Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts β-Cell Function and Survival

Abstract

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in β-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2•− production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts β-cells’ function and survival during insulitis, NOX2 might be a potential target for designing therapies against early β-cell dysfunction in the context of T1D onset.

Lipotoxicity and β-Cell Failure in Type 2 Diabetes: Oxidative Stress Linked to NADPH Oxidase and ER Stress

“…However, when inhibiting NOX with a pharmacological inhibitor, VAS2870, we saw no protection from palmitate-induced activation of UPR response [ 144 ]. Using islets exposed to proinflammatory cytokines, we have recently proposed that NOX2-induced H 2 O 2 production is ER stress dependent, as the phosphorylation of two important UPR proteins (p-eIF2-α and p-IRE1) precedes H 2 O 2 production by NOX2 and that the attenuation of ER stress delays the cytokine-induced cytosolic H 2 O 2 peak [ 189 ].…”

The loss of pancreatic islet NADPH oxidase (NOX)2 improves islet transplantation

“…Next, we first tested if ROS are produced in the cytosol of isolated islets under hypoxia. For this purpose, we made use of islets isolated from transgenic mice expressing the genetically-encoded H 2 O 2 sensor cyto-roGFP2-Orp1 [ 28 , 29 , 33 ]. Using a microplate reader (ClarioStar, BMG) we determined dynamic, real time H 2 O 2 levels during 18 h of normoxia and hypoxia in isolated islets.…”

“…Insulin secretion was performed, as previously described [ 28 ]. Briefly, WT and Nox2 −/− islets were incubated in normoxia (18% O 2 ) for 1 h followed by hypoxia (5% O 2 ) for 18 h. The supernatant was collected and kept at - 20 °C until measurement.…”

“…Of interest, NOX2 was found to be highly expressed in pancreatic β-cells [ 23 , 24 ]. We and others have previously shown that NOX2 is involved in pancreatic islet failure under lipotoxicity and cytokine exposure [ [25] , [26] , [27] , [28] ] and more importantly, this enzyme acts as a negative regulator of glucose-induced insulin secretion [ 23 , 24 ].…”

CARD9 Mediates Pancreatic Islet Beta-Cell Dysfunction Under the Duress of Hyperglycemic Stress

“…Chronic exposure of pancreatic beta-cells to metabolic stress conditions, such as hyperglycemia, has been shown to increase the generation of intracellular reactive oxygen species (ROS; oxidative stress), endoplasmic reticulum (ER) stress, and mitochondrial dysregulation (mito stress) culminating in defective glucose-stimulated insulin secretion (GSIS) and apoptotic demise of the effete beta cell [ 1 – 10 ]. Published evidence implicates NADPH oxidases, specifically the phagocyte-like NADPH oxidase (Nox2), as contributors of oxidative stress in pancreatic beta cells following exposure to diabetogenic conditions [ 1 , 11 – 16 ]. Regulatory roles of NADPH oxidases in islet beta cell dysfunction were confirmed in insulin-secreting beta cell lines, rodent islets and human islets under in vitro conditions, and in islets from animal models as well as humans with T2DM [ 1 , 11 , 17 , 18 ].…”