potassium responsiveness. Calcium influx was enhanced during glucose and tolbutamide treatments, suggesting that DMSO's mechanism of action is upstream of calcium-dependent insulin granule exocytosis. Based on these studies, investigators should use caution when conducting experiments with DMSO in the MIN6-K8 cell line and should report all DMSO concentrations when used as a solvent.

Spike-adding structure in fold/hom bursters

Square-wave or fold/hom bursting is typical of many excitable dynamical systems, such as pancreatic or other endocrine cells. Besides, it is also found in a great variety of fast-slow systems coming from other neural models, chemical reactions, laser dynamics, and so on. We focus on the spike-adding process and its connection with the homoclinic structure of the system. The creation of new fast spikes on a bursting neuron is an important phenomenon as it increases the duty cycle of the neuron. Here we mainly work with the Hindmarsh-Rose neuron model, a prototype of fold/hom bursting, but also with the pancreatic β-cell model, where, as already known from the literature, homoclinic bifurcations play an important role in bursting dynamics. Based on several numerical simulations, we present a theoretical scheme that provides a complete scenario of bifurcations involved in the spike-adding process and their connection with the homoclinic bifurcations on the parametric space. The global scheme explains the different phenomena of the spike-adding processes presented in literature (continuous and chaotic processes after Terman analysis) and moreover, it also indicates where each kind of spike-adding process occurs. Different elements are involved in the theoretical scheme, such as homoclinic isolas, canard orbits, inclination and orbit flip codimension-two bifurcation points and several pencils of period doubling and fold bifurcations, all of them illustrated with different numerical techniques. Some of these bifurcations needed in the process may be not visible on some numerical simulations because the organizing points are in different parametric planes due to the high dimension of the whole parameter space, but their effects are present. Therefore, we introduce a mechanism of the spike-adding process in fold/hom bursters in the whole space of parameters, even if apparently no role is played by the “far-away” homoclinic bifurcations. This fact is illustrated showing how the theoretical scheme provides a theoretical explanation to the different interspike-interval bifurcation diagrams (IBD) that have appeared in the literature for different models.

Synchronization in a fractional-order model of pancreatic β-cells

β-cells in the pancreas can be described by a model of coupled biological oscillators with communication links, which can synchronize their electrical activities, giving rise to a square-wave bursting-like insulin release. In fact, β-cells play a vital role in analyzing and characterizing diabetes conditions. This research work studies the synchronization between two fractional-order pancreatic β-cells. Numerically, the fractional-order model of the pancreatic β-cell is analyzed using an algorithm derived from the Grunwald–Letnikov scheme. It is found that, by modifying only the fractional-order while preserving the system parameter values, different types of bursting activities can be observed. Then, synchronization in the coupled fractional-order pancreatic β-cells is studied in detail by considering different patterns of the bursting activities. Simulation results demonstrate that a complete synchronization is effectively attained by choosing a proper value for the control gain.

Mitochondrial Morphology and Function of the Pancreatic β-Cells INS-1 Model upon Chronic Exposure to Sub-Lethal Cadmium Doses.

The impact of chronic cadmium exposure and slow accumulation on the occurrence and development of diabetes is controversial for human populations. Islets of Langerhans play a prominent role in the etiology of the disease, including by their ability to secrete insulin. Conversion of glucose increase into insulin secretion involves mitochondria. A rat model of pancreatic β-cells was exposed to largely sub-lethal levels of cadmium cations applied for the longest possible time. Cadmium entered cells at concentrations far below those inducing cell death and accumulated by factors reaching several hundred folds the basal level. The mitochondria reorganized in response to the challenge by favoring fission as measured by increased circularity at cadmium levels already ten-fold below the median lethal dose. However, the energy charge and respiratory flux devoted to adenosine triphosphate synthesis were only affected at the onset of cellular death. The present data indicate that mitochondria participate in the adaptation of β-cells to even a moderate cadmium burden without losing functionality, but their impairment in the long run may contribute to cellular dysfunction, when viability and β-cells mass are affected as observed in diabetes.

Fractional dynamical behavior of electrical activity in a model of pancreatic β-cells

The electrical activity of a three dimensional fractional-order model of pancreatic β-cells is numerically studied. The dynamics of the model is understood through the Adams–Bashforth–Moulton predictor corrector scheme, which is a time-domain approach. By considering both integer-order and fractional-order models, we highlight some differences in their dynamics characteristics. It is shown that the fractional model can give rise to chaotic dynamics due to the existence of a Smale horseshoe. Some behaviors related with diabetes mellitus are also reported. The fractional model gets closer to the real biological behavior, thus provides motivation in modeling biological systems with fractional order derivatives.

