Impact of scavenging hydrogen peroxide in the endoplasmic reticulum for β cell function

in Journal of Molecular Endocrinology

Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing.

Abstract

Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H2O2 concentration might be very high. As the role of this H2O2 in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2 removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2 significantly with a threefold increase of the EC50 value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2 reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2 toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2 in the ER in spite of a reduced insulin transcription and processing.

Introduction

Biosynthesis and secretion of insulin in response to glucose are the major tasks of pancreatic β cells. Endoplasmic reticulum (ER) is highly developed in these professional secretory cells to ensure oxidative protein folding and posttranslational modification of the synthesised proteins (Harding & Ron 2002). Since insulin, the main product of protein synthesis in the β cells, is not glycosylated, the most important function of the ER is the formation of three disulfide bonds in each insulin molecule (Bulleid 2012). During this oxidative folding process the formation of each disulfide bond, thought to be catalysed by one or more members of the protein disulfide isomerase (PDI) family, and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is associated with the generation of one molecule hydrogen peroxide (H2O2) as a by-product (Tu & Weissman 2004, Ramming & Appenzeller-Herzog 2012). Through the large number of proinsulin molecules to be synthesised and processed in each β cell, an estimated one million molecules per minute (Scheuner & Kaufman 2008), the ER, along with the mitochondria and peroxisomes, represents a major site of H2O2 generation (Tu & Weissman 2004). However, the fate of the ER-generated H2O2 and its importance for the maintenance of ER homeostasis and oxidative folding efficacy is still unresolved. A number of studies indicate that this luminal H2O2 generation is mandatory for retaining and improving the oxidative protein folding capacity of the ER (Margittai et al. 2012, Ramming & Appenzeller-Herzog 2013). This H2O2 is required for the re-oxidation of the reduced PDI protein by ER-localised glutathione peroxidases (GPx7 and GPx8) and potentially also by peroxiredoxin 4 (Prdx4). On the other hand, the activation of the ER stress pathway, which may lead to apoptotic cell death, has also been associated to the fulminant ROS generation in the ER (Malhotra et al. 2008, Bhandary et al. 2012, Cao & Kaufman 2014).

To shed light on this contentious issue, we took advantage of a recently developed catalase variant, ER-Catalase N244, which shows full enzyme activity upon expression in the ER (Lortz et al. 2015). In insulin-secreting INS-1E tissue culture cells, we studied the influence of a high luminal H2O2 inactivating activity on insulin expression and secretion. Furthermore, the role of ER-derived H2O2 in the initiation of ER stress cascades and the reduction of cell viability through β cell toxic cytokines has been investigated.

Materials and methods

Tissue culture of insulin-producing cells

Insulin-secreting INS-1E tissue culture cells (kindly provided by C. Wollheim, University of Geneva Medical Center, Geneva, Switzerland) were cultured in RPMI 1640 medium supplemented with 10 mmol/l glucose, 10% (v/v) FCS, 10 mmol/l HEPES, 1 mmol/l sodium pyruvate, 50 μmol/l 2-mercaptoethanol, penicillin, and streptomycin in a humidified atmosphere at 37 °C and 5% CO2 as described previously (Asfari et al. 1992).

Lentiviral expression of ER-localised catalase in INS-1E cells

To express catalase specifically in the ER, the mutated catalase variant ER-Catalase N244 cDNA was used (Lortz et al. 2015). For this purpose, lentiviral particles were prepared as described before in detail (Zufferey et al. 1998). 4×106 293T cells were transfected with the packaging plasmid pPAX2 (11.25 μg), the envelope plasmid pcDNA-MDG (3.75 μg), and the transfer plasmid pLenti6.3/V5-MCS-ER-Catalase N244 (15 μg) by calcium phosphate precipitation. After 48 h the virus containing culture medium was collected and centrifuged for 5 min at 700 g to remove detached cells and cell debris; then the supernatant was filtered through 0.22 μm filters (Merck Millipore, Darmstadt, Germany). The INS-1E cells were infected with the purified viral supernatant for 5–6 h and thereafter the viral supernatant was replaced by fresh medium. Transduced cells were selected for catalase expression by 1 μM blasticidin (Life Technologies). These selected cells represented a mixed cell population. Thus, integration of the lentiviral construct happens randomly at various genomic locations and possible undesirable interactions with specific genes, altering β cell function, are less likely. Therefore the use of these cells, in contrast to single cell clones, can avoid unwanted clonal selection artefacts.

Quantification of catalase activity

Catalase activity was quantified as described (Tiedge et al. 1998). Briefly, whole cell extracts were prepared in 50 mmol/l potassium phosphate buffer (pH 7.8) through sonication on ice with a Braun-Sonic 125 sonifier (Braun, Melsungen, Germany). The homogenates were then centrifuged at 10 000 g and 4 °C for 10 min and the protein content of the supernatant was determined by the BCA assay (Thermo Fisher Scientific, Rockford, IL, USA). The catalase activity was measured by ultraviolet spectroscopy, monitoring the decomposition of H2O2 at 240 nm.

Immunofluorescence staining

INS-1E cells expressing ER-Catalase N244 or WT catalase without an ER-specific targeting signal were seeded at a density of 100 000 cells/well on LabTek chamber slides (Nunc, Roskilde, Denmark). After 48 h, the cells were washed twice with PBS and subsequently fixed with 4% paraformaldehyde at room temperature for 1 h. After washing, the cells were permeabilised and blocked with PBS containing 0.2% Triton X-100 and 1% BSA. The cells were incubated with primary antibodies (anti-PDI, ab5484, Abcam, Cambridge, UK, 1:100 dilution; anti-catalase, 100–4151, Rockland Immunochemicals, Inc., Limerick, PA, USA, 1:500 dilution) diluted in PBS containing 0.1% Triton X-100 and 0.1% BSA at room temperature for 1 h. Thereafter the cells were washed with PBS and incubated with specific secondary antibodies (anti-mouse-Alexa Fluor 647, or anti-rabbit-Alexa Fluor 488, Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1:200 dilutions) for 1 h in the dark. Afterwards the cells were washed and the nuclei were counterstained with 300 nM DAPI for 5 min at room temperature. Finally, the cells were washed and mounted with Mowiol/DABCO anti-photobleaching mounting media (Sigma). Stained cells were examined with an Olympus IX81 inverted microscope (Olympus) and microscopic images were post-processed using AutoDeblur and AutoVisualize (Autoquant Imaging, New York, NY, USA).

