Dataset Information

Role of calcium-independent phospholipase A(2)? in human pancreatic islet ?-cell apoptosis.

ABSTRACT: Death of ?-cells due to apoptosis is an important contributor to ?-cell dysfunction in both type 1 and type 2 diabetes mellitus. Previously, we described participation of the Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)?) in apoptosis of insulinoma cells due to ER stress. To examine whether islet ?-cells are similarly susceptible to ER stress and undergo iPLA(2)?-mediated apoptosis, we assessed the ER stress response in human pancreatic islets. Here, we report that the iPLA(2)? protein is expressed predominantly in the ?-cells of human islets and that thapsigargin-induced ER stress promotes ?-cell apoptosis, as reflected by increases in activated caspase-3 in the ?-cells. Furthermore, we demonstrate that ER stress is associated with increases in islet iPLA(2)? message, protein, and activity, iPLA(2)?-dependent induction of neutral sphingomyelinase and ceramide accumulation, and subsequent loss of mitochondrial membrane potential. We also observe that basal activated caspase-3 increases with age, raising the possibility that ?-cells in older human subjects have a greater susceptibility to undergo apoptotic cell death. These findings reveal for the first time expression of iPLA(2)? protein in human islet ?-cells and that induction of iPLA(2)? during ER stress contributes to human islet ?-cell apoptosis. We hypothesize that modulation of iPLA(2)? activity might reduce ?-cell apoptosis and this would be beneficial in delaying or preventing ?-cell dysfunction associated with diabetes.

SUBMITTER: Lei X

PROVIDER: S-EPMC3774083 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Role of calcium-independent phospholipase A(2)β in human pancreatic islet β-cell apoptosis.

Lei Xiaoyong X Zhang Sheng S Bohrer Alan A Barbour Suzanne E SE Ramanadham Sasanka S

American journal of physiology. Endocrinology and metabolism 20121016 11

Death of β-cells due to apoptosis is an important contributor to β-cell dysfunction in both type 1 and type 2 diabetes mellitus. Previously, we described participation of the Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)β) in apoptosis of insulinoma cells due to ER stress. To examine whether islet β-cells are similarly susceptible to ER stress and undergo iPLA(2)β-mediated apoptosis, we assessed the ER stress response in human pancreatic islets. Here, we report that the iPLA(2)β prote ...[more]

PMID: 23074238

Similar Datasets

Project description:The objective of these studies was to clarify the role of Ca(2+) in the mechanism of death evoked by human amylin (hA) in islet beta-cells. hA forms fibrils in vitro and islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet beta-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 microM) manner. Dying cells underwent shrinkage of the nucleus, with clumping and segregation of chromatin into masses that lay against the nuclear envelope, and internucleosomal DNA fragmentation. These cells therefore show many features of apoptosis, although aspects of the morphology might be characteristic of this particular cell type rather than of a general apoptotic nature. Aurintricarboxylic acid, an inhibitor of both Ca(2+)-dependent and Ca(2+)-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 microM. Direct measurements of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in fura-2 acetoxymethyl ester (AM)-loaded beta-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising [Ca(2+)](i). Modulators of Ca(2+) regulation were tested for their effects on hA-induced beta-cell apoptosis. Ca(2+) ionophore (A23187) and thapsigargin (an inhibitor of endoplasmic reticular Ca(2+)-ATPase activity) by themselves evoked apoptosis accompanied by increased [Ca(2+)](i). Neither the Ca(2+) channel blocker verapamil, the extracellular Ca(2+) chelator EGTA nor the cytosolic Ca(2+) buffer bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid ('BAPTA')/AM protected beta-cells from hA-evoked apoptosis. Prolonged incubation of beta-cells with a lethal dose of hA altered neither the basal [Ca(2+)](i) nor the thapsigargin-induced release of Ca(2+) from intracellular stores. Furthermore, (45)CaCl(2) uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca(2+) homoeostasis do have a significant role in certain forms of beta-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet beta-cells.

Project description:BackgroundAntimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.Methodology/principal findingsPMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity.Conclusions/significanceFor the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.

Dataset Information

Role of calcium-independent phospholipase A(2)? in human pancreatic islet ?-cell apoptosis.

Publications

Role of calcium-independent phospholipase A(2)β in human pancreatic islet β-cell apoptosis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets