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Molecular and Cellular Endocrinology xxx (2016) 1e9
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NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice Arnaldo H. de Souza a, b, Laila R.B. Santos a, 1, Leticia P. Roma b, 2, Mohammed Bensellam a, Angelo R. Carpinelli b, Jean-Christophe Jonas a, * a b
Universit e catholique de Louvain, Institute of Experimental and Clinical Research, Pole of Endocrinology, Diabetes and Nutrition, Brussels, Belgium ~o Paulo, Sa ~o Paulo, Brazil Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2016 Received in revised form 24 August 2016 Accepted 20 September 2016 Available online xxx
High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional b-cell mass in type 2 diabetes. To test that hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H2O2 (roGFP2-Orp1), on b-cell stimulus-secretion coupling events and on b-cell apoptosis. After 1e2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was ~1.7-fold higher in NOX2-KO vs. WT islets at 20e30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration ([Ca2þ]i) and no differences in cytosolic GRX1-roGFP2 oxidation. After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and b-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, [Ca2þ]i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with b-cell apoptosis, the glucose sensitivity of the NADPH, [Ca2þ]i and insulin secretion responses increased, the maximal [Ca2þ]i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of bcell survival and function. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: NOX2 roGFP sensors Pancreatic b-cell Insulin secretion Apoptosis
1. Introduction The glucose stimulation of insulin secretion (GSIS) by pancreatic
b-cells depends on the acceleration of glucose metabolism through glycolysis and the Krebs cycle with increased production of metabolic coupling factors (MacDonald et al., 2005; Maechler, 2013; Henquin, 2009), including NADPH (Ivarsson et al., 2005; Reinbothe et al., 2009) and hydrogen peroxide (H2O2) (Pi et al., 2007; Leloup et al., 2009). While the role of H2O2 as a metabolic coupling factor
* Corresponding author. UCL/SSS/IREC/EDIN Avenue Hippocrate 55, B1.55.06, B1200 Brussels, Belgium. E-mail address: [email protected] (J.-C. Jonas). 1 Present address: Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany. 2 Present address: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
is highly debated (Martens et al., 2005; Lortz et al., 2013; Rebelato et al., 2011), it is generally accepted that the high metabolic rate and relatively low expression of H2O2-detoxifying enzymes, such as catalase and glutathione peroxidase, contributes to b-cell “oxidative stress” under states of chronic hyperglycaemia (Lenzen et al., 1996; Tiedge et al., 1997; Li et al., 2009). Although complexes I and III of the mitochondrial electron-transport chain (ETC) are likely major sources of superoxide under these conditions (Green et al., 2004), the possible role of phagocyte-like NADPH oxidase family (NOX) should not be overlooked (Je zek et al., 2012; Oliveira et al., 2003). Pancreatic b-cells cultured for 1 week at high glucose show altered gene expression, function and survival, which may contribute to the decline of the functional b-cell mass in type 2 diabetes (T2D) (Bensellam et al., 2009; Jonas et al., 2009). We previously showed that the long-term glucose regulation of oxidative stress and integrated stress-response genes in rat pancreatic islets follows an asymmetric V-shaped profile parallel to
http://dx.doi.org/10.1016/j.mce.2016.09.022 0303-7207/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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that of later b-cell apoptosis and dysfunction, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations (Jonas et al., 2009; Bensellam et al., 2012; Pascal et al., 2010). Interestingly, these changes were associated with parallel changes in mitochondrial thiol oxidation, as measured using genetically-encoded probes derived from the redox sensitive Green Fluorescent Protein (roGFP1) (Takahashi et al., 2014; Roma et al., 2012). However, decreasing mitochondrial thiol oxidation and later b-cell apoptosis with ZnCl2 or MnTBAP (Manganese [III] tetrakis [4-benzoic acid] porphyrin) did not improve GSIS after prolonged culture at high or low vs. intermediate glucose, suggesting that thiol oxidation in other cell compartments, e.g. the cytosol, may be involved in b-cell dysfunction, or that thiol oxidation is not involved in GSIS. Most NADPH oxidase enzymes are multi-protein complexes that produces superoxide anions. Superoxide can then be converted by superoxide dismutase to H2O2. NOX2 is one of the critical NOX isoform consisting of several membrane-associated and cytosolic components. In pancreatic b-cells, NOX2 is the major isoform expressed and has been associated with regulation of insulin secretion, “redox regulation” and b-cell damage in diabetes (Newsholme et al., 2009). The inhibition of NOX using diphenyleneiodonium (DPI) impaired GSIS in RIN cells and rat islets (Uchizono et al., 2006; Imoto et al., 2008), but the lack of specificity of the drug raises questions about data interpretation (Jaquet et al., 2009; Li et al., 2012). Experiments with Nox2 knockout (Nox2-KO) mice indeed revealed that NOX2-mediated superoxide production reduces cyclic AMP/protein kinase A (PKA) signalling and thereby negatively affects GSIS in mouse b-cells (Li et al., 2012). On the other hand, it has also been proposed that NOX activity could lead to bcell “oxidative stress” and dysfunction in models of increased proinflammatory cytokines, chronic hyperglycaemia and diabetes (Mohammed et al., 2013; Syed et al., 2011; Xiang et al., 2010; Anvari et al., 2015). However, those studies relied on the use of poorly specific tools to assess redox changes by reactive oxygen species (ROS) and other oxidants (Pal et al., 2013; Fujikawa et al., 2013). Here, we tested the role of NOX2 in the glucose regulation of cytosolic thiol oxidation in mouse b-cells and in the alteration of their function and survival during long-term culture in a high vs. intermediate glucose concentration.
and RPMI 1640 (Invitrogen, Carlsbad, CA) without BSA. They were then washed thrice, hand-picked under a stereomicroscope and cultured at 37 C and 5% CO2 in medium containing 10 mmol/l glucose and supplemented with 2 mmol/l glutamine, 5 g/L BSA (fraction V, Roche, Basel, Switzerland), 100 U/ml penicillin and 100 mg/ml streptomycin. The islets were cultured for up to 3 weeks in RPMI containing 10 or 30 mmol/l glucose (G10 or G30) and processed for further analysis. The medium was renewed every other day. To measure insulin secretion during culture, the medium was centrifuged (2 min at 800 rpm) and the supernatant was appropriately diluted for determination of insulin concentration by RIA. 2.3. Incubation and perifusion Krebs solution After culture, islets were perifused at a flow rate of ~1 ml/min with a bicarbonate-buffered Krebs solution containing (mmol/l) NaCl (120), KCl (4.8), CaCl2 (2.5), MgCl2 (1.2), NaHCO3 (24), 1 g/l BSA, and various glucose concentrations (0.5e30) and test substances. The solutions were continuously gassed with O2/CO2 (94/6) to maintain pH ~7.4. 2.4. NAD(P)H autofluorescence After culture, NAD(P)H autofluorescence (lex 360 nm; lem 470 nm) of perifused islets was acquired every 10 s as described (Khaldi et al., 2004) and was expressed in percentage of the fluorescence level measured after 10e15 min of treatment with 10 mmol/l carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) in 30 mmol/l glucose. 2.5. Cytosolic calcium levels After culture, islets were loaded for 2 h with 2 mmol/l Fura-2 LR acetoxymethylester (Teflabs) in a medium similar to that used during culture. The Fura-2 LR fluorescence ratio (lex 340/380 nm; lem 510 nm) of perifused islets was acquired every 5 s as described (Khaldi et al., 2004). 2.6. Acute glucose stimulation of insulin secretion
2. Materials and methods 2.1. Animals NOX2-deficient [NOX2-KO] mice (Pollock et al., 1995) were purchased from The Jackson Laboratory (Bar Harbor, MA). The animals were kept in the animal facility of the Health Science Sector of UCL devoid of murine pathogens. The body weight of NOX2-KO was measured every week as an indicator of healthy state (Li et al., 2012). KO male animals were matched for sex and age with C57BL/6J (wild-type [WT]) control mice (from 3 to 12 months, means ± SEM 6.3 ± 0.5 months for WT mice and 6.2 ± 0.5 for NOX2 KO mice). Both types of mice expressed the truncated inactive form of mitochondrial nicotinamide nucleotide transhydrogenase (Fontaine and Davis, 2016), as confirmed by PCR amplification of tail DNA according to the protocol of the Jackson Laboratory (https://www.jax.org/strain/000664). All experiments were approved by the local ethics committee for animal experimentation (Project 2013/UCL/MD/9/016). 2.2. Islet isolation and culture Islets were obtained by collagenase digestion of the pancreas, purified by gradient centrifugation using Histopaque 1077 (Sigma)