Positive Feedback Amplifies the Response of Mitochondrial Membrane Potential to Glucose Concentration in Clonal Pancreatic Beta Cells

Analysis of the cellular mechanisms of metabolic disorders, including type 2 diabetes mellitus, is complicated by the large number of reactions and interactions in metabolic networks. Metabolic control analysis with appropriate modularization is a powerful method for simplifying and analyzing these networks. To analyze control of cellular energy metabolism in adherent cell cultures of the INS-1 832/13 pancreatic β-cell model we adapted our microscopy assay of absolute mitochondrial membrane potential (ΔψM) to a fluorescence microplate reader format, and applied it in conjunction with cell respirometry. In these cells the sensitive response of ΔψM to extracellular glucose concentration drives glucose-stimulated insulin secretion. Using metabolic control analysis we identified the control properties that generate this sensitive response. Force-flux relationships between ΔψM and respiration were used to calculate kinetic responses to ΔψM of processes both upstream (glucose oxidation) and downstream (proton leak and ATP turnover) of ΔψM. The analysis revealed that glucose-evoked ΔψM hyperpolarization is amplified by increased glucose oxidation activity caused by factors downstream of ΔψM. At high glucose, the hyperpolarized ΔψM is stabilized almost completely by the action of glucose oxidation, whereas proton leak also contributes to the homeostatic control of ΔψM at low glucose. These findings suggest a strong positive feedback loop in the regulation of β-cell energetics, and a possible regulatory role of proton leak in the fasting state. Analysis of islet bioenergetics from published cases of type 2 diabetes suggests that disruption of this feedback can explain the damaged bioenergetic response of β-cells to glucose. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.

Polygodial, a sesquiterpene isolated from Drimys brasiliensis (Winteraceae), triggers glucocorticoid-like effects on pancreatic β-cells

Despite its common use, the synthetic glucocorticoid dexamethasone can cause several adverse effects, such as diabetes and insulin-related metabolic impairment. Thus, research on molecules that could provide the same anti-inflammatory response with milder side effects is constant. In this work the anti-inflammatory activity of the natural sesquiterpene polygodial, extracted from the endemic Brazilian plant Drimys brasiliensis Miers (Winteraceae), was investigated. Employing a pancreatic β-cell model (INS 1E), the effect of polygodial on signaling pathways is similar to that caused by dexamethasone – both increased MKP1 and decreased ERK1/2 expression in a dose-response and time-dependent manner. Relating to such finding, nuclear translocation of the glucocorticoid receptor was also discovered to be induced by the sesquiterpene. Molecular modeling results indicated that polygodial was capable of docking to the glucocorticoid receptor, but presented preference for the Arg611 binding site rather than Thr739 when set to bind freely inside the pocket. At last, fragmentation of DNA was verified as consequence of sesquiterpene-induced cell death. Altogether, our results suggest that, like dexamethasone, polygodial interacts the glucocorticoid receptor ligand binding domain but create fewer ligand-protein interactions at the site, yielding a weaker effector response. Such property provides an advantage when regarding the adverse effects resulting from stronger affinity ligands of the glucocorticoid receptor, such as in the case of the current standard dexamethasone-based treatment. This aspect, also, turns polygodial an interesting hit compound to the development of new drugs based on its backbone structure providing less harmful anti-inflammatory treatments.

Cystine accumulation attenuates insulin release from the pancreatic β-cell due to elevated oxidative stress and decreased ATP levels.

