Protective Role of Nuclear Factor E2-Related Factor 2 against Acute Oxidative Stress-Induced Pancreatic β-Cell Damage

Oxidative stress is implicated in the pathogenesis of pancreatic β-cell dysfunction that occurs in both type 1 and type 2 diabetes. Nuclear factor E2-related factor 2 (NRF2) is a master regulator in the cellular adaptive response to oxidative stress. The present study found that MIN6 β-cells with stable knockdown of Nrf2 (Nrf2-KD) and islets isolated from Nrf2-knockout mice expressed substantially reduced levels of antioxidant enzymes in response to a variety of stressors. In scramble MIN6 cells or wild-type islets, acute exposure to oxidative stressors, including hydrogen peroxide (H2O2) and S-nitroso-N-acetylpenicillamine, resulted in cell damage as determined by decrease in cell viability, reduced ATP content, morphology changes of islets, and/or alterations of apoptotic biomarkers in a concentration- and/or time-dependent manner. In contrast, silencing of Nrf2 sensitized MIN6 cells or islets to the damage. In addition, pretreatment of MIN6 β-cells with NRF2 activators, including CDDO-Im, dimethyl fumarate (DMF), and tert-butylhydroquinone (tBHQ), protected the cells from high levels of H2O2-induced cell damage. Given that reactive oxygen species (ROS) are involved in regulating glucose-stimulated insulin secretion (GSIS) and persistent activation of NRF2 blunts glucose-triggered ROS signaling and GSIS, the present study highlights the distinct roles that NRF2 may play in pancreatic β-cell dysfunction that occurs in different stages of diabetes.


Introduction
Pancreatic -cell damage is the fundamental pathogenesis of type 1 diabetes, which is mediated by an autoimmune and inflammatory process [1]. Disruption of pancreaticcells leading to islet dysfunction and reduced -cell mass is also implicated in type 2 diabetes [2]. Oxidative stress, which is characterized by increased production of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS), is involved in the destruction of -cells in different stages of both type 1 and type 2 diabetes. Although ROS, in particular hydrogen peroxide (H 2 O 2 ), may function as intracellular secondary messenger mediating glucose-stimulated insulin secretion (GSIS) in pancreatic -cells [3][4][5], excessive and 2 Oxidative Medicine and Cellular Longevity persistent production of ROS results in oxidative damage and disrupts the function of proteins, nucleic acids, and fatty acids [6,7].
In type 1 diabetes, pancreatic -cell failure is mediated by inflammatory cytokines, including interleukin-1 (IL-1 ), tumor necrosis factor-(TNF-), and interferon-(IFN-), which may stimulate production of RNS, especially nitric oxide (NO) [8]. Similar to H 2 O 2 , NO also functions as an important messenger molecule that mediates a variety of physiological processes, including vasodilation [9], neurotransmission [10], and immunity response [11]. However, persistent elevation of endogenous or exogenous RNS may also induce cytotoxic effects in various cell types. High levels of NO disrupt energy metabolism, induce DNA damage, activate poly(ADP-ribose) polymerase (PARP), and/or dysregulate cytosolic calcium leading to cell necrosis and apoptosis [12,13].
Although ROS and RNS participate in various cellular response pathways, they both induce DNA damage and reduce cell viability at high levels [14]. Of interest, cells have acquired complicated mechanisms to localize endogenous ROS and/or RNS distribution and defend against exogenous ROS and/or RNS toxicity. Among them, transcriptional signaling through the antioxidant response element (ARE), orchestrated by the nuclear factor E2-related factor 2 (NRF2), is a major cellular defense mechanism against oxidative or electrophilic stress. Nrf2-deficient animals and cells have been shown to display reduced antioxidant response and become intolerant to oxidative or electrophilic stress-induced damage [15][16][17][18][19]. Compared with other cell types and tissues, pancreatic -cells express low levels of many antioxidant enzymes and thus are hypothesized to be susceptible to oxidative damage induced by ROS and/or RNS [20]. However, the role of NRF2-mediated antioxidant response in acute ROS or RNS-induced pancreatic -cell damage has not been thoroughly investigated.
In the current study, we hypothesized that NRF2 plays a protective role against acute oxidative stress-induced pancreatic -cell damage, a process that may occur in type 1 and type 2 diabetes. To test the hypothesis, we determined the susceptibility of Nrf2-knockout (Nrf2−/−) islets and Nrf2knockdown (Nrf2-KD) MIN6 cells to high levels of oxidative stressors-induced cell damage. In addition, we investigated the protective effects of preactivation of NRF2 on acute oxidative stress-induced cell damage.

