Transient Oxidative Stress Damages Mitochondrial Machinery Inducing Persistent β-Cell Dysfunction
2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês
10.1074/jbc.m109.024323
ISSN1083-351X
AutoresNing Li, Thierry Brun, Miriam Cnop, Daniel A. Cunha, Décio L. Eizirik, Pierre Maechler,
Tópico(s)Birth, Development, and Health
ResumoTransient exposure of β-cells to oxidative stress interrupts the transduction of signals normally coupling glucose metabolism to insulin secretion. We investigated putative persistence of effects induced by one transient oxidative stress (200 μm H2O2, 10 min) on insulin secreting cells following recovery periods of days and weeks. Three days after oxidative stress INS-1E cells and rat islets exhibited persistent dysfunction. In particular, the secretory response to 15 mm glucose was reduced by 40% in INS-1E cells stressed 3 days before compared with naïve cells. Compared with non-stressed INS-1E cells, we observed reduced oxygen consumption (−43%) and impaired glucose-induced ATP generation (−46%). These parameters correlated with increased mitochondrial reactive oxygen species formation (+60%) accompanied with down-regulation of subunits of the respiratory chain and decreased expression of genes responsible for mitochondrial biogenesis (TFAM, −24%; PGC-1α, −67%). Three weeks after single oxidative stress, both mitochondrial respiration and secretory responses were recovered. Moreover, such recovered INS-1E cells exhibited partial resistance to a second transient oxidative stress and up-regulation of UCP2 (+78%) compared with naïve cells. In conclusion, one acute oxidative stress induces β-cell dysfunction lasting over days, explained by persistent damages in mitochondrial components. Transient exposure of β-cells to oxidative stress interrupts the transduction of signals normally coupling glucose metabolism to insulin secretion. We investigated putative persistence of effects induced by one transient oxidative stress (200 μm H2O2, 10 min) on insulin secreting cells following recovery periods of days and weeks. Three days after oxidative stress INS-1E cells and rat islets exhibited persistent dysfunction. In particular, the secretory response to 15 mm glucose was reduced by 40% in INS-1E cells stressed 3 days before compared with naïve cells. Compared with non-stressed INS-1E cells, we observed reduced oxygen consumption (−43%) and impaired glucose-induced ATP generation (−46%). These parameters correlated with increased mitochondrial reactive oxygen species formation (+60%) accompanied with down-regulation of subunits of the respiratory chain and decreased expression of genes responsible for mitochondrial biogenesis (TFAM, −24%; PGC-1α, −67%). Three weeks after single oxidative stress, both mitochondrial respiration and secretory responses were recovered. Moreover, such recovered INS-1E cells exhibited partial resistance to a second transient oxidative stress and up-regulation of UCP2 (+78%) compared with naïve cells. In conclusion, one acute oxidative stress induces β-cell dysfunction lasting over days, explained by persistent damages in mitochondrial components. Pancreatic β-cells are poised to sense blood glucose to regulate insulin exocytosis and thereby glucose homeostasis. The conversion from metabolic signals to secretory responses is mediated through mitochondrial metabolism (1.Maechler P. Cell Mol. Life Sci. 2002; 59: 1803-1818Crossref PubMed Scopus (65) Google Scholar). Failure of the insulin secreting β-cells, a common characteristic of both type 1 and type 2 diabetes, derives from various origins, among them mitochondrial impairment secondary to oxidative stress is a proposed mechanism (2.Green K. Brand M.D. Murphy M.P. Diabetes. 