After culture, batches of 5 islets were incubated for 40 min in 0.5 mmol/l glucose. Islets were then incubated for 1 h in the presence of various glucose concentrations. At the end, the medium was collected for insulin measurement and the insulin and DNA contents were measured on each batch of islets. Insulin concentration was measured by RIA using rat insulin as a standard (Heding, 1972). Islet DNA content was measured by fluorimetry using SYBR Green I (Leggate et al., 2006). 2.7. Cytosolic thiol redox state, glutathione redox potential and H2O2 concentration Islets were infected with ~100 multiplicity of infection (MOI) of an adenovirus coding the redox probe roGFP1, GRX1-roGFP2 or roGFP2-Orp1 (Dooley et al., 2004; Morgan et al., 2011) under the control of the CMV promoter. After 36e48 h culture, the fluorescence ratio of the probe (lex 400/480 nm; lem 535 nm) was dynamically monitored every 30 s in perifused islets, as described (Takahashi et al., 2014; Roma et al., 2012). The results were normalized to the fluorescence ratio of the maximally-reduced and maximally-oxidized probe (set to 0 and 100%) measured after sequential addition of 10 mmol/l dithiothreitol and 100 mmol/l aldrithiol at the end of each experiment.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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2.8. Cell apoptosis After culture, cytoplasmic histone-associated DNA fragments were measured on batches of 60 islets using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics) exactly as described (Pascal et al., 2010). The percentage of apoptotic b-cells (TUNEL and insulin double positive cells) was determined on islet sections using the “In Situ Cell Death Detection Kit” (Roche Diagnostics) followed by insulin immunostaining (Duprez et al., 2012). 2.9. Islet gene mRNA levels After culture, islet RNA was extracted and reverse transcribed as previously described (Bensellam et al., 2009). Real-time PCR was performed in a CFX96 optical cycler detection system (Bio-rad, Hercules, CA, USA) using SYBR Green method for Glut2 (forward primer: 50 -CATTCTTTGGTGGGTGGC-3'; reverse primer: 50 CCTGAGTGTGTTTGGAGCG-30 ), Hmox1 (forward primer: 50 -ACAGCATGTCCCAGGATTTGT-3'; reverse primer: 50 -AAGGAGGCCATCACCAGCTT-30 ) and Tbp (forward primer: 50 ACCCTTCACCAATGACTCCTA-3'; reverse primer: 50 -ACTTCGTGCCAGAAATGCTGA-30 ). The value obtained for each gene was normalized to that for the control gene Tbp and expressed relative to the value obtained in WT islets cultured in G10. 2.10. Statistical analysis Results are means ± SEM for at least three islets preparations. Statistical significance of differences between groups were assessed
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by two-way ANOVA followed by a post-test of Bonferroni, unless specified otherwise. Differences were considered significant if P < 0.05. 3. Results 3.1. Impact of NOX2 deficiency on glucose-induced insulin secretion in mouse islets We first confirmed the effect of NOX2 inactivation on the glucose stimulation of mitochondrial metabolism, intracellular Ca2þ concentration ([Ca2þ]i), and insulin secretion in islets from male C57BL/6J mice. As reported previously (Li et al., 2012), after 1e2 days preculture, GSIS was significantly larger in NOX2-KO vs. WT islets incubated at 20 and 30 mmol/l glucose despite their almost identical insulin content and glucose-induced rises in NAD(P)H autofluorescence and [Ca2þ]i (Fig. 1). As cytosolic superoxide can be rapidly converted to H2O2 by superoxide dismutase 1, these results confirm that NOX2-derived superoxide and hydrogen peroxide are not positively involved in acute GSIS. 3.2. Lack of impact of NOX2 deficiency on cytosolic glutathione redox state in mouse islets NOX2-deficiency is expected to reduce superoxide/H2O2 production while slightly reducing NADPH consumption in the cytosol. To test whether NOX2-deficiency impacts the cytosolic thiol redox state in b-cells, we used GRX1-roGFP2, a rapid sensor of changes in GSH/GSSG ratio (glutathione redox potential, or EGSH)
Fig. 1. Acute GSIS in WT and NOX2-KO islets - A-B, after 1e2 days preculture at G10, both groups of islets were simultaneously stimulated with increasing glucose concentrations (Gn in mmol/l, as indicated at the top of each graph) during perifusion with normal Krebs medium containing 4.8 mmol/l Kþ. The experiments ended by addition of 10 mmol/l FCCP (A) or depolarization with 30 mmol/l Kþ in the presence of 250 mmol/l diazoxide (K30Dz) (B). NAD(P)H fluorescence was expressed in percentage of the level measured at the end of FCCP treatment. C-D, after 1e2 days preculture at G10, batches of 5 islets were pre-incubated 40 min in G0.5 before 1 h incubation at various glucose concentrations (total of 9 batches of 5 islets per condition). The data were normalized to the DNA content of each batch of islets. D, insulin to DNA content ratio from incubated islets. Results are means ± SE for 3 experiments. *, P < 0.05 for the glucose effects within each group (vs. the previous condition in A-B or vs. G0.5 in C); #, P < 0.05; ##, P < 0.01 for differences between WT vs. NOX2 KO islets.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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Fig. 2. Acute effects of glucose on cytosolic GRX1-roGFP2 fluorescence ratio in WT and NOX2 KO islets - A-B, after 2e4 days culture at G10, islets expressing GRX1-roGFP2 were perifused with Krebs medium containing different glucose concentrations (Gn in mmol/l) in the absence (A) or presence (B) of exogenous H2O2. Each experiment ended by sequential addition of 10 mmol/l DTT and 100 mmol/l aldrithiol (AT-2) for normalization of the traces. Results are means ± SE for 3 experiments. *, significantly different (P < 0.05 in A and P < 0.01 in B) vs. previous condition within each group. There were no significant differences between the two types of islets.
(Gutscher et al., 2008) and tested the response of the probe to acute changes in glucose concentration from 10 to 0 then 30 mmol/l. Under control conditions, omission of glucose did not significantly affect GRX1eroGFP2 fluorescence ratio, but stimulation from 0 to 30 mmol/l glucose acutely reduced the probe, hence cytosolic EGSH (Fig. 2A), in agreement with recent data obtained in re-aggregated human islets (Ferdaoussi et al., 2015). In comparison, when the probe was oxidized by a constant supply of exogenous H2O2, omission of glucose further increased the probe oxidation, an effect that was reversed upon stimulation with 30 mmol/l glucose (Fig. 2B) (Takahashi et al., 2014). However, in contrast to our initial expectations, the glucose responses in the absence or presence of exogenous H2O2 were similar in NOX2-KO and WT islets. These results suggest that NOX2 does not participate in the acute glucose regulation of cytosolic EGSH in these islets. 3.3. Lack of impact of NOX2 deficiency on cytosolic thiol oxidation in cultured mouse islets We next used cytosolic roGFP1 to measure the level of cytosolic thiol oxidation after culture under control and glucotoxic conditions. As shown in Fig. 3A, overnight culture in 30 instead of 10 mmol/l glucose significantly increased roGFP1 fluorescence ratio in mouse islets, likely reflecting an increase in cytosolic thiol
Fig. 3. Effects of glucose and NOX2 deficiency on cytosolic roGFP1 and roGFP2Orp1 fluorescence ratio in cultured mouse islets - After 2e4 days culture at G10, WT and NOX2-KO mouse islets expressing roGFP1 (A) or roGFP2-Orp1 (B) were cultured overnight at G10 or G30, as indicated on the X-axis. Then islets were perifused for 20 min with a Krebs solution containing the same glucose concentration as during culture before sequential application of 10 mmol/l DTT and 100 mmol/l aldrithiol (AT-2) for normalization of the traces. Results are means ± SE for 3 to 5 experiments. *, significant effect of glucose within each group (P < 0.001). There were no significant differences between the two types of islets.
oxidation. However, this effect was not different in NOX2-KO vs. WT islets. Similar results were obtained in islets expressing roGFP2Orp1, a probe that specifically reacts with H2O2 (Albrecht et al., 2011) (Fig. 3B). These results suggest that, although prolonged exposure to high vs. intermediate glucose concentrations increased cytosolic thiol oxidation and H2O2 concentration in islet cells, this effect did not depend on NOX2 expression.