Establishment of Stable Cell
Lines and Measurement of Luciferase Activity. MIN6 cells were transduced with Lentivirus containing shRNA against Nrf2 (SHVRSNM 010902, Sigma), scrambled nontarget negative control (SHC002V, Sigma), or ARE-luciferase reporter (SABiosciences, Frederick, MD) as previously described [17]. The luciferase activity of ARE-luciferase reporter cells was determined by Luciferase Reporter Assay System (Promega, Madison, WI) and normalized to cell viability with the same treatment as detailed in our previous studies [22].

Animals and Islet
Isolation. Nrf2−/− mice developed as described previously [23] were kindly provided by Dr. Masayuki Yamamoto (Tohoku University, Japan). Wild-type (Nrf2+/+) and Nrf2−/− mice were maintained on normal chow diet. All animal experiments were approved by the Institutional Animal Care and Use Committee of China Medical University. Pancreatic islets were isolated from 8-12week-old Nrf2+/+ and Nrf2−/− mice by in situ collagenase P (Roche, Switzerland) perfusion of pancreas under a dissecting microscope, as described previously [4]. Isolated islets were cultured in RPMI 1640 supplemented with 10 mmol/l glucose, 10% FBS, 25 mmol/l HEPES, 2 mmol/l L-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin. All the isolated islets were cultured for 48 hrs before in vitro experiments.

Measurement of Intracellular Glutathione (GSH).
MIN6 cells were sonicated in cold PBS immediately after collection. The whole cell extracts were obtained by centrifugation at 12,000 ×g for 5 min, and protein concentration was determined by a BCA assay kit (Pierce Biotechnology, Rockford, IL). Following an immediate deproteinization with metaphosphoric acid (final concentration at 5%), levels of total GSH were measured using a BIOXYTECH GSH/GSSG-412 kit (OxisResearch, Portland, OR) according to the manufacturer's protocol. Of note, the levels of oxidized GSH (GSSG) in MIN6 cells were too low to be measured.

Determination of Intracellular
Peroxide. Levels of intracellular peroxide were measured by flow cytometry (Becton Dickinson FACSort, Becton Dickinson, San Jose, CA) using a fluorescent probe 5-(and-6)-chloromethyl-2 ,7 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA, Molecular Probes, Eugene, OR) as described previously [4]. The loading concentration of CM-H 2 DCFDA was 2 M and the preloading times were 30 min. In the measurements, dead cells and clumps were eliminated based upon Forward Scatter versus Side Scatter measurements, and untreated cells provided a source of comparison.

Cell Viability and Cytotoxicity Assay.
Cell viability was determined as described previously [26]. Briefly, 10,000 cells per well were plated into a 96-well plate. Following a 24 hr culture, medium was replaced with fresh medium containing various stressors at the appropriate concentrations. Cells were treated for indicated time with stressors and followed by an immediate measurement for cell viability by using the nonradioactive cell proliferation assay kit (Promega, Madison, WI). Intracellular ATP levels were measured using an ATP Bioluminescent Somatic Cell Assay Kit (Sigma).

Statistical Analyses.
All statistical analyses were performed using Graphpad Prism 4 (GraphPad Software, San Diego, CA). Statistical significance was defined as < 0.05. Data are expressed as mean ± SD. For comparisons between and among groups, Student's -test and one-way ANOVA with Bonferroni post hoc testing were performed, respectively.