2004; 53: S110-S118Crossref PubMed Google Scholar). Oxidative stress is characterized by a persistent imbalance between excessive production of reactive oxygen species (ROS) 3The abbreviations used are: ROSreactive oxygen speciesSODsuperoxide dismutaseCATcatalaseGPxglutathione peroxidasemtDNAmitochondrial DNABSAbovine serum albuminFCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazonePBSphosphate-buffered salineTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingBrdUrdbromodeoxyuridineΔΨmmitochondrial membrane potential.3The abbreviations used are: ROSreactive oxygen speciesSODsuperoxide dismutaseCATcatalaseGPxglutathione peroxidasemtDNAmitochondrial DNABSAbovine serum albuminFCCPcarbonyl cyanide p-trifluoromethoxyphenylhydrazonePBSphosphate-buffered salineTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingBrdUrdbromodeoxyuridineΔΨmmitochondrial membrane potential. and limited antioxidant defenses. Examples of ROS include superoxide (O2−̇), hydroxyl radical (OH⋅), and hydrogen peroxide (H2O2). Superoxide can be converted to less reactive H2O2 by superoxide dismutase (SOD) and then to oxygen and water by catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxin, which constitute antioxidant defenses. Increased oxidative stress and free radical damages have been proposed to participate in the diabetic state (3.Yu B.P. Physiol. Rev. 1994; 74: 139-162Crossref PubMed Scopus (2186) Google Scholar). In type 1 diabetes, ROS are implicated in β-cell dysfunction caused by autoimmune reactions and inflammatory cytokines (4.Rabinovitch A. Diabetes Metab. Rev. 1998; 14: 129-151Crossref PubMed Scopus (415) Google Scholar). In the context of type 2 diabetes, excessive ROS could promote deficient insulin synthesis (5.Evans J.L. Goldfine I.D. Maddux B.A. Grodsky G.M. Endocr. Rev. 2002; 23: 599-622Crossref PubMed Scopus (1716) Google Scholar, 6.Robertson R.P. Harmon J. Tran P.O. Tanaka Y. 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However, uncontrolled increase of oxidants, or reduction of their detoxification, leads to free radical-mediated chain reactions ultimately triggering pathogenic events. Pancreatic β-cells are relatively weak in expressing free radical-quenching enzymes SOD, CAT, and GPx (10.Tiedge M. Lortz S. Drinkgern J. Lenzen S. Diabetes. 1997; 46: 1733-1742Crossref PubMed Google Scholar, 11.Welsh N. Margulis B. Borg L.A. Wiklund H.J. Saldeen J. Flodström M. Mello M.A. Andersson A. Pipeleers D.G. Hellerström C. Mol. Med. 1995; 1: 806-820Crossref PubMed Google Scholar), rendering those cells particularly susceptible to oxidative attacks (12.Maechler P. Jornot L. Wollheim C.B. J. Biol. Chem. 1999; 274: 27905-27913Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Mitochondria are not only the main source of cellular oxidants, they are also the primary target of ROS (13.Turrens J.F. J. Physiol. 2003; 552: 335-344Crossref PubMed Scopus (3415) Google Scholar, 14.Li N. Frigerio F. Maechler P. Biochem. Soc Trans. 2008; 36: 930-934Crossref PubMed Scopus (82) Google Scholar). reactive oxygen species superoxide dismutase catalase glutathione peroxidase mitochondrial DNA bovine serum albumin carbonyl cyanide p-trifluoromethoxyphenylhydrazone phosphate-buffered saline terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling bromodeoxyuridine mitochondrial membrane potential. reactive oxygen species superoxide dismutase catalase glutathione peroxidase mitochondrial DNA bovine serum albumin carbonyl cyanide p-trifluoromethoxyphenylhydrazone phosphate-buffered saline terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling bromodeoxyuridine mitochondrial membrane potential. Mitochondria are essential for pancreatic β-cell function, and damages to these organelles are well known to markedly alter glucose-stimulated insulin secretion (15.Maechler P. de Andrade P.B. Biochem. Soc Trans. 2006; 34: 824-827Crossref PubMed Scopus (33) Google Scholar). The mitochondrial genome constitutes one of the targets, encoding for 13 polypeptides essential for the integrity of electron transport chain (16.Wallace D.C. Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2578) Google Scholar). Damages to mitochondrial DNA (mtDNA) induce mutations that in turn may favor ROS generation, although the contribution of mtDNA mutations to ROS generation remains unclear. We previously reported that patient-derived mitochondrial A3243G mutation, causing mitochondrial inherited diabetes, is responsible for defective mitochondrial metabolism associated with elevated ROS levels and reduced antioxidant enzyme expression (17.de Andrade P.B. Rubi B. Frigerio F. van den Ouweland J.M. Maassen J.A. Maechler P. Diabetologia. 2006; 49: 1816-1826Crossref PubMed Scopus (69) Google Scholar). On the other hand, mtDNA mutator mice exhibit accelerated aging without changes in superoxide levels in embryonic fibroblasts (18.Trifunovic A. Hansson A. Wredenberg A. 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Turnbull D.M. Curr. Opin. Clin. Nutr. Metab. Care. 2000; 3: 473-478Crossref PubMed Scopus (48) Google Scholar). These age-related mitochondrial changes are foreseen to play a role in the late onset diabetes. In a rat model of intrauterine growth retardation, a vicious cycle between accumulation of mtDNA mutations and elevation of ROS production has been associated to β-cell abnormalities and the onset of type 2 diabetes in adulthood (23.Simmons R.A. Suponitsky-Kroyter I. Selak M.A. J. Biol. Chem. 2005; 280: 28785-28791Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Similarly, mitochondrion-derived ROS impair β-cell function in the Zucker diabetic fatty rat (24.Bindokas V.P. Kuznetsov A. Sreenan S. Polonsky K.S. Roe M.W. Philipson L.H. J. Biol. Chem. 2003; 278: 9796-9801Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Altogether, these observations point to ROS action as a triggering event inducing mitochondrial dysfunction and ultimately resulting in the loss of the secretory response in β-cells (14.Li N. Frigerio F. Maechler P. Biochem. Soc Trans. 2008; 36: 930-934Crossref PubMed Scopus (82) Google Scholar). In vitro, oxidative stress applied to β-cells rapidly interrupts the transduction of signals normally coupling glucose metabolism to insulin secretion (12.Maechler P. Jornot L. Wollheim C.B. J. Biol. Chem. 1999; 274: 27905-27913Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 25.Krippeit-Drews P. Kramer C. Welker S. Lang F. Ammon H.P. Drews G. J. Physiol. 1999; 514: 471-481Crossref PubMed Scopus (96) Google Scholar). Specifically, we reported that INS-1E β-cells and rat islets subjected to a 10-min H2O2 exposure exhibit impaired secretory response associated with mitochondrial dysfunction appearing already during the first minutes of oxidative stress (12.Maechler P. Jornot L. Wollheim C.B. J. Biol. Chem. 1999; 274: 27905-27913Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). In the context of the mitochondrial theory of aging (21.Harman D. J. Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6326) Google Scholar, 26.Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (464) Google Scholar), it is important to know whether transient exposure to H2O2 could possibly induce persistent modifications of mitochondrial function. Cells surviving an oxidative stress might carry defects leading to progressive loss of β-cell function. In the present study, we asked the simple but unanswered question if a short transient oxidative stress could induce durable alterations of the mitochondria and thereby chronically impair β-cell function. INS-1E β-cells and rat islets were transiently exposed to H2O2 for 10 min and analyzed after days and weeks of standard tissue culture. INS-1E cells were used as a well differentiated clone derived from rat insulinoma INS-1 cells and cultured as detailed previously (26.Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (464) Google Scholar). Pancreatic islets were isolated by collagenase digestion from Wistar rats weighting 200–250 g, hand-picked and cultured free-floating in RPMI 1640 medium before experiments (27.Carobbio S. Ishihara H. Fernandez-Pascual S. Bartley C. Martin-Del-Rio R. Maechler P. Diabetologia. 