3.4. Lack of impact of NOX2 deficiency on the glucotoxic alterations of b-cell function in cultured mouse islets Prolonged exposure to high glucose concentrations induces bcell dysfunction and apoptosis, thereby contributing to the gradual decrease in functional b-cell mass in T2D patients. Human and rat islets cultured in high glucose for 1 week exhibit b-cell dysfunction and apoptosis, but an associated increase in mitochondrial oxidative stress was only documented in rat islets (Duprez et al., 2012; Brun et al., 2015). However, in mouse islets, glucotoxicity and cell apoptosis can only be seen after 3 weeks of culture (Pascal et al., 2010). In agreement with previous observations that culture at G10 optimally preserves rodent b-cell function and survival, WT islets cultured for 1 or 3 weeks at G10 displayed similar rises in NAD(P)H autofluorescence, [Ca2þ]i and insulin secretion upon acute stepwise glucose stimulation (Fig. 4A, C, E and 5A, C, E). The islet insulin content ratio was, however, approximately reduced by 50% after 1 or 3 weeks of culture as compared with islets cultured for 1 or 2 days (compare Figs. 4E and 5E with Fig. 1D; see also ESM table 4 for details on the islet insulin to DNA content ratio under each condition tested). These responses were almost identical in islets from NOX2-KO mice, except for the ~1.4-fold larger insulin secretion in NOX2-KO islets incubated for 1 h at 30 mmol/l glucose after 1 week culture in G10 (Fig. 4E). In agreement with previous studies in mouse islets (Pascal et al., 2008, 2010), 1e3 weeks of culture in G30 markedly increased the glucose sensitivity of WT islets upon subsequent glucose stimulation, as shown by the shift to the left of the glucose concentrationresponse curves for changes in NAD(P)H autofluorescence, [Ca2þ]i and insulin secretion. (Figs. 4 and 5). It also reduced the maximal amplitude of glucose-stimulated [Ca2þ]i rise. However, the maximal secretory response to glucose was not reduced. These responses were almost identical in NOX2-KO islets (Suppl. tables 13), suggesting that NOX2 does not contribute to the alterations of bcell stimulus secretion coupling by prolonged culture at high vs. intermediate glucose concentration in male C57BL/6J mouse islets.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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3.5. Lack of impact of NOX2 deficiency on the glucotoxic alteration of b-cell survival in cultured mouse islets Despite the lack of impact of NOX2 deficiency on the glucotoxic alterations of stimulus-secretion coupling events, the small, albeit not significant, reduction in cytosolic thiol oxidation in NOX2-KO vs. WT islets led us to test the effects of 3 weeks of culture at G10 or G30 on b-cell apoptosis in both islet types. In agreement with previous studies in rat and mouse islets (Jonas et al., 2009), the level of islet cell DNA fragmentation and b-cell apoptosis were minimum after prolonged culture at G10, with less than 2% of apoptotic b-cells
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in both groups of islets (Fig. 6AeB and ESM Fig. 2). In comparison, there was a significant increase in islet DNA fragmentation (~5fold) and b-cell apoptosis (~4-fold) after 3 weeks of culture at G30 vs. G10, confirming the glucotoxic stimulation of b-cell apoptosis. These changes were not associated with changes in the proportion of b-cells among islet cells, as measured by immunohistochemistry on islet sections (Fig. 6C). Again, NOX2 deficiency did not significantly alter islet DNA fragmentation and b-cell apoptosis after culture at either G10 or G30, excluding NOX2 as a critical regulator of mouse pancreatic b-cell survival during culture under control and glucotoxic conditions (Fig. 6 and ESM Fig. 2).
Fig. 4. Lack of impact of NOX2 deficiency on islet glucose responsiveness after 1 week culture in high glucose e After 1e3 days culture at G10, mouse islets were cultured for 1 week at G10 or G30, as indicated at the top of the figure. AeD, after culture, WT and NOX2-KO islets were perifused with normal Krebs medium containing 4.8 mmol/l Kþ and increasing glucose concentrations (Gn in mmol/l, as specified in each graph) before addition of 10 mmol/l FCCP (AeB), or depolarization with 30 mmol/l Kþ in the presence of 250 mmol/l diazoxide (K30Dz) (CeD). NAD(P)H fluorescence was expressed in percentage of the level measured at the end of FCCP treatment, which triggers rapid oxidation of mitochondrial NADH by ETC complex I. E,F, after 7e10 days culture and 40 min preincubation in G0.5, islets were incubated in batches of 5 for 1 h at various glucose concentrations. The rate of insulin secretion and the islet insulin content were measured at the end of the incubation and normalized to the islet DNA content. Results are means ± SE for 3 experiments. *, P < 0.05 for the glucose effects within each group (vs. previous condition in A-D or vs. G0.5 in E-F); #, P < 0.05 for differences between WT vs. NOX2 KO islets.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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A.H. de Souza et al. / Molecular and Cellular Endocrinology xxx (2016) 1e9
Fig. 5. Lack of impact of NOX2 deficiency on islet glucose responsiveness after 3 weeks culture in high glucose e After 1e3 days culture at G10, mouse islets were cultured for 3 weeks at G10 or G30, as indicated at the top of the figure. AeD, after culture, WT and NOX2-KO islets were perifused with normal Krebs medium containing 4.8 mmol/l Kþ and increasing glucose concentrations (Gn in mmol/l, as specified in each graph) before addition of 10 mmol/l FCCP (AeB), or depolarization with 30 mmol/l Kþ in the presence of 250 mmol/l diazoxide (K30Dz) (CeD). NAD(P)H fluorescence was expressed in percentage of the level measured at the end of FCCP treatment. E,F, after culture and 40 min preincubation in G0.5, islets were incubated in batches of 5 for 1 h at various glucose concentrations. The rate of insulin secretion and the islet insulin content were measured at the end of the incubation and normalized to the islet DNA content. Results are means ± SE for 3 to 4 experiments. *, P < 0.05 for the glucose effects within each group (vs. previous condition in A-D or vs. G0.5 in E-F). There were no significant differences between the two types of islets.
3.6. Lack of impact of NOX2 deficiency on the glucotoxic alteration of b-cell gene mRNA levels in cultured mouse islets
result from its compensation by increased expression of another Nox isoform.
Glut2 mRNA levels are typically reduced, while Hmox1 mRNA levels are increased under glucotoxic conditions. We therefore measured the effect of 3 weeks of culture in G10 or G30 on these mRNA levels in both islet types as a further evaluation of the impact of NOX2 on b-cell glucotoxicity. As expected, Glut2 mRNA levels decreased and Hmox1 mRNA levels increased after culture in G30 vs. G10, but these effects were similar in WT and Nox2-KO islets (Fig. 7). Finally, we also measured the mRNA levels of Nox1-4. As shown in ESM table 5, Nox1-4 mRNA levels were not affected by long term culture in G30 vs. G10. In addition, Nox1, 3 and 4 mRNA levels were not detectably increased in Nox2-KO islets, showing that the lack of impact of Nox2-KO on b-cell glucotoxicity does not
4. Discussion In this study, we demonstrate that, although NOX2 deficiency increases acute GSIS by male C57BL/6J mouse islets, the enzyme does not detectably contribute to the glucotoxic induction of cytosolic thiol oxidation and rise in H2O2, b-cell dysfunction and apoptosis during prolonged cultured at 30 vs. 10 mmol/l glucose. 4.1. Short-term glucose effects Previous studies on the role of NOX in the regulation of GSIS relied on the use of chemical inhibitors (DPI or apocynin) or
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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Fig. 7. Nox2 deficiency does not affect Glut2 and Hmox1 mRNA levels after prolonged culture in the presence of intermediate or high glucose concentrations. After 1e3 days preculture at G10, mouse islets were cultured for 3 weeks at G10 or G30. Changes in gene/Tbp mRNA ratio for (A) Glut2 and (B) Hmox1. Results are means ± SE from 3 to 4 experiments. *, P < 0.05; **,P < 0.001 for the effect of glucose. There were no significant differences between the two types of islets.
Fig. 6. NOX2 deficiency does not affect islet cell apoptosis after prolonged culture at intermediate or high glucose concentrations. After 1e3 days preculture at G10, mouse islets were cultured for 3 weeks at G10 or G30, as indicated on the X-axis. A, DNA fragmentation was expressed relative to the value measured in WT islets cultured in G10. B, percentage of apoptotic b-cells (TUNEL-positive/DAPI-positive nuclei in insulin-positive cells). C, percentage of b -cells in islet sections. Results are means ± SE for 3 experiments (AeC) plus data from each islet section analysed (B and C). *, P < 0.05; **, P < 0.001 for the glucose effects within each group. There were no significant differences between the two types of islets.
play a positive role in acute GSIS can be excluded in this experimental model. The negative effect of NOX2 on GSIS has been attributed to superoxide-mediated inactivation of cAMP/protein kinase A signalling and, hence, reduced amplification of insulin secretion (Li et al., 2012). In that study, superoxide-mediated nitroblue tetrazolium (NBT) and dihydroethidine (DHE) oxidation was ~50% lower in NOX2-KO vs. WT islets incubated at high glucose (Li et al., 2012). However, although cytosolic GRX1-roGFP2 can be indirectly oxidized by menadione that triggers superoxide production (Takahashi et al., 2014; Chuang et al., 2002), cytosolic EGSH did not differ between islet types under any condition tested. These results are not necessarily incompatible, for several reasons. First, NBT and DHE could be oxidized in the extracellular space within islets, whereas GRX1-roGFP2 only senses what happens in the compartment where it is expressed. Second, in contrast with NBT and DHE that are not reduced after their oxidation by superoxide, changes in GRX1-roGFP2 fluorescence ratio monitor changes in EGSH that result from the dynamic equilibrium between glutathione oxidation by ROS and electrophiles and its reduction by glutathionereductase using NADPH as a cofactor (Schwarzl€ ander et al., 2016). Finally, GRX1-roGFP2 measurements could miss EGSH variations in sub-regions of b-cells, e.g. in the population of vesicles containing endosomes/lysosomes and insulin granules in which NOX2 was shown to reside in human b-cells (Li et al., 2012). The acute glucosemediated reduction of GRX1-roGFP2 fluorescence ratio we observed therefore does not exclude a hypothetical increase in ROS production at high glucose, but indicates that it is more than compensated for by a concomitant increase in glutathione reductive capacity.