Stable Knockdown of Nrf2 in MIN6 Cells Results in Attenuated Antioxidant
Response. To investigate the role of NRF2 in acute oxidative stress-induced -cell damage, a line of MIN6 cells with stable silencing of Nrf2 was developed. As shown in Figure 1(a), lentiviral shRNA-mediated stable knockdown of Nrf2 in MIN6 cells resulted in 70% reduction in mRNA expression of Nrf2 compared to the control cells that were expressed scrambled nontarget negative control shRNA (scramble). In agreement with the reduction of Nrf2 mRNA, Nrf2-KD cells displayed substantially reduced protein expression of NRF2 under vehicle and arseniteor tBHQ-challenged conditions (Figure 1(b)). In scramble cells, the protein levels of NRF2 in nuclear fractions were dramatically increased by arsenite or tBHQ treatments, whereas the inductions were almost totally diminished in Nrf2-KD cells (Figure 1(b)). In addition, Nrf2-KD cells showed reduced expression of two major ARE-dependent genes glutamate-cysteine ligase catalytic subunit (Gclc) and NAD(P)H quinone oxidoreductase 1 (Nqo1) under basal or tBHQ-challenged conditions (Figure 1(c)). Furthermore, silencing of Nrf2 resulted in significantly decreased intracellular levels of GSH (Figure 1(d)) and elevation of intracellular ROS (Figure 1(e)). reduction in cell viability (Figure 2(a)) and ATP content (Figure 2(b)). In response to glucose oxidase, which is a mild and long-lasting H 2 O 2 -generating system that catalyzes the oxidation of glucose to produce H 2 O 2 , Nrf2-KD MIN6 cells also showed an elevated susceptibility to its cytotoxicity (Figure 2 Figure 1(c)) and arsenite [17].

Nrf2-Deficient -Cells
To extend the findings above, the susceptibility of scramble and Nrf2-KD MIN6 cells to a NO-releasing compound SNAP-induced cytotoxicity was determined. Compared to Scr cells, Nrf2-KD MIN6 cells were more vulnerable to SNAPinduced reduction in cell viability (Figure 4(a)) and apoptosis (Figure 4(b)).
To validate the key findings observed in MIN6 cells, H 2 O 2 -induced cell damage was determined in cultured islets isolated from wild-type and Nrf2−/− mice. As shown in Figure 5, Nrf2−/− islets showed reduced expression of Nqo1 and Gclc (Figure 5(a)), confirming that the ARE-dependent transcription was attenuated in the tissues. Consistent with the conclusions obtained in MIN6 cells, Nrf2−/− islets exhibited more serious damage than Nrf2+/+ islets as determined by morphology changes in response to H 2 O 2 exposure (Figures 5(b) and 5(c)).