Quantification of cell viability after H2O2 treatment

After 24 h seeding of 40 000 cells/well of a 96-well plate, cells were incubated with the indicated H2O2 concentrations (INS-1E: 0–125 μM, INS-1E ER-Catalase: 0–1250 μM) for 2 h in 20 mmol/l HEPES-supplemented Krebs–Ringer bicarbonate medium with 5 mmol/l glucose. After removal of the H2O2 containing medium the cells were incubated for another 22 h in fresh RPMI 1640 medium and thereafter cell viability was determined by a microplate-based MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) (Mosmann 1983). Briefly, the incubation medium was carefully removed and replaced by MTT-solution (0.5 mg/ml). After 1 h incubation at 37 °C, the MTT solution was carefully removed and the intracellularly generated water-insoluble formazan crystals were dissolved in DMSO. The absorbance was quantified using a microplate photometer (Powerwave 340, BioTek Instruments, Winooski, VT, USA).

Quantification of cell viability after exposure to pro-inflammatory cytokines

Cells were seeded at a density of 25 000 cells/well in 100 μl culture medium onto 96-well plates and allowed to attach for 24 h before they were incubated for 72 h with 600 U/ml human IL1β or a combination of cytokines (cytokine mixture) consisting of 60 U/ml IL1β, 185 U/ml human TNFα, and 14 U/ml IFNγ (PromoCell, Heidelberg, Germany). Thereafter the cell viability was determined by using a microplate-based MTT assay.

Quantification of ER stress after exposure to pro-inflammatory cytokines

Cells were seeded on 6 cm tissue culture plates at a density of 1×10 6 cells and allowed to attach for a period of 24 h. Thereafter the cells were exposed for 16 h to 600 U/ml human IL1β or a combination of cytokines (cytokine mixture) consisting of 60 U/ml IL1β, 185 U/ml human TNFα, and 14 U/ml IFNγ. After cytokine incubation total RNA was isolated as previously described (Tiedge et al. 1998). For cDNA synthesis, random hexamers were used to prime the reaction of the RevertAid H- M-MuLV reverse transcriptase (Life Technologies). The reactions were performed with GoTaq qPCR Master Mix (Promega) in a ViiA 7 real-time PCR system (Life Technologies). Samples were denatured at 94 °C for 3 min followed by 40 PCR cycles. Each cycle comprised a melting step at 94 °C for 30 s, an annealing step at 60 °C for 30 s, and an extension step at 72 °C for 30 s. Optimal parameters for the PCR reactions were empirically defined and the purity and specificity of the amplified PCR product in each experiment was verified by melting curve analysis. All transcripts showed Ct-values, which were at least ten Ct-values lower than the blank values. Each PCR amplification was performed in triplicate. Data are expressed as relative gene expression after normalisation against the geometric mean of the housekeeping genes Tuba4a, Actb and Ppia with qbasePLUS (Biogazelle, Zulte, Belgium). The primer sequences are listed in Table 1.

Table 1

List of primers for the detection of ER stress-related genes by RT-qPCR

GeneGene symbolOligonucleotide sequence (5′–3′)
ER stress specific genes
Activating transcription factor 4Atf4Forward: 5′-AATGGCTGGCTATGGATGGG-3′
Reverse: 5′-TCTGTCCCGGAAAAGGCATC-3′
Activating transcription factor 6Atf6Forward: 5′-GAGAGGTGTCTGTTTCGGGG-3′
Reverse: 5′-CGTGGACTCCCAGTCTTCAC-3′
C/EBP homologous proteinChopForward: 5′-CCAGCAGAGGTCACAAGCAC-3′
Reverse: 5′-CGCACTGACCACTCTGTTTC-3′
Glucose-regulated protein, 78 kDaGrp78/BipForward: 5′-CCACCAGGATGCAGACATTG-3′
Reverse: 5′-AGGGCCTCCACTTCCATAGA-3′
Housekeeping genes
α-tubulin 4aTuba4aForward: 5′-ACGTGAGACGTACATCCCAAACTCA-3′
Reverse: 5′-CTCCCAGCAGGCATTGCCCA-3′
β-actinActbForward: 5′-GCGTCCACCCGCGAGTACAA-3′
Reverse: 5′-TTGCAATGCCGGAGCCGTT-3′
Peptidylprolyl isomerase A (cyclophilin A)PpiaForward: 5′-TTGCAGACGCCGCTGTCTCTT-3′
Reverse: 5′-TGGAACTTTGTCTGCAAACAGCTCG-3′

Quantification of glucose-induced insulin secretion, insulin and proinsulin content

INS-1E cells were seeded in six-well plates at a density of 0.5×106 cells and grown for 48 h. Then the cells were incubated for 1 h in bicarbonate-buffered Krebs–Ringer solution without glucose, supplemented with 0.1% albumin. Thereafter the cells were stimulated for 2 h either with 3, 10, or 30 mmol/l glucose. After incubation, medium was removed and gently centrifuged to remove detached cells. Secreted insulin in the supernatant and insulin content of the incubated cells were determined by RIA using rat insulin as standard and the resulting values were normalised to DNA content. Proinsulin content was quantified by a rat proinsulin specific ELISA according to the manufacturer's protocol (Mercodia, Uppsala, Sweden).

Statistical analyses

Data are expressed as means±s.e.m. Statistical analyses were performed using ANOVA plus Bonferroni test for multiple comparisons or unpaired two-tailed Student's t-test. The EC50 values for the H2O2-induced toxicity were calculated from the concentration-response curves fitted by the sigmoidal concentration-response logarithm. All analyses were performed with the GraphPad Prism 5.03 Software (Graphpad, San Diego, CA, USA).