The pancreatic β-cell has reduced antioxidant defences making it more susceptible to oxidative stress. In cystinosis, a lysosomal storage disorder, an altered redox state may contribute to cellular dysfunction. This rare disease is caused by an abnormal lysosomal cystine transporter, cystinosin, which causes excessive accumulation of cystine in the lysosome. Cystinosis associated kidney damage and dysfunction leads to the Fanconi syndrome and ultimately end-stage renal disease. Following kidney transplant, cystine accumulation in other organs including the pancreas leads to multi-organ dysfunction. In this study, a Ctns gene knockdown model of cystinosis was developed in the BRIN-BD11 rat clonal pancreatic β-cell line using Ctns-targeting siRNA. Additionally there was reduced cystinosin expression, while cell cystine levels were similarly elevated to the cystinotic state. Decreased levels of chronic (24 h) and acute (20 min) nutrient-stimulated insulin secretion were observed. This decrease may be due to depressed ATP generation particularly from glycolysis. Increased ATP production and the ATP/ADP ratio are essential for insulin secretion. Oxidised glutathione levels were augmented, resulting in a lower [glutathione/oxidised glutathione] redox potential. Additionally, the mitochondrial membrane potential was reduced, apoptosis levels were elevated, as were markers of oxidative stress, including reactive oxygen species, superoxide and hydrogen peroxide. Furthermore, the basal and activated phosphorylated forms of the redox-sensitive transcription factor NF-κB were increased in cells with silenced Ctns. From this study, the cystinotic-like pancreatic β-cell model demonstrated that the altered oxidative status of the cell, resulted in depressed mitochondrial function and pathways of ATP production, causing reduced nutrient-stimulated insulin secretion.

PH Dependence of Amylin Fibrillization

In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic β-cells that make insulin and amylin. The aggregative misfolding of amylin is pH-dependent, and exposure of the hormone to acidic and basic environments could be physiologically important. Amylin has two ionizable residues between pH 3 and 9: the α-amino group and His18. Our approach to measuring the pKa values for these sites has been to look at the pH dependence of fibrillization in amylin variants that have only one of the two groups. The α-amino group at the unstructured N-terminus of amylin has a pKa near 8.0, similar to the value in random coil models. By contrast, His18, which is involved in the intermolecular β-sheet structure of the fibrils, has a pKa that is lowered to 5.0 in the fibrils compared to the random coil value of 6.5. The lowered pKa of His18 is due to the hydrophobic environment of the residue, and electrostatic repulsion between positively charged His18 residues on neighboring amylin molecules in the fibril. His18 acts as an electrostatic switch inhibiting fibrillization in its charged state. The presence of a charged side chain at position 18 also affects fibril morphology and lowers amylin cytotoxicity toward a MIN6 mouse model of pancreatic β-cells. In addition to the two expected pKa values, we detected an apparent pKa of ~4.0 for the amylin-derived peptide NAc-SNNFGAILSS-NH2, which has no titratable groups. This pKa is due to the pH-induced ionization of the dye thioflavin T. By using alternative methods to follow fibrillization such as the dye Nile Red or turbidimetry, we were able to distinguish between the titration of the dye and groups on the peptide. Large differences in reaction kinetics were observed between the different methods at acidic pH, because of charges on the ThT dye, which hinder fibril formation much like the charges on the protein.

Do plants mediate their anti-diabetic effects through anti-oxidant and anti-apoptotic actions? an in vitro assay of 3 Indian medicinal plants

BackgroundBoth experimental and clinical studies suggest that oxidative stress plays a major role in the pathogenesis of both types of diabetes mellitus. This oxidative stress leads to β-cell destruction by apoptosis. Hence exploring agents modulating oxidative stress is an effective strategy in the treatment of both Type I and Type II diabetes. Plants are a major source of anti-oxidants and exert protective effects against oxidative stress in biological systems. Phyllanthus emblica, Curcuma longa and Tinospora cordifolia are three such plants widely used in Ayurveda for their anti-hyperglycemic activity. Additionally their anti-oxidant properties have been scientifically validated in various experimental in vitro and in vivo models. Hence the present in vitro study was planned to assess whether the anti-hyperglycemic effects of the hydro-alcoholic extracts of Phyllanthus emblica (Pe) and Curcuma longa (Cl) and aqueous extract of Tinospora cordifolia (Tc) are mediated through their antioxidant and/or anti-apoptotic property in a streptozotocin induced stress model.MethodsRINm5F cell line was used as a model of pancreatic β-cells against stress induced by streptozotocin (2 mM). Non-toxic concentrations of the plant extracts were identified using MTT assay. Lipid peroxidation through MDA release, modulation of apoptosis and insulin release were the variables measured to assess streptozotocin induced damage and protection afforded by the plant extracts.ResultsAll 3 plants extracts significantly inhibited MDA release from RIN cells indicating protective effect against STZ induced oxidative damage. They also exhibited a dose dependent anti-apoptotic effect as seen by a decrease in the sub G0 population in response to STZ. None of the plant extracts affected insulin secretion from the cells to a great extent.ConclusionThe present study thus demonstrated that the protective effect of the selected medicinal plants against oxidative stress induced by STZ in vitro, which was exerted through their anti-oxidant and anti-apoptotic actions.