Discussion
The impairment of pancreatic -cell function is critical in the pathophysiology of both type 1 and type 2 diabetes. Amidst the various mechanisms proposed for -cell dysfunction and their roles in the progression of -cell damage, oxidative stress has been proposed as a common denominator [27]. The present study found that acute exposure of MIN6 -cells or isolated mouse islets to the most common oxidative stressors, Intact islets (% of Veh) Oxidative stress in pancreatic -cells is generally induced by glucotoxicity, lipotoxicity, and/or inflammation in different stages of diabetes [27]. Although ROS and RNS are critical signaling molecules mediating a variety of signaling Oxidative Medicine and Cellular Longevity cascades [14], they both lead to cell damage at high levels. Partially due to low basal expression of many antioxidant enzymes, -cells are relatively vulnerable to oxidative damage induced by excessive ROS and/or RNS [20]. In the present study, we found that H 2 O 2 may induce cell damage in MIN6 cells and cultured islets at a concentration as low as 50 M, which is dramatically lower than the concentrations we used in human keratinocytes to activate NRF2 [25]. Under oxidative stress, most of cells may upregulate their intracellular antioxidant capacity by transcriptional induction of many antioxidant and phase II detoxification enzymes via NRF2mediated antioxidant response and thus protect the cells from oxidative damage [15,18,19,[28][29][30]. Our previous studies have elucidated that NRF2 also plays a critical role in pancreatic -cell defense against oxidative/electrophilic stress [17]. Abolishment of the NRF2-mediated antioxidant response by targeted disruption of the Nrf2 gene in MIN6 cells and mouse islets increased their susceptibility to environmental oxidative stressor arsenic-induced cytotoxicity and/or apoptosis [17]. Preactivation of NRF2 with tBHQ significantly protects MIN6 cells from arsenic-induced acute cytotoxicity in Nrf2dependent manner [17]. Recently, Yagishita et al. presented detailed in vivo evidence from four genetically engineered mouse models to demonstrate that NRF2 induction prevents oxidative and nitrosative stress-induced oxidative damage in pancreatic -cells [31]. In the current study we used the most common oxidative stressors and two genetically engineered cell models to confirm that NRF2-mediated antioxidant response protects pancreatic -cell against oxidative stressinduced cell damage in vitro and ex vivo. These new findings provided additional evidence to support that NRF2-mediated antioxidant response protects pancreatic -cell against acute ROS or RNS-induced cell damage. Given that NRF2-mediated antioxidant response plays critical role in cell defense, NRF2 has been considered as a valuable therapeutic target. CDDO-Im is one of the most potent synthetic triterpenoids shown to activate NRF2 and induce phase II detoxifying and antioxidant enzymes. It was firstly investigated in protecting against aflatoxininduced tumorigenesis in liver [32,33]. Recently, it has been used for the treatment of chronic kidney disease, cancer, and other diseases [34][35][36]. DMF is a newly found NRF2 activator and has been used for the treatment of psoriasis [37] and more recently for multiple sclerosis [38]. CDDO-Im and DMF have been reported to protect endothelial cells and prevent vascular injury via NRF2 activation [39,40]. In the present study, we found that CDDO-Im and DMF pretreatments may dramatically enhance the activity of AREdependent transcription in pancreatic -cells and protect the cells against acute oxidative stress-induced cell damage. Together with the protective effect by another well-known NRF2 activator, tBHQ, we conclude that preactivation of NRF2 may protect pancreatic -cells from acute oxidative stress-induced cell damage.
Although ROS and RNS have been demonstrated to be destructive factors at high levels, they also function as physiological signaling molecules mediating a variety of physiological processes, including GSIS in -cells [3,4]. In the case where ROS serve as cell signaling molecules, persistent elevation of endogenous antioxidants could diminish such a signal. Thus, persistent activation of NRF2-mediated antioxidant response has the potential to cause an undesirable effect. Our previous study showed that persistent NRF2 activation by prolonged arsenic exposure blunts glucose-stimulated ROS signaling to mediate GSIS in pancreatic -cells [22]. Therefore, we have proposed that NRF2-mediated antioxidant response plays paradoxical roles in -cell function. On the one hand, this pathway protects cells from oxidative damage and possible cell death, thus minimizing oxidative damagerelated impairment in -cell dysfunction; on the other hand, the induction of endogenous antioxidants in the presence of oxidative stress may blunt ROS signal, resulting in reduced GSIS [41,42]. Considering that glucose metabolism-derived ROS are involved in regulating GSIS in -cells and persistent activation of NRF2 blunts glucose-triggered ROS signaling and GSIS, we conclude that NRF2 may play distinct roles in pancreatic -cell dysfunction that occurs in different stages of diabetes.

Conclusions
The present study demonstrated that NRF2-mediated antioxidant response protects pancreatic -cells from oxidative stress-induced cell damage. In light of the inhibitory effect of NRF2-mediated antioxidant response on ROS signaling in -cell GSIS, the present study highlights distinct roles that NRF2 may play in pancreatic -cell dysfunction that occurs in different stages of diabetes. Thus, more detailed investigations focusing on the exact mode of ROS and various endogenous antioxidants in regulating GSIS andcell survival during different stages of diabetes are needed.