Results

Subcellular localisation of ER-Catalase N244 and quantification of ER-Catalase N244 enzyme activity and its effect on H2O2 induced toxicity in INS-1E cells

To document the functional expression of ER-Catalase N244 in insulin-secreting INS-1E cells, the enzyme activity of cell lysates was quantified after lentiviral transduction. ER-Catalase-N244 expressing INS-1E cells showed with 148.5±4.9 U/mg protein a significantly higher enzyme activity than untransfected control cells (10.3±3.2 U/mg protein, P<0.001) (data not shown). As the ER-specific expression is crucial, an immunofluorescence staining was performed. The co-staining of catalase and PDI in Fig. 1 documents a colocalisation in the ER confirming the ER-specific expression of ER-Catalase N244 in the INS-1E cells. In contrast, INS-1E cells overexpressing the unmodified WT catalase showed no clear colocalisation of catalase and PDI (Fig. 1, lower panel). Thus, only the ER-targeted catalase variant ER-Catalase N244 was expressed in the ER, while the native catalase protein was localised outside the ER, presumably in the cytosol and the peroxisomes. To demonstrate the in vivo functionality of the expressed ER-Catalase N244 and to provide an estimate of its H2O2 inactivating capacity, INS-1E control and ER-Catalase N244 expressing cells were incubated with increasing H2O2 concentrations and the cell viability was determined thereafter. Both cell clones showed a concentration-dependent decrease of their cell viability with an EC50 value of 45.6 μM H2O2 for the control cells (Fig. 2) and a significantly higher EC50 value of 132 μM for the ER-Catalase N244 expressing cells (P<0.001, Fig. 2). These differences in the EC50 values were especially obvious at concentrations up to 100 μM, which is the most relevant range for physiological and pathological processes. At 50 μM H2O2, the ER-Catalase expressing cells showed no decrease in cell viability and at 100 μM a residual cell viability of still 75% could be detected, whereas the control cells showed at the same H2O2 concentrations only a residual viability of 42 and 2%, respectively (Fig. 2).

Figure 1
Figure 1

Subcellular localisation of ER-Catalase N244 and WT catalase in insulin-secreting INS-1E cells. Cells were seeded on LabTek chamber slides. After fixation and permeabilisation the cells were stained with specific antibodies for catalase (green) and for the ER-specific protein disulfide isomerase (PDI, red), followed by counterstaining with DAPI (blue). A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-15-0132.

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-15-0132

Figure 2
Figure 2

Effects of ER-Catalase N244 expression on cytotoxicity after incubation of INS-1E cells with H2O2. INS-1E control and ER-Catalase N244 expressing cells were incubated for 2 h with the indicated H2O2 concentrations in Krebs–Ringer medium, and after removal of the H2O2-containing medium for another 22 h in fresh medium. Thereafter, the viability of the cells was determined by the MTT assay and expressed as % of untreated cells. Data are expressed as mean±s.e.m. of six to eight independent experiments. ***P<0.001 compared with control cells (unpaired two-tailed Student's t-test).

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-15-0132

Effects of ER-Catalase N244 on the ER stress response after exposure of INS-1E cells to pro-inflammatory cytokines

To determine the influence of ER-specific detoxification of H2O2 on the initiation of ER stress in response to β cell toxic cytokines, the gene expression of three major mediators of ER stress, namely Chop, Atf4, and Atf6, and of the ER-chaperon Grp78/Bip was quantified by RT-qPCR. As shown in Fig. 3A, a 16 h exposure to 600 U/ml IL1β alone resulted in a sixfold and incubation with a cytokine mixture (60 U/ml IL1β, 185 U/ml TNFα, and 14 U/ml IFNγ) in a 20-fold increase of Chop gene expression in INS-1E control cells. Both, Atf4 and Atf6, were induced by the cytokine treatment, but to a much lower extent compared with Chop. However, Atf4 gene expression was induced by IL1β (2.4-fold) and the cytokine mixture (4.2-fold), as well as Atf6 (1.2- and 2.2-fold respectively) (Fig. 3B and C). At variance from the increased expression of the investigated ER stress mediators Chop, Atf4, and Atf6, the expression of the ER located chaperon Grp78/Bip was significantly reduced by IL1β alone and the cytokine mixture. After both incubations Bip expression was reduced approx. 40%.

Figure 3
Figure 3

Effects of ER-Catalase N244 expression on the induction of ER stress genes after incubation of INS-1E cells with cytokines. INS-1E control and ER-Catalase N244 expressing cells were incubated for 16 h under control conditions, with 600 U/ml IL1β or with a cytokine mixture, composed of 60 U/ml IL1β, 185 U/ml TNFα, and 14 U/ml IFNγ. After incubation, total RNA was isolated and the expression of Chop (A), Atf4 (B), Atf6 (C), and Grp78/Bip (D) was analysed by RT-qPCR with specific primers. The expression levels were normalised to the housekeeping genes Tuba4a, Actb, and Ppia. The gene expression level of untreated control cells was set as 100%. Data are expressed as means±s.e.m. of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with untreated cells (ANOVA plus Bonferroni test for multiple comparisons).

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-15-0132

The expression level of all four investigated ER stress related genes in ER-Catalase N244 expressing INS-1E cells under control conditions and after cytokine exposure was comparable to that in control cells and no significant differences could be detected. These data indicate that expression of ER-Catalase N244 in INS-1E cells and thereby reduction of luminal H2O2 did not alter the expression of the ER stress-induced genes Chop, Atf4, and Atf6 and of the ER located chaperon Grp78/Bip after cytokine exposure.

Effects of ER-Catalase N244 on cell viability after exposure of INS-1E cells to pro-inflammatory cytokines

After having shown that ER-specific inactivation of H2O2 had no influence on the cytokine-induced expression of ER stress genes, we also analysed the effect of ER-Catalase N244 expression on cytokine-mediated cellular toxicity.

Therefore transfected and untransfected insulin-secreting INS-1E cells were incubated again with 600 U/ml IL1β or with the cytokine mixture (60 U/ml IL1β, 185 U/ml TNFα, and 14 U/ml IFNγ). After a 72 h incubation with IL1β a substantial 85% loss of viability could be detected for the INS-1E control cells (P<0.001). With the cytokine mixture this loss of viability was only slightly augmented (Fig. 4). The viability of ER-Catalase N244 expressing INS-1E cells after cytokine treatment did not significantly differ from untransfected control cells.