Effects of Heparin on Amylin Fibrillization

Amylin is an endocrine hormone that helps regulate blood glucose levels and controls appetite. In patients with type 2 diabetes, amylin misfolds into amyloid plaques that have been implicated in the destruction of the pancreatic β-cells that synthesize insulin and amylin. The plaques found in situ contain not only amylin but heparan sulfate proteoglycans that attach to the fibrils through their glycosaminoglycan chains. We investigated the interaction of amylin with heparin fragments of defined length, that model the glycosaminoglycan chains associated with amyloid deposits. We found that heparin enhances fibrillization in a manner that depends on the length of the polysaccharide fragments. We used NMR to establish that the negatively charged heparin fragments bind to the positively charged N-terminal half of amylin. We used FRET to determine that heparin associates with amylin fibrils rather than enhancing fibrillization catalytically. We used TIRFM to show that fluorescein-labeled heparin is co-localized with amylin fibrils. The mechanism of binding appears to involve electrostatic complementation between the negatively charged heparin helix structure and the positively charged cross-β sheet structure of amylin fibrils. To see how heparin affects the biological function of amylin, we used a fluorimetric assay of cytotoxicity towards a mouse model of pancreatic β-cells. With heparin fragments longer than 20 saccharides, cell death was similar to when amylin was added alone. By contrast, short heparin fragments of 2 to 8 saccharides protect against cytotoxicity and as such may be leads for drugs to treat type 2 diabetes.

Mathematical modeling demonstrates how multiple slow processes can provide adjustable control of islet bursting

Pancreatic islets exhibit bursting oscillations that give rise to oscillatory Ca2+ entry and insulin secretion from β-cells. These oscillations are driven by a slowly activating K+ current, Kslow, which is composed of two components: an ATP-sensitive K+ current and a Ca2+-activated K+ current through SK4 channels. Using a mathematical model of pancreatic β-cells, we analyze how the factors that comprise Kslow can contribute to bursting. We employ the dominance factor technique developed recently to do this and demonstrate that the contributions that the slow processes make to bursting are non-obvious and often counter-intuitive, and that their contributions vary with parameter values and are thus adjustable.

Mechanism of Amylin Fibrillization Enhancement by Heparin

We characterized the interaction of amylin with heparin fragments of defined length, which model the glycosaminoglycan chains associated with amyloid deposits found in type 2 diabetes. Binding of heparin fragments to the positively charged N-terminal half of monomeric amylin depends on the concentration of negatively charged saccharides but is independent of oligosaccharide length. By contrast, amylin fibrillogenesis has a sigmoidal dependence on heparin fragment length, with an enhancement observed for oligosaccharides longer than four monomers and a leveling off of effects beyond 12 monomers. The length dependence suggests that the negatively charged helical structure of heparin electrostatically complements the positively charged surface of the fibrillar amylin cross-β structure. Fluorescence resonance energy transfer and total internal reflection fluorescence microscopy experiments indicate that heparin associates with amylin fibrils, rather than enhancing fibrillogenesis catalytically. Short heparin fragments containing two- or eight-saccharide monomers protect against amylin cytotoxicity toward a MIN6 mouse cell model of pancreatic β-cells.

SiRNA‐mediated knock‐down of ZnT3 and ZnT8 affects production and secretion of insulin and apoptosis in INS‐1E cells

Zinc is essential for the crystallization of insulin in pancreatic β-cells and is thought to induce apoptosis in a dose-dependent manner, thereby regulating β-cell mass. Therefore, a tight intracellular regulation of Zn²(+) is required. The zinc-transporter family SLC30A is an important factor in the regulation of zinc homeostasis. The aim of this study was to examine the effect of the zinc transporters ZnT3 and ZnT8 on insulin metabolism and apoptosis. Both these proteins are present in pancreatic β-cells and have been linked to diabetes. The objective of our study was to perform a considerable siRNA-mediated knock-down of ZnT3 and ZnT8 in INS-1E cells, a pancreatic β-cell model, and afterwards examine the impact on cell viability and insulin metabolism. Increased levels of apoptosis were observed after knock-down of both ZnT3 and ZnT8. Insulin secretion was significantly reduced by ZnT3 knock-down, whereas knock-down of ZnT8 resulted in increased intracellular content of insulin accompanied by a relatively lowered secretion. Both zinc transporters in this way seem to play a role in β-cell survival and the ability of these cells to react appropriately to surrounding glucose concentrations.