4.2. Long-term glucose effects silencing p47phox (Imoto et al., 2008; Morgan et al., 2009), a regulatory protein that plays a pivotal role in NOX2 activation (Quinn and Gauss, 2004). In these studies, NOX2 inhibition led to a decrease in acute GSIS that was attributed to alterations of glucoseinduced rise in [Ca2þ]i, suggesting a positive role of superoxide in GSIS (Imoto et al., 2008; Morgan et al., 2009). In this study, however, we confirm the observation by Li and colleagues (Li et al., 2012) that NOX2 deficiency increases GSIS during short-term incubation at or above 20 mmol/l glucose without affecting the islet insulin content nor the glucose-induced rises in [Ca2þ]i. Our results thereby strengthen the view that NOX2 is a negative modulator of maximal GSIS acting at a step distal to Ca2þ influx (Li et al., 2012). Thus, the hypothesis that NOX2-derived superoxide and H2O2 may
We previously showed that prolonged culture at G30 vs. G10 induces b-cell apoptosis, alterations of stimulus-secretion coupling events, impaired GSIS and increased mitochondrial oxidative stress markers in rodent pancreatic islets (Bensellam et al., 2009; Pascal et al., 2010; Duprez et al., 2012). However, antioxidant agents able to reduce mitochondrial oxidative stress improved b-cell survival without restoring GSIS, suggesting that the latter is more sensitive to low levels of oxidative stress, or that it resulted from redox imbalance in other cell compartments, e.g. in the cytosol (Duprez et al., 2012). In this context, it was previously suggested that elevated NOX activity under high glucose conditions might be a mechanism of b-cell oxidative stress in diabetes (Newsholme et al., 2009).
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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In the present study, the increase of both cytosolic thiol oxidation and H2O2 concentration after prolonged culture at G30 vs. G10 was followed by glucotoxic alterations of b-cell stimulussecretion coupling events (although maximal GSIS was not reduced for an unclear reason) and increased b-cell apoptosis. These data suggest that cytosolic redox imbalance could contribute to oxidative stress induced by high glucose in mouse islets. However, although NOX2 deficiency increased acute GSIS in islets cultured for a few days under control conditions (see previous section), it failed to reduce the glucose-induced oxidation of roGFP1 and roGFP2-Orp1 or the glucotoxic alterations of b-cell survival and function after long-term culture at G30. The lack of protective effect of NOX2 deficiency on the oxidation of roGFP-derived probes contrasts with a recent report showing that chemical inhibition of NOX2 with gp91-ds-tat reduced the stimulation of DCF oxidation and NOX2 activity (measured by luminescence assay) by 20 mmol/l glucose in lysates of INS-1 832/13, or INS-1 subclone 832/13 cells (Sidarala et al., 2015). Again, it should be stressed that these measurements were done at the whole cell levels and that the fluorescence ratio of roGFPderived probes is not only influenced by ROS but also by the reductive capacity of NADPH-dependent antioxidant enzymes. In this context, our data on NAD(P)H autofluorescence do not allow to conclude whether cytosolic NADPH was higher in Nox2-KO vs. WT islets, but the lack of differences in the oxidation of GRX1roGFP2 between both islets types does not support that hypothesis. Nevertheless, our results show that NOX2-derived superoxide does not detectably contribute to cytosolic thiol/ glutathione oxidation and H2O2 concentration in intact living islet cells, nor to the glucotoxic alterations of b-cell function and survival. Our results, however, do not rule out the possibility that ETC complex III-mediated superoxide production in the mitochondrial intermembrane space could contribute to cytosolic oxidative stress and b-cell glucotoxicity. In addition, we do not exclude the possibility that NOX2 could play a role in in vivo bcell dysfunction and apoptosis in T2D (Cnop et al., 2005), as chronic exposure to elevated levels of free fatty acids (FFAs) have been shown to increase the expression and activity of NOX enzymes in insulin-secreting cells and rat islets (Yuan et al., 2010; Morgan et al., 2007). 4.3. A note of caution One possible limitation of this study lies in the C57BL/6J genetic background of NOX2-KO mice and their WT controls. This strain of mice, which is widely used in transgenesis, indeed expresses an inactive form of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial matrix enzyme involved in NADPH production. As NNT inactivation was previously found to aggravate the phenotype of superoxide dismutase 2 KO mice (Huang et al., 2006), it remains to be seen if NOX2 deficiency has the same effect on GSIS and lack of contribution to b-cell glucotoxicity when expressed in mice that express functional NNT. Also, the changes in fluorescence ratio of roGFP-derived probes shown in this paper have to be taken as representative only of what happens in the absence of functional NNT, as we recently showed that NNT has a large impact on the glucose regulation of mitochondrial EGSH, hence to a smaller extent on cytosolic EGSH as well (Santos et al., 2015). 5. Conclusion Long-term culture of mouse islets at high vs. intermediate glucose concentration triggers cytosolic redox imbalance that may contribute to the late impairment of b-cell function and survival. NOX2-containing NADPH oxidase is a negative modulator of GSIS
during short-term incubation at 20e30 mmol/l glucose, but this effect is independent from detectable alterations in cytosolic thiol or glutathione redox state. Finally, NOX2 activation is not involved in the alterations of b-cell function and survival after long-term culture at high vs. intermediate glucose concentrations. Acknowledgments We thank Dr Tobias Dick (German Cancer Research Center, Heidelberg, Germany) for providing the plasmid coding roGFP2Orp1. We thank Denis Charlier and Fabien Knockaert for expert technical help. We also thank Heeyoung Chae for help in islet isolation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.mce.2016.09.022. Funding ~o de Amparo a Pesquisa do AHS was recipient of a Fundaça Estado de S~ ao Paulo (Scholarship FAPESP 2011/21299-9) and Bolsa gio de Pesquisa no Exterior (Scholarship BEPE/FAPESP 2013/ Esta 18232-5). MB is recipient of a “MOVE-IN Louvain” incoming Postdoc fellowship. JCJ is Research Director of the Fonds de la Recherche Scientifique-FNRS, Brussels, Belgium. es 12/ This study was funded by Actions de Recherche Concerte française de Belgique and Grant 17-047 from the Communaute dicale to 3.4521.12 from the Fonds de la Recherche Scientifique Me JCJ. Author's contributions AHS, LPR, ARC and JCJ conceived and designed the experiments. AHS, LPR, LRBS and MB performed the experiments and analysed the data. AHS and JCJ wrote the paper. All other coauthors edited and approved the paper. References Albrecht, S.C., Barata, A.G., Grosshans, J., Teleman, A.A., Dick, T.P., 2011. In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis. Cell Metab. 14 (6), 819e829. €m, P., Walum, E., Welsh, N., 2015. The novel NADPH oxidase 4 Anvari, E., Wikstro inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice. Free Radic. Res. 49 (11), 1308e1318. Bensellam, M., Van Lommel, L., Overbergh, L., Schuit, F.C., Jonas, J.C., 2009. Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations. Diabetologia 52 (3), 463e476. Bensellam, M., Laybutt, D.R., Jonas, J.C., 2012. The molecular mechanisms of pancreatic b-cell glucotoxicity: recent findings and future research directions. Mol. Cell Endocrinol. 364 (1e2), 1e27. Brun, T., Li, N., Jourdain, A.A., Gaudet, P., Duhamel, D., Meyer, J., Bosco, D., Maechler, P., 2015. Diabetogenic milieus induce specific changes in mitochondrial transcriptome and differentiation of human pancreatic islets. Hum. Mol. Genet. 24 (18), 5270e5284. Chuang, Y.Y., Chen, Y., Gadisetti, Chandramouli, V.R., Cook, J.A., Coffin, D., Tsai, M.H., DeGraff, W., Yan, H., Zhao, S., Russo, A., Liu, E.T., Mitchell, J.B., 2002. Gene expression after treatment with hydrogen peroxide, menadione, or t-butyl hydroperoxide in breast cancer cells. Cancer Res. 62 (21), 6246e6254. € rns, A., Lenzen, S., Eizirik, D.L., 2005. Mechanisms Cnop, M., Welsh, N., Jonas, J.C., Jo of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes 54 (2), S97eS107. Dooley, C.T., Dore, T.M., Hanson, G.T., Jackson, W.C., Remington, S.J., Tsien, R.Y., 2004. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 279 (21), 22284e22293. Duprez, J., Roma, L.P., Close, A.F., Jonas, J.C., 2012. Protective antioxidant and antiapoptotic effects of ZnCl2 in rat pancreatic islets cultured in low and high glucose concentrations. PloS one 7 (10), e46831. Ferdaoussi, M., Dai, X., Jensen, M.V., Wang, R., Peterson, B.S., Huang, C., Ilkayeva, O., Smith, N., Miller, N., Hajmrle, C., Spigelman, A.F., Wright, R.C., Plummer, G.,