Figure 4
Figure 4

Effects of ER-Catalase N244 expression on cytotoxicity after incubation of INS-1E cells with cytokines. INS-1E control and ER-Catalase N244 expressing cells were incubated for 72 h under control conditions, with 600 U/ml IL1β or with a cytokine mixture, consisting of 60 U/ml IL1β, 185 U/ml TNFα, and 14 U/ml IFNγ. The cell viability was determined thereafter by the MTT viability assay and expressed as % of untreated cells. Data are expressed as means±s.e.m. of five independent experiments. ***P<0.001 compared with untreated cells (ANOVA plus Bonferroni test for multiple comparisons).

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-15-0132

Effects of ER-Catalase N244 on glucose-induced insulin secretion and insulin content of INS-1E cells

To study the influence of ER-specific H2O2 deprivation on glucose-induced insulin secretion, INS-1E cells were incubated for 2 h either with 3, 10, or 30 mM glucose and thereafter the amount of secreted insulin was quantified in the supernatant. Basal insulin secretion of INS-1E control cells at 3 mM glucose was 0.6 ng/(μg/DNA per h) and was more than fourfold elevated by 10 and 30 mM glucose to ∼2.8 ng/(μg/DNA per h) (10 mM, P<0.001; 30 mM, P<0.005) (Fig. 5A). The insulin content of the INS-1E control cells was 28 ng/μg DNA and the proinsulin content was 5–6 ng/μg DNA, both without significant differences between the different glucose concentrations (Fig. 5B and C). The rate of glucose-induced insulin secretion from the ER-Catalase N244 expressing INS-1E cells was comparable to that of control INS-1E cells at all glucose concentrations, including the rate of basal insulin secretion at 3 mM (Fig. 5A). Although the proinsulin content in the ER-Catalase N244 transfected INS-1E cells remained unchanged at all three glucose concentrations, the insulin content was significantly lower and represented only two-thirds of that of untransfected control cells (Fig. 5B and C). Thus, the ratio between insulin and proinsulin content in control INS-1E cells was almost twice as high as in ER-Catalase N244 expressing cells (Fig. 5D).

Figure 5
Figure 5

Effects of ER-Catalase N244 expression on glucose-induced insulin secretion, insulin content, and proinsulin content in insulin-secreting INS-1E cells. After 1 h incubation at 0 mmol/l glucose INS-1E control and ER-Catalase N244 expressing cells were incubated separately for 2 h with the indicated glucose concentrations. Thereafter insulin secretion (A) and insulin content (B) were determined by radioimmunoassay. The proinsulin content was quantified by a proinsulin-specific ELISA (C). The insulin content to proinsulin content ratio was calculated from the presented content data (D). Data are expressed as means±s.e.m. of 12 (insulin secretion and content) or nine (proinsulin content) independent experiments. **P<0.01 and ***P<0.001 compared with cells incubated at 3 mmol/l glucose (ANOVA plus Bonferroni test for multiple comparisons). #P<0.05 and ##P<0.01 compared with control cells incubated with the same glucose concentration (unpaired two-tailed Student's t-test).

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-15-0132

Effects of ER-Catalase N244 on insulin gene expression in INS-1E cells

To determine whether the observed decreased insulin content might be a result of a lower insulin biosynthesis, the mRNA expression level of the insulin 1 and insulin 2 genes was quantified. Although the insulin 1 gene expression was the same in both cell clones, the expression of the insulin 2 gene was, in agreement with the reduced insulin content of ER-Catalase expressing INS-1E cells, 30% lower than in control cells (Fig. 6).

Figure 6
Figure 6

Effects of ER-Catalase N244 expression on insulin 1 and 2 gene expression in insulin-secreting INS-1E cells. INS-1E control (open bars) and ER-Catalase N244 (black bars) expressing cells were incubated for 24 h under control conditions and thereafter total RNA was isolated and gene expression was analysed by RT-qPCR with insulin 1 and insulin 2 specific primer sets. The expression levels were normalised to the housekeeping genes Tuba4a, Actb, and Ppia. Data are expressed as means±s.e.m. of five independent experiments. *P<0.05 compared with control cells (unpaired two-tailed Student's t-test).

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-15-0132

Discussion

H2O2 generation in the ER through PDI and ERO1, the main enzymes of the oxidative protein folding machinery, is a physiological process in all secretory cells including pancreatic β cells (Harding et al. 2003, Gross et al. 2006). Therefore, the question is how cells can cope with these high luminal H2O2 concentrations and if those huge amounts of H2O2 may support the folding of nascent proteins. Both aspects are particularly relevant for insulin-secreting cells to satisfy the high insulin biosynthesis demand, which accounts for roughly 50% of protein synthesis under physiological conditions (Schuit et al. 1991). States of increased insulin demand, such as insulin resistance in type 2 diabetes, require a further increase of the insulin secretory capacity (DeFronzo et al. 1989). In addition, the expression of H2O2-degrading enzymes such as catalase and glutathione peroxidase and also of the ER-resident H2O2-metabolising enzyme Prdx4 is very low in insulin-secreting cells (Tiedge et al. 1998, Mehmeti et al. 2014). Thus, a possible effect of luminal H2O2 on oxidative protein folding is likely to be particularly prominent in insulin-secreting cells.

Moreover, there is increasing evidence that ER stress induced by β cell toxic cytokines may raise ROS production in the ER (Malhotra & Kaufman 2007). Although the involvement of nitric oxide and the disturbance of the calcium homeostasis in the ER have been identified as crucial mediators (Oyadomari et al. 2001, Cardozo et al. 2005), the relevance of luminal H2O2 for these processes is still unsolved.

We therefore investigated the importance of ER-specific H2O2 degradation in insulin-secreting INS-1E cells for insulin gene expression, insulin content and secretion, as well as for the activation of ER stress cascades through β cell toxic cytokines.

