Oxidative stress is hypothesized to play a role in pancreatic β-cell damage, potentially contributing to β-cell dysfunction and death in both type 1 and type 2 diabetes. Oxidative stress arises when ...naturally occurring reactive oxygen species (ROS) are produced at levels that overwhelm the antioxidant capacity of the cell. ROS, including superoxide and hydrogen peroxide, are primarily produced by electron leak during mitochondrial oxidative metabolism. Additionally, peroxynitrite, an oxidant generated by the reaction of superoxide and nitric oxide, may also cause β-cell damage during autoimmune destruction of these cells. β-cells are thought to be susceptible to oxidative damage based on reports that they express low levels of antioxidant enzymes compared to other tissues. Furthermore, markers of oxidative damage are observed in islets from diabetic rodent models and human patients. However, recent studies have demonstrated high expression of various isoforms of peroxiredoxins, thioredoxin, and thioredoxin reductase in β-cells and have provided experimental evidence supporting a role for these enzymes in promoting β-cell function and survival in response to a variety of oxidative stressors. This mini-review will focus on the mechanism by which thioredoxins and peroxiredoxins detoxify ROS and on the protective roles of these enzymes in β-cells. Additionally, we speculate about the role of this antioxidant system in promoting insulin secretion.
Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. Here, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in ...glial cells. We show that ER stress is present in the central nervous system (CNS) concomitant with inflammation and astrogliosis in the multiple sclerosis (MS) mouse model of experimental autoimmune encephalomyelitis (EAE). Astrocytes do not easily succumb to ER stress but rather activate an inflammatory program involving activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of interleukin 6 (IL-6) and several chemokines. Moreover, the activation of STAT3 signaling is dependent on PERK, a central component of the ER stress response, which we show is phosphorylated by JAK1. Disruption of PERK abrogates ER stress-induced activation of STAT3 and subsequent gene expression. Additionally, ER-stressed astrocytes, via paracrine signaling, can stimulate activation of microglia, leading to production of IL-6 and oncostatin M (OSM). These IL-6 cytokines can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK/JAK1/STAT3 signaling pathway that elicits a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation, which may be relevant in diseases such as MS.
Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease characterized by the destruction of insulin‐secreting pancreatic β cells. In humans with T1D and in nonobese diabetic (NOD) mice (a ...murine model for human T1D), autoreactive T cells cause β‐cell destruction, as transfer or deletion of these cells induces or prevents disease, respectively. CD4+ and CD8+ T cells use distinct effector mechanisms and act at different stages throughout T1D to fuel pancreatic β‐cell destruction and disease pathogenesis. While these adaptive immune cells employ distinct mechanisms for β‐cell destruction, one central means for enhancing their autoreactivity is by the secretion of proinflammatory cytokines, such as IFN‐γ, TNF‐α, and IL‐1. In addition to their production by diabetogenic T cells, proinflammatory cytokines are induced by reactive oxygen species (ROS) via redox‐dependent signaling pathways. Highly reactive molecules, proinflammatory cytokines are produced upon lymphocyte infiltration into pancreatic islets and induce disease pathogenicity by directly killing β cells, which characteristically possess low levels of antioxidant defense enzymes. In addition to β‐cell destruction, proinflammatory cytokines are necessary for efficient adaptive immune maturation, and in the context of T1D they exacerbate autoimmunity by intensifying adaptive immune responses. The first half of this review discusses the mechanisms by which autoreactive T cells induce T1D pathogenesis and the importance of ROS for efficient adaptive immune activation, which, in the context of T1D, exacerbates autoimmunity. The second half provides a comprehensive and detailed analysis of (1) the mechanisms by which cytokines such as IL‐1 and IFN‐γ influence islet insulin secretion and apoptosis and (2) the key free radicals and transcription factors that control these processes.