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NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice Arnaldo H. de Souza a, b, Laila R.B. Santos a, 1, Leticia P. Roma b, 2, Mohammed Bensellam a, Angelo R. Carpinelli b, Jean-Christophe Jonas a, * a b
Universit e catholique de Louvain, Institute of Experimental and Clinical Research, Pole of Endocrinology, Diabetes and Nutrition, Brussels, Belgium ~o Paulo, Sa ~o Paulo, Brazil Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2016 Received in revised form 24 August 2016 Accepted 20 September 2016 Available online xxx
High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional b-cell mass in type 2 diabetes. To test that hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H2O2 (roGFP2-Orp1), on b-cell stimulus-secretion coupling events and on b-cell apoptosis. After 1e2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was ~1.7-fold higher in NOX2-KO vs. WT islets at 20e30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration ([Ca2þ]i) and no differences in cytosolic GRX1-roGFP2 oxidation. After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and b-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, [Ca2þ]i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with b-cell apoptosis, the glucose sensitivity of the NADPH, [Ca2þ]i and insulin secretion responses increased, the maximal [Ca2þ]i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of bcell survival and function. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: NOX2 roGFP sensors Pancreatic b-cell Insulin secretion Apoptosis
1. Introduction The glucose stimulation of insulin secretion (GSIS) by pancreatic
b-cells depends on the acceleration of glucose metabolism through glycolysis and the Krebs cycle with increased production of metabolic coupling factors (MacDonald et al., 2005; Maechler, 2013; Henquin, 2009), including NADPH (Ivarsson et al., 2005; Reinbothe et al., 2009) and hydrogen peroxide (H2O2) (Pi et al., 2007; Leloup et al., 2009). While the role of H2O2 as a metabolic coupling factor
* Corresponding author. UCL/SSS/IREC/EDIN Avenue Hippocrate 55, B1.55.06, B1200 Brussels, Belgium. E-mail address: [email protected] (J.-C. Jonas). 1 Present address: Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany. 2 Present address: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
is highly debated (Martens et al., 2005; Lortz et al., 2013; Rebelato et al., 2011), it is generally accepted that the high metabolic rate and relatively low expression of H2O2-detoxifying enzymes, such as catalase and glutathione peroxidase, contributes to b-cell “oxidative stress” under states of chronic hyperglycaemia (Lenzen et al., 1996; Tiedge et al., 1997; Li et al., 2009). Although complexes I and III of the mitochondrial electron-transport chain (ETC) are likely major sources of superoxide under these conditions (Green et al., 2004), the possible role of phagocyte-like NADPH oxidase family (NOX) should not be overlooked (Je zek et al., 2012; Oliveira et al., 2003). Pancreatic b-cells cultured for 1 week at high glucose show altered gene expression, function and survival, which may contribute to the decline of the functional b-cell mass in type 2 diabetes (T2D) (Bensellam et al., 2009; Jonas et al., 2009). We previously showed that the long-term glucose regulation of oxidative stress and integrated stress-response genes in rat pancreatic islets follows an asymmetric V-shaped profile parallel to
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Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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that of later b-cell apoptosis and dysfunction, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations (Jonas et al., 2009; Bensellam et al., 2012; Pascal et al., 2010). Interestingly, these changes were associated with parallel changes in mitochondrial thiol oxidation, as measured using genetically-encoded probes derived from the redox sensitive Green Fluorescent Protein (roGFP1) (Takahashi et al., 2014; Roma et al., 2012). However, decreasing mitochondrial thiol oxidation and later b-cell apoptosis with ZnCl2 or MnTBAP (Manganese [III] tetrakis [4-benzoic acid] porphyrin) did not improve GSIS after prolonged culture at high or low vs. intermediate glucose, suggesting that thiol oxidation in other cell compartments, e.g. the cytosol, may be involved in b-cell dysfunction, or that thiol oxidation is not involved in GSIS. Most NADPH oxidase enzymes are multi-protein complexes that produces superoxide anions. Superoxide can then be converted by superoxide dismutase to H2O2. NOX2 is one of the critical NOX isoform consisting of several membrane-associated and cytosolic components. In pancreatic b-cells, NOX2 is the major isoform expressed and has been associated with regulation of insulin secretion, “redox regulation” and b-cell damage in diabetes (Newsholme et al., 2009). The inhibition of NOX using diphenyleneiodonium (DPI) impaired GSIS in RIN cells and rat islets (Uchizono et al., 2006; Imoto et al., 2008), but the lack of specificity of the drug raises questions about data interpretation (Jaquet et al., 2009; Li et al., 2012). Experiments with Nox2 knockout (Nox2-KO) mice indeed revealed that NOX2-mediated superoxide production reduces cyclic AMP/protein kinase A (PKA) signalling and thereby negatively affects GSIS in mouse b-cells (Li et al., 2012). On the other hand, it has also been proposed that NOX activity could lead to bcell “oxidative stress” and dysfunction in models of increased proinflammatory cytokines, chronic hyperglycaemia and diabetes (Mohammed et al., 2013; Syed et al., 2011; Xiang et al., 2010; Anvari et al., 2015). However, those studies relied on the use of poorly specific tools to assess redox changes by reactive oxygen species (ROS) and other oxidants (Pal et al., 2013; Fujikawa et al., 2013). Here, we tested the role of NOX2 in the glucose regulation of cytosolic thiol oxidation in mouse b-cells and in the alteration of their function and survival during long-term culture in a high vs. intermediate glucose concentration.
and RPMI 1640 (Invitrogen, Carlsbad, CA) without BSA. They were then washed thrice, hand-picked under a stereomicroscope and cultured at 37 C and 5% CO2 in medium containing 10 mmol/l glucose and supplemented with 2 mmol/l glutamine, 5 g/L BSA (fraction V, Roche, Basel, Switzerland), 100 U/ml penicillin and 100 mg/ml streptomycin. The islets were cultured for up to 3 weeks in RPMI containing 10 or 30 mmol/l glucose (G10 or G30) and processed for further analysis. The medium was renewed every other day. To measure insulin secretion during culture, the medium was centrifuged (2 min at 800 rpm) and the supernatant was appropriately diluted for determination of insulin concentration by RIA. 2.3. Incubation and perifusion Krebs solution After culture, islets were perifused at a flow rate of ~1 ml/min with a bicarbonate-buffered Krebs solution containing (mmol/l) NaCl (120), KCl (4.8), CaCl2 (2.5), MgCl2 (1.2), NaHCO3 (24), 1 g/l BSA, and various glucose concentrations (0.5e30) and test substances. The solutions were continuously gassed with O2/CO2 (94/6) to maintain pH ~7.4. 2.4. NAD(P)H autofluorescence After culture, NAD(P)H autofluorescence (lex 360 nm; lem 470 nm) of perifused islets was acquired every 10 s as described (Khaldi et al., 2004) and was expressed in percentage of the fluorescence level measured after 10e15 min of treatment with 10 mmol/l carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) in 30 mmol/l glucose. 2.5. Cytosolic calcium levels After culture, islets were loaded for 2 h with 2 mmol/l Fura-2 LR acetoxymethylester (Teflabs) in a medium similar to that used during culture. The Fura-2 LR fluorescence ratio (lex 340/380 nm; lem 510 nm) of perifused islets was acquired every 5 s as described (Khaldi et al., 2004). 2.6. Acute glucose stimulation of insulin secretion
2. Materials and methods 2.1. Animals NOX2-deficient [NOX2-KO] mice (Pollock et al., 1995) were purchased from The Jackson Laboratory (Bar Harbor, MA). The animals were kept in the animal facility of the Health Science Sector of UCL devoid of murine pathogens. The body weight of NOX2-KO was measured every week as an indicator of healthy state (Li et al., 2012). KO male animals were matched for sex and age with C57BL/6J (wild-type [WT]) control mice (from 3 to 12 months, means ± SEM 6.3 ± 0.5 months for WT mice and 6.2 ± 0.5 for NOX2 KO mice). Both types of mice expressed the truncated inactive form of mitochondrial nicotinamide nucleotide transhydrogenase (Fontaine and Davis, 2016), as confirmed by PCR amplification of tail DNA according to the protocol of the Jackson Laboratory (https://www.jax.org/strain/000664). All experiments were approved by the local ethics committee for animal experimentation (Project 2013/UCL/MD/9/016). 2.2. Islet isolation and culture Islets were obtained by collagenase digestion of the pancreas, purified by gradient centrifugation using Histopaque 1077 (Sigma)