In the present study, we could show that insulin-secreting INS-1E tissue culture cells were successfully protected against H2O2-induced loss of viability through the expression of ER-Catalase N244. The threefold increase of the EC50 value for H2O2 toxicity documents convincingly the high H2O2 inactivation capacity of ER-Catalase N244, also when compared with the overexpression of Prdx4 (Mehmeti et al. 2012, Mehmeti et al. 2014) or the targeted expression of a WT catalase cDNA in the ER (Margittai et al. 2012).

This high luminal catalase activity did not change the expression of classical ER stress genes under control conditions and, more importantly, did not counteract the cytokine-induced initiation of the ER stress pathway. Furthermore, the expression of the luminally located protein Grp78/Bip, serving as a central sensor for ER stress due to its function as a molecular chaperone and the activation of the ER stress pathway through its dissociation from IRE1, PERK, and ATF6 (Bertolotti et al. 2000, Schröder & Kaufman 2005), was downregulated by the used cytokines independently of the ER-Catalase N244 expression.

These observations indicate either that the luminal H2O2 concentration is not altered by pro-inflammatory cytokines or that the luminal H2O2 is not involved in the cytokine-mediated ER stress response. Additionally, ER-Catalase N244 could not antagonise the IL1β- or cytokine mixture-induced cell death, indicating that generation of luminal H2O2 at variance to mitochondrial H2O2 is not involved in this process (Gurgul-Convey et al. 2011, Mehmeti et al. 2011).

Although a stimulatory function of H2O2 on insulin translation and secretion has been considered, the question of a direct influence of H2O2 on glucose-induced insulin secretion is yet unresolved. As an explanation, the unusually low expression of H2O2-inactivating enzymes in insulin-secreting cells (Tiedge et al. 1998) has been put forward (Pi et al. 2007, Leloup et al. 2009, Zito et al. 2010). On the other hand, it has been considered that stimulation of insulin biosynthesis in states of increased insulin demand such as insulin resistance in the early stage of type 2 diabetes is accompanied by increased H2O2 generation in the ER, causing defective insulin secretion, β cell dysfunction and ultimately glucose intolerance (Back & Kaufman 2012). However, an effective removal of H2O2 in the ER (present study) as well as in mitochondria by a mitochondrially located catalase (Lortz et al. 2013) did not affect glucose-induced insulin secretion. These results do not provide support for a significant role of H2O2 in glucose-stimulated insulin secretion.

Nevertheless, we could observe a reduction of insulin 2 mRNA expression, insulin content and a decrease of the insulin/proinsulin ratio due to an unchanged proinsulin content. This is in contrast to previous findings after Prdx4 overexpression, which increased insulin 1 and 2 mRNA expression, insulin content and glucose-induced insulin secretion (Mehmeti et al. 2014). These results indicate that efficient removal of H2O2 as such by ER-Catalase N244 does not favour proinsulin protein folding. ER-Catalase N244 expression could not replicate the positive effects of Prdx4 overexpression on gene expression, biosynthesis and secretion of insulin (Mehmeti et al. 2014).

In conclusion, the present results show that effective elimination of luminal H2O2 by ER-Catalase N244 left glucose-induced insulin secretion unaffected, even if insulin gene expression and insulin content were reduced. Cellular toxicity induced by pro-inflammatory cytokines and accompanying ER stress were also not abolished.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work has been supported by a grant from the European Union (BetaBAT, Grant Agreement 277713) in the Framework Programme 7.

Author contribution statement

S Lo and I M contributed to the design, conduct, data analysis and manuscript preparation. S Le contributed to the planning, design, data analysis, and manuscript preparation. All authors approved the final manuscript.

Acknowledgements

The skilful technical assistance of Maren Böger is gratefully acknowledged.

References

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  • BertolottiAZhangYHendershotLMHardingHPRonD2000Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nature Cell Biology2326332. (doi:10.1038/35014014).

    • Search Google Scholar
    • Export Citation
  • BhandaryBMarahattaAKimHRChaeHJ2012An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. International Journal of Molecular Sciences14434456. (doi:10.3390/ijms14010434).

    • Search Google Scholar
    • Export Citation
  • BulleidNJ2012Disulfide bond formation in the mammalian endoplasmic reticulum. Cold Spring Harbor Perspectives in Biology4a013219. (doi:10.1101/cshperspect.a013219).

    • Search Google Scholar
    • Export Citation
  • CaoSSKaufmanRJ2014Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxidants & Redox Signaling21396413. (doi:10.1089/ars.2014.5851).

    • Search Google Scholar
    • Export Citation
  • CardozoAKOrtisFStorlingJFengYMRasschaertJTonnesenMVan EylenFMandrup-PoulsenTHerchuelzAEizirikDL2005Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic β-cells. Diabetes54452461. (doi:10.2337/diabetes.54.2.452).

    • Search Google Scholar
    • Export Citation
  • DeFronzoRAFerranniniESimonsonDC1989Fasting hyperglycemia in non-insulin-dependent diabetes mellitus: contributions of excessive hepatic glucose production and impaired tissue glucose uptake. Metabolism38387395. (doi:10.1016/0026-0495(89)90129-7).

    • Search Google Scholar
    • Export Citation
  • GrossESevierCSHeldmanNVituEBentzurMKaiserCAThorpeCFassD2006Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p. PNAS103299304. (doi:10.1073/pnas.0506448103).

    • Search Google Scholar
    • Export Citation
  • Gurgul-ConveyEMehmetiILortzSLenzenS2011Cytokine toxicity in insulin-producing cells is mediated by nitro-oxidative stress-induced hydroxyl radical formation in mitochondria. Journal of Molecular Medicine89785798. (doi:10.1007/s00109-011-0747-1).

    • Search Google Scholar
    • Export Citation
  • HardingHPRonD2002Endoplasmic reticulum stress and the development of diabetes: a review. Diabetes51 (Suppl 3) S455S461. (doi:10.2337/diabetes.51.2007.S455).

    • Search Google Scholar
    • Export Citation
  • HardingHPZhangYZengHNovoaILuPDCalfonMSadriNYunCPopkoBPaulesR2003An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Molecular Cell11619633. (doi:10.1016/S1097-2765(03)00105-9).