Oxidative stress is thought to promote pancreatic β-cell dysfunction and contribute to both type 1 and type 2 diabetes. Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are ...mediators of oxidative stress that arise largely from electron leakage during oxidative phosphorylation. Reports that β-cells express low levels of antioxidant enzymes, including catalase and GSH peroxidases, have supported a model in which β-cells are ill-equipped to detoxify ROS. This hypothesis seems at odds with the essential role of β-cells in the control of metabolic homeostasis and organismal survival through exquisite coupling of oxidative phosphorylation, a prominent ROS-producing pathway, to insulin secretion. Using glucose oxidase to deliver H2O2 continuously over time and Amplex Red to measure extracellular H2O2 concentration, we found here that β-cells can remove micromolar levels of this oxidant. This detoxification pathway utilizes the peroxiredoxin/thioredoxin antioxidant system, as selective chemical inhibition or siRNA-mediated depletion of thioredoxin reductase sensitized β-cells to continuously generated H2O2. In contrast, when delivered as a bolus, H2O2 induced the DNA damage response, depleted cellular energy stores, and decreased β-cell viability independently of thioredoxin reductase inhibition. These findings show that β-cells have the capacity to detoxify micromolar levels of H2O2 through a thioredoxin reductase–dependent mechanism and are not as sensitive to oxidative damage as previously thought.
Viral infection has been identified as an environmental factor that may play a role in initiating the destruction of pancreatic β-cells during the development of autoimmune diabetes. We have ...identified a pro-inflammatory response activated in pancreatic islets in response to infection with encephalomyocarditis virus (EMCV). In response to the local production of cytokines such as IL-1 by resident islet macrophages, β-cells produce nitric oxide, a free radical with known antiviral activity. Nitric oxide has been characterized both as a potent antiviral molecule and as a mediator of β-cell dysfunction and damage and so we investigated the role of nitric oxide in controlling β-cell viability in the context of EMCV infection. We find that nitric oxide, either supplied exogenously with donor compounds or produced endogenously in response to cytokine treatment, attenuates EMCV replication and protects β-cells against EMCV-mediated death. These protective actions of nitric oxide are restricted to conditions in which nitric oxide is produced at low micromolar levels (consistent with the levels of nitric oxide generated by iNOS) and are associated with the ability of nitric oxide to impair mitochondrial oxidation and decrease ATP levels in β-cells. Treatment with rotenone, an inhibitor of complex I of the electron transport chain, likewise attenuates EMCV replication in β-cells, which are reliant on oxidative metabolism for ATP generation, but not in fibroblasts, which can sustain ATP levels via glycolysis alone. These findings suggest a mechanism of protection restricted to β-cells where nitric oxide production attenuates viral replication by inhibiting oxidative metabolism and depleting cellular stores of ATP, supporting a protective role for nitric oxide in the response of β-cells to viral infection.
Disclosure
J. Stafford: None. J.A. Corbett: None.
Funding
American Heart Association (17PRE3253000); National Institutes of Health (DK052194, AI44458)
Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic β-cell damage during the ...pathogenesis of autoimmune diabetes. While β-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in β-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of β-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that β-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (
) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (
) had no effect. Together, these results suggest that β-cells use cytoplasmic
as a primary defense mechanism against both ROS and RNS.
Cytokines released in and around pancreatic islets during islet inflammation are believed to contribute to impaired β cell function and β cell death during the development of diabetes. Nitric oxide, ...produced by β cells in response to cytokine exposure, controls many of the responses of β cells during islet inflammation. Recent Advances: Although nitric oxide has been shown to inhibit insulin secretion and oxidative metabolism and induce DNA damage in β cells, it also activates protective pathways that promote recovery of insulin secretion and oxidative metabolism and repair of damaged DNA. Recent studies have identified a novel role for nitric oxide in selectively regulating the DNA damage response in β cells.
Does nitric oxide mediate cytokine-induced β cell damage, or is nitric oxide produced by β cells in response to cytokines to protect β cells from damage?