After culture, batches of 5 islets were incubated for 40 min in 0.5 mmol/l glucose. Islets were then incubated for 1 h in the presence of various glucose concentrations. At the end, the medium was collected for insulin measurement and the insulin and DNA contents were measured on each batch of islets. Insulin concentration was measured by RIA using rat insulin as a standard (Heding, 1972). Islet DNA content was measured by fluorimetry using SYBR Green I (Leggate et al., 2006). 2.7. Cytosolic thiol redox state, glutathione redox potential and H2O2 concentration Islets were infected with ~100 multiplicity of infection (MOI) of an adenovirus coding the redox probe roGFP1, GRX1-roGFP2 or roGFP2-Orp1 (Dooley et al., 2004; Morgan et al., 2011) under the control of the CMV promoter. After 36e48 h culture, the fluorescence ratio of the probe (lex 400/480 nm; lem 535 nm) was dynamically monitored every 30 s in perifused islets, as described (Takahashi et al., 2014; Roma et al., 2012). The results were normalized to the fluorescence ratio of the maximally-reduced and maximally-oxidized probe (set to 0 and 100%) measured after sequential addition of 10 mmol/l dithiothreitol and 100 mmol/l aldrithiol at the end of each experiment.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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2.8. Cell apoptosis After culture, cytoplasmic histone-associated DNA fragments were measured on batches of 60 islets using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics) exactly as described (Pascal et al., 2010). The percentage of apoptotic b-cells (TUNEL and insulin double positive cells) was determined on islet sections using the “In Situ Cell Death Detection Kit” (Roche Diagnostics) followed by insulin immunostaining (Duprez et al., 2012). 2.9. Islet gene mRNA levels After culture, islet RNA was extracted and reverse transcribed as previously described (Bensellam et al., 2009). Real-time PCR was performed in a CFX96 optical cycler detection system (Bio-rad, Hercules, CA, USA) using SYBR Green method for Glut2 (forward primer: 50 -CATTCTTTGGTGGGTGGC-3'; reverse primer: 50 CCTGAGTGTGTTTGGAGCG-30 ), Hmox1 (forward primer: 50 -ACAGCATGTCCCAGGATTTGT-3'; reverse primer: 50 -AAGGAGGCCATCACCAGCTT-30 ) and Tbp (forward primer: 50 ACCCTTCACCAATGACTCCTA-3'; reverse primer: 50 -ACTTCGTGCCAGAAATGCTGA-30 ). The value obtained for each gene was normalized to that for the control gene Tbp and expressed relative to the value obtained in WT islets cultured in G10. 2.10. Statistical analysis Results are means ± SEM for at least three islets preparations. Statistical significance of differences between groups were assessed
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by two-way ANOVA followed by a post-test of Bonferroni, unless specified otherwise. Differences were considered significant if P < 0.05. 3. Results 3.1. Impact of NOX2 deficiency on glucose-induced insulin secretion in mouse islets We first confirmed the effect of NOX2 inactivation on the glucose stimulation of mitochondrial metabolism, intracellular Ca2þ concentration ([Ca2þ]i), and insulin secretion in islets from male C57BL/6J mice. As reported previously (Li et al., 2012), after 1e2 days preculture, GSIS was significantly larger in NOX2-KO vs. WT islets incubated at 20 and 30 mmol/l glucose despite their almost identical insulin content and glucose-induced rises in NAD(P)H autofluorescence and [Ca2þ]i (Fig. 1). As cytosolic superoxide can be rapidly converted to H2O2 by superoxide dismutase 1, these results confirm that NOX2-derived superoxide and hydrogen peroxide are not positively involved in acute GSIS. 3.2. Lack of impact of NOX2 deficiency on cytosolic glutathione redox state in mouse islets NOX2-deficiency is expected to reduce superoxide/H2O2 production while slightly reducing NADPH consumption in the cytosol. To test whether NOX2-deficiency impacts the cytosolic thiol redox state in b-cells, we used GRX1-roGFP2, a rapid sensor of changes in GSH/GSSG ratio (glutathione redox potential, or EGSH)
Fig. 1. Acute GSIS in WT and NOX2-KO islets - A-B, after 1e2 days preculture at G10, both groups of islets were simultaneously stimulated with increasing glucose concentrations (Gn in mmol/l, as indicated at the top of each graph) during perifusion with normal Krebs medium containing 4.8 mmol/l Kþ. The experiments ended by addition of 10 mmol/l FCCP (A) or depolarization with 30 mmol/l Kþ in the presence of 250 mmol/l diazoxide (K30Dz) (B). NAD(P)H fluorescence was expressed in percentage of the level measured at the end of FCCP treatment. C-D, after 1e2 days preculture at G10, batches of 5 islets were pre-incubated 40 min in G0.5 before 1 h incubation at various glucose concentrations (total of 9 batches of 5 islets per condition). The data were normalized to the DNA content of each batch of islets. D, insulin to DNA content ratio from incubated islets. Results are means ± SE for 3 experiments. *, P < 0.05 for the glucose effects within each group (vs. the previous condition in A-B or vs. G0.5 in C); #, P < 0.05; ##, P < 0.01 for differences between WT vs. NOX2 KO islets.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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Fig. 2. Acute effects of glucose on cytosolic GRX1-roGFP2 fluorescence ratio in WT and NOX2 KO islets - A-B, after 2e4 days culture at G10, islets expressing GRX1-roGFP2 were perifused with Krebs medium containing different glucose concentrations (Gn in mmol/l) in the absence (A) or presence (B) of exogenous H2O2. Each experiment ended by sequential addition of 10 mmol/l DTT and 100 mmol/l aldrithiol (AT-2) for normalization of the traces. Results are means ± SE for 3 experiments. *, significantly different (P < 0.05 in A and P < 0.01 in B) vs. previous condition within each group. There were no significant differences between the two types of islets.
(Gutscher et al., 2008) and tested the response of the probe to acute changes in glucose concentration from 10 to 0 then 30 mmol/l. Under control conditions, omission of glucose did not significantly affect GRX1eroGFP2 fluorescence ratio, but stimulation from 0 to 30 mmol/l glucose acutely reduced the probe, hence cytosolic EGSH (Fig. 2A), in agreement with recent data obtained in re-aggregated human islets (Ferdaoussi et al., 2015). In comparison, when the probe was oxidized by a constant supply of exogenous H2O2, omission of glucose further increased the probe oxidation, an effect that was reversed upon stimulation with 30 mmol/l glucose (Fig. 2B) (Takahashi et al., 2014). However, in contrast to our initial expectations, the glucose responses in the absence or presence of exogenous H2O2 were similar in NOX2-KO and WT islets. These results suggest that NOX2 does not participate in the acute glucose regulation of cytosolic EGSH in these islets. 3.3. Lack of impact of NOX2 deficiency on cytosolic thiol oxidation in cultured mouse islets We next used cytosolic roGFP1 to measure the level of cytosolic thiol oxidation after culture under control and glucotoxic conditions. As shown in Fig. 3A, overnight culture in 30 instead of 10 mmol/l glucose significantly increased roGFP1 fluorescence ratio in mouse islets, likely reflecting an increase in cytosolic thiol
Fig. 3. Effects of glucose and NOX2 deficiency on cytosolic roGFP1 and roGFP2Orp1 fluorescence ratio in cultured mouse islets - After 2e4 days culture at G10, WT and NOX2-KO mouse islets expressing roGFP1 (A) or roGFP2-Orp1 (B) were cultured overnight at G10 or G30, as indicated on the X-axis. Then islets were perifused for 20 min with a Krebs solution containing the same glucose concentration as during culture before sequential application of 10 mmol/l DTT and 100 mmol/l aldrithiol (AT-2) for normalization of the traces. Results are means ± SE for 3 to 5 experiments. *, significant effect of glucose within each group (P < 0.001). There were no significant differences between the two types of islets.
oxidation. However, this effect was not different in NOX2-KO vs. WT islets. Similar results were obtained in islets expressing roGFP2Orp1, a probe that specifically reacts with H2O2 (Albrecht et al., 2011) (Fig. 3B). These results suggest that, although prolonged exposure to high vs. intermediate glucose concentrations increased cytosolic thiol oxidation and H2O2 concentration in islet cells, this effect did not depend on NOX2 expression.