    • Search Google Scholar
    • Export Citation
  • LeloupCTourrel-CuzinCMagnanCKaracaMCastelJCarneiroLColombaniALKtorzaACasteillaLPenicaudL2009Mitochondrial reactive oxygen species are obligatory signals for glucose-induced insulin secretion. Diabetes58673681. (doi:10.2337/db07-1056).

    • Search Google Scholar
    • Export Citation
  • LortzSGurgul-ConveyENaujokOLenzenS2013Overexpression of the antioxidant enzyme catalase does not interfere with the glucose responsiveness of insulin-secreting INS-1E cells and rat islets. Diabetologia56774782. (doi:10.1007/s00125-012-2823-7).

    • Search Google Scholar
    • Export Citation
  • LortzSLenzenSMehmetiI2015N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum. Free Radical Biology & Medicine807783. (doi:10.1016/j.freeradbiomed.2014.11.024).

    • Search Google Scholar
    • Export Citation
  • MalhotraJDKaufmanRJ2007Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword?Antioxidants & Redox Signaling922772293. (doi:10.1089/ars.2007.1782).

    • Search Google Scholar
    • Export Citation
  • MalhotraJDMiaoHZhangKWolfsonAPennathurSPipeSWKaufmanRJ2008Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. PNAS1051852518530. (doi:10.1073/pnas.0809677105).

    • Search Google Scholar
    • Export Citation
  • MargittaiELowPStillerIGrecoAGarcia-ManteigaJMPengoNBenedettiASitiaRBanhegyiG2012Production of H(2)O(2) in the endoplasmic reticulum promotes in vivo disulfide bond formation. Antioxidants & Redox Signaling1610881099. (doi:10.1089/ars.2011.4221).

    • Search Google Scholar
    • Export Citation
  • MehmetiIGurgul-ConveyELenzenSLortzS2011Induction of the intrinsic apoptosis pathway in insulin-secreting cells is dependent on oxidative damage of mitochondria but independent of caspase-12 activation. Biochimica et Biophysica Acta181318271835. (doi:10.1016/j.bbamcr.2011.06.022).

    • Search Google Scholar
    • Export Citation
  • MehmetiILortzSLenzenS2012The H(2)O(2)-sensitive HyPer protein targeted to the endoplasmic reticulum as a mirror of the oxidizing thiol-disulfide milieu. Free Radical Biology & Medicine5314511458. (doi:10.1016/j.freeradbiomed.2012.08.010).

    • Search Google Scholar
    • Export Citation
  • MehmetiILortzSElsnerMLenzenS2014Peroxiredoxin 4 improves insulin biosynthesis and glucose-induced insulin secretion in insulin-secreting INS-1E cells. Journal of Biological Chemistry2892690426913. (doi:10.1074/jbc.M114.568329).

    • Search Google Scholar
    • Export Citation
  • MosmannT1983Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods655563. (doi:10.1016/0022-1759(83)90303-4).

    • Search Google Scholar
    • Export Citation
  • OyadomariSTakedaKTakiguchiMGotohTMatsumotoMWadaIAkiraSArakiEMoriM2001Nitric oxide-induced apoptosis in pancreatic β cells is mediated by the endoplasmic reticulum stress pathway. PNAS981084510850. (doi:10.1073/pnas.191207498).

    • Search Google Scholar
    • Export Citation
  • PiJBaiYZhangQWongVFloeringLMDanielKReeceJMDeeneyJTAndersenMECorkeyBE2007Reactive oxygen species as a signal in glucose-stimulated insulin secretion. Diabetes5617831791. (doi:10.2337/db06-1601).

    • Search Google Scholar
    • Export Citation
  • RammingTAppenzeller-HerzogC2012The physiological functions of mammalian endoplasmic oxidoreductin 1: on disulfides and more. Antioxidants & Redox Signaling1611091118. (doi:10.1089/ars.2011.4475).

    • Search Google Scholar
    • Export Citation
  • RammingTAppenzeller-HerzogC2013Destroy and exploit: catalyzed removal of hydroperoxides from the endoplasmic reticulum. International Journal of Cell Biology2013180906. (doi:10.1155/2013/180906).

    • Search Google Scholar
    • Export Citation
  • ScheunerDKaufmanRJ2008The unfolded protein response: a pathway that links insulin demand with β-cell failure and diabetes. Endocrine Reviews29317333. (doi:10.1210/er.2007-0039).

    • Search Google Scholar
    • Export Citation
  • SchröderMKaufmanRJ2005The mammalian unfolded protein response. Annual Review of Biochemistry74739789. (doi:10.1146/annurev.biochem.73.011303.074134).

    • Search Google Scholar
    • Export Citation
  • SchuitFCKiekensRPipeleersDG1991Measuring the balance between insulin synthesis and insulin release. Biochemical and Biophysical Research Communications17811821187. (doi:10.1016/0006-291X(91)91017-7).

    • Search Google Scholar
    • Export Citation
  • TiedgeMLortzSMundayRLenzenS1998Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species. Diabetes4715781585. (doi:10.2337/diabetes.47.10.1578).

    • Search Google Scholar
    • Export Citation
  • TuBPWeissmanJS2004Oxidative protein folding in eukaryotes: mechanisms and consequences. Journal of Cell Biology164341346. (doi:10.1083/jcb.200311055).

    • Search Google Scholar
    • Export Citation
  • ZitoEMeloEPYangYWahlanderANeubertTARonD2010Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin. Molecular Cell40787797. (doi:10.1016/j.molcel.2010.11.010).

    • Search Google Scholar
    • Export Citation
  • ZuffereyRDullTMandelRJBukovskyAQuirozDNaldiniLTronoD1998Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. Journal of Virology7298739880.

    • Search Google Scholar
    • Export Citation

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    Subcellular localisation of ER-Catalase N244 and WT catalase in insulin-secreting INS-1E cells. Cells were seeded on LabTek chamber slides. After fixation and permeabilisation the cells were stained with specific antibodies for catalase (green) and for the ER-specific protein disulfide isomerase (PDI, red), followed by counterstaining with DAPI (blue). A full colour version of this figure is available at http://dx.doi.org/10.1530/JME-15-0132.

