Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells

Abstract

It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

Anti‐angiogenic effect of quercetin and its 8‐methyl pentamethyl ether derivative in human microvascular endothelial cells

“…After treatments, the cells were detached by scraping, collected by centrifugation and lysed as previously described . The membranes were incubated with primary antibodies (4°C, o/n) and then with secondary antibodies (1 hour, room temperature).…”

“…The imbalance among the molecules that coordinate the neoangiogenesis is often the cause of inflammatory, immune, infectious and malignant disorders . The newly formed blood vessels are fenestrated, permeable, tortuous and heterogeneous both in their structure and efficiency of perfusion .…”

“…The imbalance among the molecules that coordinate the neoangiogenesis is often the cause of inflammatory, immune, infectious and malignant disorders. 38 The newly formed blood vessels are fenestrated, permeable, tortuous and heterogeneous both in their structure and efficiency of perfusion. 43 Aberrant angiogenesis, in fact, is responsible for several ocular diseases, such as choroidal subretinal neovascularization, retinopathy of prematurity, proliferative diabetic retinopathy 4 and retinoblastoma.…”

“…After treatments, the cells were detached by scraping, collected by centrifugation and lysed as previously described. 38 The membranes were incubated with primary antibodies (4°C, o/n) and then with secondary antibodies (1 hour, room temperature). The membranes, after anti p-VEGFR2, VEGFR2, p-AKT, AKT, p-ERK and ERK antibodies, were successfully stripped and re-probed for β-actin, to test the equal protein loading.…”

Short Duration Alagebrium Chloride Therapy Prediabetes Does Not Inhibit Progression to Autoimmune Diabetes in an Experimental Model

“…Arachidonic acid is a product generated from this reaction and can be further metabolised into proinflammatory lipid mediators. Within β-cells, calcium-independent phospholipase appears to be involved in glucose stimulated insulin secretion [ 74 , 75 ], proliferation and apoptosis in INS-1 cells [ 75 ] and human islets [ 76 ] and has thus been suggested as a target to improve β-cell longevity [ 76 ].…”