3.4. Lack of impact of NOX2 deficiency on the glucotoxic alterations of b-cell function in cultured mouse islets Prolonged exposure to high glucose concentrations induces bcell dysfunction and apoptosis, thereby contributing to the gradual decrease in functional b-cell mass in T2D patients. Human and rat islets cultured in high glucose for 1 week exhibit b-cell dysfunction and apoptosis, but an associated increase in mitochondrial oxidative stress was only documented in rat islets (Duprez et al., 2012; Brun et al., 2015). However, in mouse islets, glucotoxicity and cell apoptosis can only be seen after 3 weeks of culture (Pascal et al., 2010). In agreement with previous observations that culture at G10 optimally preserves rodent b-cell function and survival, WT islets cultured for 1 or 3 weeks at G10 displayed similar rises in NAD(P)H autofluorescence, [Ca2þ]i and insulin secretion upon acute stepwise glucose stimulation (Fig. 4A, C, E and 5A, C, E). The islet insulin content ratio was, however, approximately reduced by 50% after 1 or 3 weeks of culture as compared with islets cultured for 1 or 2 days (compare Figs. 4E and 5E with Fig. 1D; see also ESM table 4 for details on the islet insulin to DNA content ratio under each condition tested). These responses were almost identical in islets from NOX2-KO mice, except for the ~1.4-fold larger insulin secretion in NOX2-KO islets incubated for 1 h at 30 mmol/l glucose after 1 week culture in G10 (Fig. 4E). In agreement with previous studies in mouse islets (Pascal et al., 2008, 2010), 1e3 weeks of culture in G30 markedly increased the glucose sensitivity of WT islets upon subsequent glucose stimulation, as shown by the shift to the left of the glucose concentrationresponse curves for changes in NAD(P)H autofluorescence, [Ca2þ]i and insulin secretion. (Figs. 4 and 5). It also reduced the maximal amplitude of glucose-stimulated [Ca2þ]i rise. However, the maximal secretory response to glucose was not reduced. These responses were almost identical in NOX2-KO islets (Suppl. tables 13), suggesting that NOX2 does not contribute to the alterations of bcell stimulus secretion coupling by prolonged culture at high vs. intermediate glucose concentration in male C57BL/6J mouse islets.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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3.5. Lack of impact of NOX2 deficiency on the glucotoxic alteration of b-cell survival in cultured mouse islets Despite the lack of impact of NOX2 deficiency on the glucotoxic alterations of stimulus-secretion coupling events, the small, albeit not significant, reduction in cytosolic thiol oxidation in NOX2-KO vs. WT islets led us to test the effects of 3 weeks of culture at G10 or G30 on b-cell apoptosis in both islet types. In agreement with previous studies in rat and mouse islets (Jonas et al., 2009), the level of islet cell DNA fragmentation and b-cell apoptosis were minimum after prolonged culture at G10, with less than 2% of apoptotic b-cells
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in both groups of islets (Fig. 6AeB and ESM Fig. 2). In comparison, there was a significant increase in islet DNA fragmentation (~5fold) and b-cell apoptosis (~4-fold) after 3 weeks of culture at G30 vs. G10, confirming the glucotoxic stimulation of b-cell apoptosis. These changes were not associated with changes in the proportion of b-cells among islet cells, as measured by immunohistochemistry on islet sections (Fig. 6C). Again, NOX2 deficiency did not significantly alter islet DNA fragmentation and b-cell apoptosis after culture at either G10 or G30, excluding NOX2 as a critical regulator of mouse pancreatic b-cell survival during culture under control and glucotoxic conditions (Fig. 6 and ESM Fig. 2).
Fig. 4. Lack of impact of NOX2 deficiency on islet glucose responsiveness after 1 week culture in high glucose e After 1e3 days culture at G10, mouse islets were cultured for 1 week at G10 or G30, as indicated at the top of the figure. AeD, after culture, WT and NOX2-KO islets were perifused with normal Krebs medium containing 4.8 mmol/l Kþ and increasing glucose concentrations (Gn in mmol/l, as specified in each graph) before addition of 10 mmol/l FCCP (AeB), or depolarization with 30 mmol/l Kþ in the presence of 250 mmol/l diazoxide (K30Dz) (CeD). NAD(P)H fluorescence was expressed in percentage of the level measured at the end of FCCP treatment, which triggers rapid oxidation of mitochondrial NADH by ETC complex I. E,F, after 7e10 days culture and 40 min preincubation in G0.5, islets were incubated in batches of 5 for 1 h at various glucose concentrations. The rate of insulin secretion and the islet insulin content were measured at the end of the incubation and normalized to the islet DNA content. Results are means ± SE for 3 experiments. *, P < 0.05 for the glucose effects within each group (vs. previous condition in A-D or vs. G0.5 in E-F); #, P < 0.05 for differences between WT vs. NOX2 KO islets.
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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Fig. 5. Lack of impact of NOX2 deficiency on islet glucose responsiveness after 3 weeks culture in high glucose e After 1e3 days culture at G10, mouse islets were cultured for 3 weeks at G10 or G30, as indicated at the top of the figure. AeD, after culture, WT and NOX2-KO islets were perifused with normal Krebs medium containing 4.8 mmol/l Kþ and increasing glucose concentrations (Gn in mmol/l, as specified in each graph) before addition of 10 mmol/l FCCP (AeB), or depolarization with 30 mmol/l Kþ in the presence of 250 mmol/l diazoxide (K30Dz) (CeD). NAD(P)H fluorescence was expressed in percentage of the level measured at the end of FCCP treatment. E,F, after culture and 40 min preincubation in G0.5, islets were incubated in batches of 5 for 1 h at various glucose concentrations. The rate of insulin secretion and the islet insulin content were measured at the end of the incubation and normalized to the islet DNA content. Results are means ± SE for 3 to 4 experiments. *, P < 0.05 for the glucose effects within each group (vs. previous condition in A-D or vs. G0.5 in E-F). There were no significant differences between the two types of islets.
3.6. Lack of impact of NOX2 deficiency on the glucotoxic alteration of b-cell gene mRNA levels in cultured mouse islets
result from its compensation by increased expression of another Nox isoform.
Glut2 mRNA levels are typically reduced, while Hmox1 mRNA levels are increased under glucotoxic conditions. We therefore measured the effect of 3 weeks of culture in G10 or G30 on these mRNA levels in both islet types as a further evaluation of the impact of NOX2 on b-cell glucotoxicity. As expected, Glut2 mRNA levels decreased and Hmox1 mRNA levels increased after culture in G30 vs. G10, but these effects were similar in WT and Nox2-KO islets (Fig. 7). Finally, we also measured the mRNA levels of Nox1-4. As shown in ESM table 5, Nox1-4 mRNA levels were not affected by long term culture in G30 vs. G10. In addition, Nox1, 3 and 4 mRNA levels were not detectably increased in Nox2-KO islets, showing that the lack of impact of Nox2-KO on b-cell glucotoxicity does not
4. Discussion In this study, we demonstrate that, although NOX2 deficiency increases acute GSIS by male C57BL/6J mouse islets, the enzyme does not detectably contribute to the glucotoxic induction of cytosolic thiol oxidation and rise in H2O2, b-cell dysfunction and apoptosis during prolonged cultured at 30 vs. 10 mmol/l glucose. 4.1. Short-term glucose effects Previous studies on the role of NOX in the regulation of GSIS relied on the use of chemical inhibitors (DPI or apocynin) or
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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Fig. 7. Nox2 deficiency does not affect Glut2 and Hmox1 mRNA levels after prolonged culture in the presence of intermediate or high glucose concentrations. After 1e3 days preculture at G10, mouse islets were cultured for 3 weeks at G10 or G30. Changes in gene/Tbp mRNA ratio for (A) Glut2 and (B) Hmox1. Results are means ± SE from 3 to 4 experiments. *, P < 0.05; **,P < 0.001 for the effect of glucose. There were no significant differences between the two types of islets.
Fig. 6. NOX2 deficiency does not affect islet cell apoptosis after prolonged culture at intermediate or high glucose concentrations. After 1e3 days preculture at G10, mouse islets were cultured for 3 weeks at G10 or G30, as indicated on the X-axis. A, DNA fragmentation was expressed relative to the value measured in WT islets cultured in G10. B, percentage of apoptotic b-cells (TUNEL-positive/DAPI-positive nuclei in insulin-positive cells). C, percentage of b -cells in islet sections. Results are means ± SE for 3 experiments (AeC) plus data from each islet section analysed (B and C). *, P < 0.05; **, P < 0.001 for the glucose effects within each group. There were no significant differences between the two types of islets.
play a positive role in acute GSIS can be excluded in this experimental model. The negative effect of NOX2 on GSIS has been attributed to superoxide-mediated inactivation of cAMP/protein kinase A signalling and, hence, reduced amplification of insulin secretion (Li et al., 2012). In that study, superoxide-mediated nitroblue tetrazolium (NBT) and dihydroethidine (DHE) oxidation was ~50% lower in NOX2-KO vs. WT islets incubated at high glucose (Li et al., 2012). However, although cytosolic GRX1-roGFP2 can be indirectly oxidized by menadione that triggers superoxide production (Takahashi et al., 2014; Chuang et al., 2002), cytosolic EGSH did not differ between islet types under any condition tested. These results are not necessarily incompatible, for several reasons. First, NBT and DHE could be oxidized in the extracellular space within islets, whereas GRX1-roGFP2 only senses what happens in the compartment where it is expressed. Second, in contrast with NBT and DHE that are not reduced after their oxidation by superoxide, changes in GRX1-roGFP2 fluorescence ratio monitor changes in EGSH that result from the dynamic equilibrium between glutathione oxidation by ROS and electrophiles and its reduction by glutathionereductase using NADPH as a cofactor (Schwarzl€ ander et al., 2016). Finally, GRX1-roGFP2 measurements could miss EGSH variations in sub-regions of b-cells, e.g. in the population of vesicles containing endosomes/lysosomes and insulin granules in which NOX2 was shown to reside in human b-cells (Li et al., 2012). The acute glucosemediated reduction of GRX1-roGFP2 fluorescence ratio we observed therefore does not exclude a hypothetical increase in ROS production at high glucose, but indicates that it is more than compensated for by a concomitant increase in glutathione reductive capacity.