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    Effects of ER-Catalase N244 expression on cytotoxicity after incubation of INS-1E cells with H2O2. INS-1E control and ER-Catalase N244 expressing cells were incubated for 2 h with the indicated H2O2 concentrations in Krebs–Ringer medium, and after removal of the H2O2-containing medium for another 22 h in fresh medium. Thereafter, the viability of the cells was determined by the MTT assay and expressed as % of untreated cells. Data are expressed as mean±s.e.m. of six to eight independent experiments. ***P<0.001 compared with control cells (unpaired two-tailed Student's t-test).

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    Effects of ER-Catalase N244 expression on the induction of ER stress genes after incubation of INS-1E cells with cytokines. INS-1E control and ER-Catalase N244 expressing cells were incubated for 16 h under control conditions, with 600 U/ml IL1β or with a cytokine mixture, composed of 60 U/ml IL1β, 185 U/ml TNFα, and 14 U/ml IFNγ. After incubation, total RNA was isolated and the expression of Chop (A), Atf4 (B), Atf6 (C), and Grp78/Bip (D) was analysed by RT-qPCR with specific primers. The expression levels were normalised to the housekeeping genes Tuba4a, Actb, and Ppia. The gene expression level of untreated control cells was set as 100%. Data are expressed as means±s.e.m. of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 compared with untreated cells (ANOVA plus Bonferroni test for multiple comparisons).

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    Effects of ER-Catalase N244 expression on cytotoxicity after incubation of INS-1E cells with cytokines. INS-1E control and ER-Catalase N244 expressing cells were incubated for 72 h under control conditions, with 600 U/ml IL1β or with a cytokine mixture, consisting of 60 U/ml IL1β, 185 U/ml TNFα, and 14 U/ml IFNγ. The cell viability was determined thereafter by the MTT viability assay and expressed as % of untreated cells. Data are expressed as means±s.e.m. of five independent experiments. ***P<0.001 compared with untreated cells (ANOVA plus Bonferroni test for multiple comparisons).

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    Effects of ER-Catalase N244 expression on glucose-induced insulin secretion, insulin content, and proinsulin content in insulin-secreting INS-1E cells. After 1 h incubation at 0 mmol/l glucose INS-1E control and ER-Catalase N244 expressing cells were incubated separately for 2 h with the indicated glucose concentrations. Thereafter insulin secretion (A) and insulin content (B) were determined by radioimmunoassay. The proinsulin content was quantified by a proinsulin-specific ELISA (C). The insulin content to proinsulin content ratio was calculated from the presented content data (D). Data are expressed as means±s.e.m. of 12 (insulin secretion and content) or nine (proinsulin content) independent experiments. **P<0.01 and ***P<0.001 compared with cells incubated at 3 mmol/l glucose (ANOVA plus Bonferroni test for multiple comparisons). #P<0.05 and ##P<0.01 compared with control cells incubated with the same glucose concentration (unpaired two-tailed Student's t-test).

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    Effects of ER-Catalase N244 expression on insulin 1 and 2 gene expression in insulin-secreting INS-1E cells. INS-1E control (open bars) and ER-Catalase N244 (black bars) expressing cells were incubated for 24 h under control conditions and thereafter total RNA was isolated and gene expression was analysed by RT-qPCR with insulin 1 and insulin 2 specific primer sets. The expression levels were normalised to the housekeeping genes Tuba4a, Actb, and Ppia. Data are expressed as means±s.e.m. of five independent experiments. *P<0.05 compared with control cells (unpaired two-tailed Student's t-test).

  • AsfariMJanjicDMedaPLiGHalbanPAWollheimCB1992Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology130167178. (doi:10.1210/endo.130.1.1370150).

    • Search Google Scholar
    • Export Citation
  • BackSHKaufmanRJ2012Endoplasmic reticulum stress and type 2 diabetes. Annual Review of Biochemistry81767793. (doi:10.1146/annurev-biochem-072909-095555).

    • Search Google Scholar
    • Export Citation
  • BertolottiAZhangYHendershotLMHardingHPRonD2000Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nature Cell Biology2326332. (doi:10.1038/35014014).

    • Search Google Scholar
    • Export Citation
  • BhandaryBMarahattaAKimHRChaeHJ2012An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. International Journal of Molecular Sciences14434456. (doi:10.3390/ijms14010434).

    • Search Google Scholar
    • Export Citation
  • BulleidNJ2012Disulfide bond formation in the mammalian endoplasmic reticulum. Cold Spring Harbor Perspectives in Biology4a013219. (doi:10.1101/cshperspect.a013219).

    • Search Google Scholar
    • Export Citation
  • CaoSSKaufmanRJ2014Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxidants & Redox Signaling21396413. (doi:10.1089/ars.2014.5851).

    • Search Google Scholar
    • Export Citation
  • CardozoAKOrtisFStorlingJFengYMRasschaertJTonnesenMVan EylenFMandrup-PoulsenTHerchuelzAEizirikDL2005Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic β-cells. Diabetes54452461. (doi:10.2337/diabetes.54.2.452).

    • Search Google Scholar
    • Export Citation
  • DeFronzoRAFerranniniESimonsonDC1989Fasting hyperglycemia in non-insulin-dependent diabetes mellitus: contributions of excessive hepatic glucose production and impaired tissue glucose uptake. Metabolism38387395. (doi:10.1016/0026-0495(89)90129-7).

    • Search Google Scholar
    • Export Citation
  • GrossESevierCSHeldmanNVituEBentzurMKaiserCAThorpeCFassD2006Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p. PNAS103299304. (doi:10.1073/pnas.0506448103).

    • Search Google Scholar
    • Export Citation
  • Gurgul-ConveyEMehmetiILortzSLenzenS2011Cytokine toxicity in insulin-producing cells is mediated by nitro-oxidative stress-induced hydroxyl radical formation in mitochondria. Journal of Molecular Medicine89785798. (doi:10.1007/s00109-011-0747-1).