β cells appear to be the only islet endocrine cell type capable of responding to proinflammatory cytokines with the production of nitric oxide, and these terminally differentiated cells have a limited capacity to regenerate. It is likely that there is a physiological purpose for this response, and understanding this could open new areas of study regarding the loss of functional β cell mass during diabetes development.
Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet ...macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.
Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models, leading to a serious concern regarding the safety of pediatric anesthesia. However, if and how ...ketamine induces human neural cell toxicity is unknown. Recapitulation of neurogenesis from human embryonic stem cells (hESCs) in vitro allows investigation of the toxic effects of ketamine on neural stem cells (NSCs) and developing neurons, which is impossible to perform in humans. In the present study, we assessed the influence of ketamine on the hESC-derived NSCs and neurons.
hESCs were directly differentiated into neurons via NSCs. NSCs and 2-week-old neurons were treated with varying doses of ketamine for different durations. NSC proliferation capacity was analyzed by Ki67 immunofluorescence staining and bromodeoxyuridine assay. Neuroapoptosis was analyzed by TUNEL staining and caspase 3 activity measurement. The mitochondria-related neuronal apoptosis pathway including mitochondrial membrane potential, cytochrome c distribution within cells, mitochondrial fission, and reactive oxygen species (ROS) production were also investigated.
Ketamine (100 µM) increased NSC proliferation after 6-hour exposure. However, significant neuronal apoptosis was only observed after 24 hours of ketamine treatment. In addition, ketamine decreased mitochondrial membrane potential and increased cytochrome c release from mitochondria into cytosol. Ketamine also enhanced mitochondrial fission as well as ROS production compared with no-treatment control. Importantly, Trolox, a ROS scavenger, significantly attenuated the increase of ketamine-induced ROS production and neuronal apoptosis.
These data for the first time demonstrate that (1) ketamine increases NSC proliferation and causes neuronal apoptosis; (2) mitochondria are involved in ketamine-induced neuronal toxicity, which can be prevented by Trolox; and (3) the stem cell-associated neurogenesis system may provide a simple and promising in vitro model for rapidly screening anesthetic neurotoxicity and studying the underlying mechanisms as well as prevention strategies to avoid this toxic effect.
Human CD19 antigen is a 95-kDa type I membrane glycoprotein in the immunoglobulin superfamily whose expression is limited to the various stages of B-cell development and differentiation and is ...maintained in the majority of B-cell malignancies, including leukemias and non-Hodgkin lymphomas of B-cell origin. Coupled with its differential and favorable expression profile, CD19 has rapid internalization kinetics and is not shed into the circulation, making it an ideal target for the development of antibody-drug conjugates (ADCs) to treat B-cell malignancies. ADCT-402 (loncastuximab tesirine) is a novel CD19-targeted ADC delivering SG3199, a highly cytotoxic DNA minor groove interstrand crosslinking pyrrolobenzodiazepine (PDB) dimer warhead. It showed potent and highly targeted in vitro cytotoxicity in CD19-expressing human cell lines. ADCT-402 was specifically bound, internalized, and trafficked to lysosomes in CD19-expressing cells and, following release of the PBD warhead, resulted in formation of DNA crosslinks that persisted for 36 hours. Bystander killing of CD19− cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent antitumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA crosslinks and γ-H2AX DNA damage response were measured in tumors by 24 hours after single dose administration, whereas matched peripheral blood mononuclear cells showed no evidence of DNA damage. Pharmacokinetic analysis in rat and cynomolgus monkey showed excellent stability and tolerability of ADCT-402 in vivo. Together, these impressive data were used to support the clinical testing of this novel ADC in patients with CD19-expressing B-cell malignancies.
•ADCT-402 is a CD19-targeted ADC delivering SG3199, a cytotoxic DNA minor groove interstrand crosslinking PDB dimer warhead.•ADCT-402 has potent and selective antitumor activity against CD19-expressing hematological malignancies warranting clinical development.
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