4.2. Long-term glucose effects silencing p47phox (Imoto et al., 2008; Morgan et al., 2009), a regulatory protein that plays a pivotal role in NOX2 activation (Quinn and Gauss, 2004). In these studies, NOX2 inhibition led to a decrease in acute GSIS that was attributed to alterations of glucoseinduced rise in [Ca2þ]i, suggesting a positive role of superoxide in GSIS (Imoto et al., 2008; Morgan et al., 2009). In this study, however, we confirm the observation by Li and colleagues (Li et al., 2012) that NOX2 deficiency increases GSIS during short-term incubation at or above 20 mmol/l glucose without affecting the islet insulin content nor the glucose-induced rises in [Ca2þ]i. Our results thereby strengthen the view that NOX2 is a negative modulator of maximal GSIS acting at a step distal to Ca2þ influx (Li et al., 2012). Thus, the hypothesis that NOX2-derived superoxide and H2O2 may
We previously showed that prolonged culture at G30 vs. G10 induces b-cell apoptosis, alterations of stimulus-secretion coupling events, impaired GSIS and increased mitochondrial oxidative stress markers in rodent pancreatic islets (Bensellam et al., 2009; Pascal et al., 2010; Duprez et al., 2012). However, antioxidant agents able to reduce mitochondrial oxidative stress improved b-cell survival without restoring GSIS, suggesting that the latter is more sensitive to low levels of oxidative stress, or that it resulted from redox imbalance in other cell compartments, e.g. in the cytosol (Duprez et al., 2012). In this context, it was previously suggested that elevated NOX activity under high glucose conditions might be a mechanism of b-cell oxidative stress in diabetes (Newsholme et al., 2009).
Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022
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In the present study, the increase of both cytosolic thiol oxidation and H2O2 concentration after prolonged culture at G30 vs. G10 was followed by glucotoxic alterations of b-cell stimulussecretion coupling events (although maximal GSIS was not reduced for an unclear reason) and increased b-cell apoptosis. These data suggest that cytosolic redox imbalance could contribute to oxidative stress induced by high glucose in mouse islets. However, although NOX2 deficiency increased acute GSIS in islets cultured for a few days under control conditions (see previous section), it failed to reduce the glucose-induced oxidation of roGFP1 and roGFP2-Orp1 or the glucotoxic alterations of b-cell survival and function after long-term culture at G30. The lack of protective effect of NOX2 deficiency on the oxidation of roGFP-derived probes contrasts with a recent report showing that chemical inhibition of NOX2 with gp91-ds-tat reduced the stimulation of DCF oxidation and NOX2 activity (measured by luminescence assay) by 20 mmol/l glucose in lysates of INS-1 832/13, or INS-1 subclone 832/13 cells (Sidarala et al., 2015). Again, it should be stressed that these measurements were done at the whole cell levels and that the fluorescence ratio of roGFPderived probes is not only influenced by ROS but also by the reductive capacity of NADPH-dependent antioxidant enzymes. In this context, our data on NAD(P)H autofluorescence do not allow to conclude whether cytosolic NADPH was higher in Nox2-KO vs. WT islets, but the lack of differences in the oxidation of GRX1roGFP2 between both islets types does not support that hypothesis. Nevertheless, our results show that NOX2-derived superoxide does not detectably contribute to cytosolic thiol/ glutathione oxidation and H2O2 concentration in intact living islet cells, nor to the glucotoxic alterations of b-cell function and survival. Our results, however, do not rule out the possibility that ETC complex III-mediated superoxide production in the mitochondrial intermembrane space could contribute to cytosolic oxidative stress and b-cell glucotoxicity. In addition, we do not exclude the possibility that NOX2 could play a role in in vivo bcell dysfunction and apoptosis in T2D (Cnop et al., 2005), as chronic exposure to elevated levels of free fatty acids (FFAs) have been shown to increase the expression and activity of NOX enzymes in insulin-secreting cells and rat islets (Yuan et al., 2010; Morgan et al., 2007). 4.3. A note of caution One possible limitation of this study lies in the C57BL/6J genetic background of NOX2-KO mice and their WT controls. This strain of mice, which is widely used in transgenesis, indeed expresses an inactive form of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial matrix enzyme involved in NADPH production. As NNT inactivation was previously found to aggravate the phenotype of superoxide dismutase 2 KO mice (Huang et al., 2006), it remains to be seen if NOX2 deficiency has the same effect on GSIS and lack of contribution to b-cell glucotoxicity when expressed in mice that express functional NNT. Also, the changes in fluorescence ratio of roGFP-derived probes shown in this paper have to be taken as representative only of what happens in the absence of functional NNT, as we recently showed that NNT has a large impact on the glucose regulation of mitochondrial EGSH, hence to a smaller extent on cytosolic EGSH as well (Santos et al., 2015). 5. Conclusion Long-term culture of mouse islets at high vs. intermediate glucose concentration triggers cytosolic redox imbalance that may contribute to the late impairment of b-cell function and survival. NOX2-containing NADPH oxidase is a negative modulator of GSIS
during short-term incubation at 20e30 mmol/l glucose, but this effect is independent from detectable alterations in cytosolic thiol or glutathione redox state. Finally, NOX2 activation is not involved in the alterations of b-cell function and survival after long-term culture at high vs. intermediate glucose concentrations. Acknowledgments We thank Dr Tobias Dick (German Cancer Research Center, Heidelberg, Germany) for providing the plasmid coding roGFP2Orp1. We thank Denis Charlier and Fabien Knockaert for expert technical help. We also thank Heeyoung Chae for help in islet isolation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.mce.2016.09.022. Funding ~o de Amparo a Pesquisa do AHS was recipient of a Fundaça Estado de S~ ao Paulo (Scholarship FAPESP 2011/21299-9) and Bolsa gio de Pesquisa no Exterior (Scholarship BEPE/FAPESP 2013/ Esta 18232-5). MB is recipient of a “MOVE-IN Louvain” incoming Postdoc fellowship. JCJ is Research Director of the Fonds de la Recherche Scientifique-FNRS, Brussels, Belgium. es 12/ This study was funded by Actions de Recherche Concerte française de Belgique and Grant 17-047 from the Communaute dicale to 3.4521.12 from the Fonds de la Recherche Scientifique Me JCJ. Author's contributions AHS, LPR, ARC and JCJ conceived and designed the experiments. AHS, LPR, LRBS and MB performed the experiments and analysed the data. AHS and JCJ wrote the paper. All other coauthors edited and approved the paper. References Albrecht, S.C., Barata, A.G., Grosshans, J., Teleman, A.A., Dick, T.P., 2011. In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis. Cell Metab. 14 (6), 819e829. €m, P., Walum, E., Welsh, N., 2015. The novel NADPH oxidase 4 Anvari, E., Wikstro inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice. Free Radic. Res. 49 (11), 1308e1318. Bensellam, M., Van Lommel, L., Overbergh, L., Schuit, F.C., Jonas, J.C., 2009. Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations. Diabetologia 52 (3), 463e476. Bensellam, M., Laybutt, D.R., Jonas, J.C., 2012. The molecular mechanisms of pancreatic b-cell glucotoxicity: recent findings and future research directions. Mol. Cell Endocrinol. 364 (1e2), 1e27. Brun, T., Li, N., Jourdain, A.A., Gaudet, P., Duhamel, D., Meyer, J., Bosco, D., Maechler, P., 2015. Diabetogenic milieus induce specific changes in mitochondrial transcriptome and differentiation of human pancreatic islets. Hum. Mol. Genet. 24 (18), 5270e5284. Chuang, Y.Y., Chen, Y., Gadisetti, Chandramouli, V.R., Cook, J.A., Coffin, D., Tsai, M.H., DeGraff, W., Yan, H., Zhao, S., Russo, A., Liu, E.T., Mitchell, J.B., 2002. Gene expression after treatment with hydrogen peroxide, menadione, or t-butyl hydroperoxide in breast cancer cells. Cancer Res. 62 (21), 6246e6254. € rns, A., Lenzen, S., Eizirik, D.L., 2005. Mechanisms Cnop, M., Welsh, N., Jonas, J.C., Jo of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities. Diabetes 54 (2), S97eS107. Dooley, C.T., Dore, T.M., Hanson, G.T., Jackson, W.C., Remington, S.J., Tsien, R.Y., 2004. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 279 (21), 22284e22293. Duprez, J., Roma, L.P., Close, A.F., Jonas, J.C., 2012. Protective antioxidant and antiapoptotic effects of ZnCl2 in rat pancreatic islets cultured in low and high glucose concentrations. PloS one 7 (10), e46831. Ferdaoussi, M., Dai, X., Jensen, M.V., Wang, R., Peterson, B.S., Huang, C., Ilkayeva, O., Smith, N., Miller, N., Hajmrle, C., Spigelman, A.F., Wright, R.C., Plummer, G.,
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Please cite this article in press as: de Souza, A.H., et al., NADPH oxidase-2 does not contribute to b-cell glucotoxicity in cultured pancreatic islets from C57BL/6J mice, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.022