    • Search Google Scholar
    • Export Citation
  • HardingHPRonD2002Endoplasmic reticulum stress and the development of diabetes: a review. Diabetes51 (Suppl 3) S455S461. (doi:10.2337/diabetes.51.2007.S455).

    • Search Google Scholar
    • Export Citation
  • HardingHPZhangYZengHNovoaILuPDCalfonMSadriNYunCPopkoBPaulesR2003An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Molecular Cell11619633. (doi:10.1016/S1097-2765(03)00105-9).

    • Search Google Scholar
    • Export Citation
  • LeloupCTourrel-CuzinCMagnanCKaracaMCastelJCarneiroLColombaniALKtorzaACasteillaLPenicaudL2009Mitochondrial reactive oxygen species are obligatory signals for glucose-induced insulin secretion. Diabetes58673681. (doi:10.2337/db07-1056).

    • Search Google Scholar
    • Export Citation
  • LortzSGurgul-ConveyENaujokOLenzenS2013Overexpression of the antioxidant enzyme catalase does not interfere with the glucose responsiveness of insulin-secreting INS-1E cells and rat islets. Diabetologia56774782. (doi:10.1007/s00125-012-2823-7).

    • Search Google Scholar
    • Export Citation
  • LortzSLenzenSMehmetiI2015N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum. Free Radical Biology & Medicine807783. (doi:10.1016/j.freeradbiomed.2014.11.024).

    • Search Google Scholar
    • Export Citation
  • MalhotraJDKaufmanRJ2007Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword?Antioxidants & Redox Signaling922772293. (doi:10.1089/ars.2007.1782).

    • Search Google Scholar
    • Export Citation
  • MalhotraJDMiaoHZhangKWolfsonAPennathurSPipeSWKaufmanRJ2008Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. PNAS1051852518530. (doi:10.1073/pnas.0809677105).

    • Search Google Scholar
    • Export Citation
  • MargittaiELowPStillerIGrecoAGarcia-ManteigaJMPengoNBenedettiASitiaRBanhegyiG2012Production of H(2)O(2) in the endoplasmic reticulum promotes in vivo disulfide bond formation. Antioxidants & Redox Signaling1610881099. (doi:10.1089/ars.2011.4221).

    • Search Google Scholar
    • Export Citation
  • MehmetiIGurgul-ConveyELenzenSLortzS2011Induction of the intrinsic apoptosis pathway in insulin-secreting cells is dependent on oxidative damage of mitochondria but independent of caspase-12 activation. Biochimica et Biophysica Acta181318271835. (doi:10.1016/j.bbamcr.2011.06.022).

    • Search Google Scholar
    • Export Citation
  • MehmetiILortzSLenzenS2012The H(2)O(2)-sensitive HyPer protein targeted to the endoplasmic reticulum as a mirror of the oxidizing thiol-disulfide milieu. Free Radical Biology & Medicine5314511458. (doi:10.1016/j.freeradbiomed.2012.08.010).

    • Search Google Scholar
    • Export Citation
  • MehmetiILortzSElsnerMLenzenS2014Peroxiredoxin 4 improves insulin biosynthesis and glucose-induced insulin secretion in insulin-secreting INS-1E cells. Journal of Biological Chemistry2892690426913. (doi:10.1074/jbc.M114.568329).

    • Search Google Scholar
    • Export Citation
  • MosmannT1983Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods655563. (doi:10.1016/0022-1759(83)90303-4).

    • Search Google Scholar
    • Export Citation
  • OyadomariSTakedaKTakiguchiMGotohTMatsumotoMWadaIAkiraSArakiEMoriM2001Nitric oxide-induced apoptosis in pancreatic β cells is mediated by the endoplasmic reticulum stress pathway. PNAS981084510850. (doi:10.1073/pnas.191207498).

    • Search Google Scholar
    • Export Citation
  • PiJBaiYZhangQWongVFloeringLMDanielKReeceJMDeeneyJTAndersenMECorkeyBE2007Reactive oxygen species as a signal in glucose-stimulated insulin secretion. Diabetes5617831791. (doi:10.2337/db06-1601).

    • Search Google Scholar
    • Export Citation
  • RammingTAppenzeller-HerzogC2012The physiological functions of mammalian endoplasmic oxidoreductin 1: on disulfides and more. Antioxidants & Redox Signaling1611091118. (doi:10.1089/ars.2011.4475).

    • Search Google Scholar
    • Export Citation
  • RammingTAppenzeller-HerzogC2013Destroy and exploit: catalyzed removal of hydroperoxides from the endoplasmic reticulum. International Journal of Cell Biology2013180906. (doi:10.1155/2013/180906).

    • Search Google Scholar
    • Export Citation
  • ScheunerDKaufmanRJ2008The unfolded protein response: a pathway that links insulin demand with β-cell failure and diabetes. Endocrine Reviews29317333. (doi:10.1210/er.2007-0039).

    • Search Google Scholar
    • Export Citation
  • SchröderMKaufmanRJ2005The mammalian unfolded protein response. Annual Review of Biochemistry74739789. (doi:10.1146/annurev.biochem.73.011303.074134).

    • Search Google Scholar
    • Export Citation
  • SchuitFCKiekensRPipeleersDG1991Measuring the balance between insulin synthesis and insulin release. Biochemical and Biophysical Research Communications17811821187. (doi:10.1016/0006-291X(91)91017-7).

    • Search Google Scholar
    • Export Citation
  • TiedgeMLortzSMundayRLenzenS1998Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species. Diabetes4715781585. (doi:10.2337/diabetes.47.10.1578).

    • Search Google Scholar
    • Export Citation
  • TuBPWeissmanJS2004Oxidative protein folding in eukaryotes: mechanisms and consequences. Journal of Cell Biology164341346. (doi:10.1083/jcb.200311055).

    • Search Google Scholar
    • Export Citation
  • ZitoEMeloEPYangYWahlanderANeubertTARonD2010Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin. Molecular Cell40787797. (doi:10.1016/j.molcel.2010.11.010).

    • Search Google Scholar
    • Export Citation
  • ZuffereyRDullTMandelRJBukovskyAQuirozDNaldiniLTronoD1998Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. Journal of Virology7298739880.

    • Search Google Scholar
    • Export Citation