2004; 47: 266-276Crossref PubMed Scopus (76) Google Scholar). INS-1E cells were seeded in Falcon (OmniLab, Mettmenstetten, Switzerland) multiwell plates and Petri dishes treated with polyornithine (Sigma-Aldrich). After 2 days of culture, cells were exposed to 200 μm H2O2 (Sigma-Aldrich) and 10 min later 100 units/ml CAT (Sigma-Aldrich) were added to neutralize H2O2. The 200 μm concentration of H2O2 was selected according to: (i) the dose-response performed previously showing direct effects above 100 μm (12.Maechler P. Jornot L. Wollheim C.B. J. Biol. Chem. 1999; 274: 27905-27913Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar); (ii) the dose-response performed on INS-1E cells analyzed 3 days after the 10-min oxidative stress (see supplemental Figs. S1 and S2). Following the H2O2 and CAT treatment, cells were further cultured in normal complete media and, where indicated, passaged once a week until analysis. Control cells were cultured for the same period of time as stressed cells. Regarding primary cells, rat pancreatic islets were isolated, exposed to oxidative stress as described above, and cultured before hand-picking for analyses. Insulin secretion assay was performed as detailed previously (26.Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (464) Google Scholar). In brief, cells were preincubated in glucose-free Krebs-Ringer bicarbonate HEPES buffer (KRBH, in mM: 135 NaCl, 3.6 KCl, 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, 1.5 CaCl2, 10 HEPES, pH 7.4) containing 0.1% bovine serum albumin (KRBH/BSA). Next, cells were incubated for 30 min at 37 °C in KRBH/BSA containing basal 2.5 mm or stimulatory 15 mm glucose concentrations. Non-nutrient-stimulated secretion was induced by 30 mm KCl (at 2.5 mm glucose). Cultured rat islets were hand-picked for either static insulin secretion or perifusion experiments. For perifusion protocols, cultured islets were placed by 10 in chambers of 250-μl volume (Brandel, Gaithersburg, MD), and then perifused at a flow rate of 1 ml/min (27.Carobbio S. Ishihara H. Fernandez-Pascual S. Bartley C. Martin-Del-Rio R. Maechler P. Diabetologia. 2004; 47: 266-276Crossref PubMed Scopus (76) Google Scholar) with KRBH/BSA medium containing sequentially basal 2.8 mm glucose followed by stimulatory 16.7 mm glucose. Insulin was collected, and insulin levels were determined by radioimmunoassay (Linco, St. Charles, MO). The mitochondrial membrane potential (ΔΨm) was measured as described (26.Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (464) Google Scholar) in INS-1E cells loaded with Rhodamine-123 (Molecular Probes, Eugene, OR) and monitored in a plate-reader fluorometer (Fluostar Optima, BMG Lab Technologies, Offenburg, Germany). At indicated time points, glucose was raised to 15 mm, and then 1 μm of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, Sigma-Aldrich) was added. Cellular ATP levels were monitored in cells expressing the ATP-sensitive bioluminescent probe luciferase after transduction with the viral construct AdCAG-Luc the day before measurements (28.Ishihara H. Maechler P. Gjinovci A. Herrera P.L. Wollheim C.B. Nat. Cell Biol. 2003; 5: 330-335Crossref PubMed Scopus (325) Google Scholar, 29.Maechler P. Wang H. Wollheim C.B. FEBS Lett. 1998; 422: 328-332Crossref PubMed Scopus (82) Google Scholar). In the presence of 100 μm luciferin, cells were stimulated with 15 mm glucose and 2 mm NaN3 was added at the end as a mitochondrial poison. Luminescence was monitored in a plate-reader luminometer (26.Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (464) Google Scholar). Total cellular ATP concentrations were determined according to manufacturer's instructions (ATP Bioluminescence Assay Kit HS II, Roche Applied Science) following cell incubation for 10 min with KRBH at 2.5 mm and 15 mm glucose. Oxygen consumption measurements were performed either on live cell suspension or isolated mitochondria. At indicated times cells were trypsinized and left to recover (60 min, 37 °C) before stimulation with 15 mm glucose. For isolated mitochondria, INS-1E cells were washed with PBS, scraped, collected in mitochondria isolation buffer (250 mm sucrose, 20 mm Tris/HCl, and 2 mm EGTA at pH 7.4) supplemented with 0.5% BSA, and pelleted by centrifugation (10 min at 1000 rpm). Pellets were then resuspended with mitochondria isolation buffer and homogenized in a 3-ml Teflon glass potter with 500 rpm rotation rate for 20 up-and-down strokes, followed by centrifugation for 8 min at 1500 × g to pull down nuclei. Supernatants were centrifuged for 10 min at 12,000 × g, and the resulting pellets were collected with respiration buffer (200 mm sucrose, 50 mm KCl, 20 mm Tris/HCl, 1 mm MgCl2, and 5 mm KH2PO4 at pH 7.0). After protein determination, 100 μg of mitochondria were used for measurements of oxygen consumption using a Clark-type electrode (Rank Brothers Ltd., Cambridge, UK). Succinate (5 mm) and 150 μm ADP-induced oxygen consumptions were performed on intact mitochondria directly after isolation. Oxygen consumption induced by NADH (5 mm) was measured in permeabilized mitochondria following snap-freezing in liquid nitrogen and thawing four times. This procedure weakened mitochondrial membrane shortly before experiments and thereby permitted uncoupled respiration measurements. Complex I activity was measured as the rate of NADH-driven electron flux through complex I using a variation of the Amplex Red assay (Molecular Probes), which detects mitochondrial H2O2 by monitoring peroxidase-catalyzed oxidation of Amplex Red to resorufin (30.Keeney P.M. Xie J. Capaldi R.A. Bennett Jr., J.P. J. Neurosci. 2006; 26: 5256-5264Crossref PubMed Scopus (556) Google Scholar). Mitochondria were isolated and resuspended with respiration buffer as described above for oxygen consumption. Mitochondria were then snap-frozen in liquid nitrogen and thawed four times for permeabilization. Mitochondrial samples at a final protein concentration of 400 μg/ml were supplemented with SOD1 (40 units/ml, Sigma-Aldrich), horseradish peroxidase (0.1 unit/ml, Molecular Probes), and Amplex Red (50 μm, Molecular Probes). The reaction mixture in Krebs-Ringer Phosphate (KRPG, 145 mm NaCl, 5.7 mm Na2HPO4, 4.86 mm KCl, 0.54 mm CaCl2, 1.22 mm MgSO4, and 5.5 mm glucose at pH 7.35) with or without 5 mm NADH or 5 mm succinate was incubated at 37 °C with readings every min at 544 nm excitation and 590 nm emission. Rates of NADH-driven electron flux through complex I were calculated over the linear increasing period, and the end plateau values of each reaction were treated as NADH- or succinate-induced mitochondrial ROS generation. For negative controls of basal activity, CAT (200 units/ml) was added at the beginning of the trace. Mitochondrial isolation was performed as described above. Here, 10 mm triethanolamine and 0.1 mg/ml digitonin were added in mitochondria isolation buffer to weaken cell membrane. Resulting pellets were collected and resuspended with mitochondria buffer (250 mm sucrose, 10 mm Tris/HCl, and 0.2 mm EDTA at pH 7.8) supplemented with protease inhibitor mixture. For primary cells, 3 days after the 10-min oxidant exposure cell lysates were obtained and supplemented with protease inhibitor mixture. Protein samples (10 μg/lane) were subjected to 10–20% SDS-PAGE gradient gel before transfer onto polyvinylidene fluoride membrane. Membrane was then blocked overnight at 4 °C with 3% (w/v) gelatin protein (Top Block, Juro, Switzerland) in mitochondria PBS (1.4 mm KH2PO4, 8 mm Na2HPO4, 140 mm NaCl, and 2.7 mm KCl at pH 7.3), incubated in mitochondria PBS containing 0.6% Top block with 0.05% Tween 20 and premixed mouse monoclonal antibodies against five subunits of oxidative phosphorylation complexes (MS604 1:250; MitoSciences, Eugene, OR) at room temperature for 2 h. After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse antibody (1:2000, Amersham Biosciences), and immunoreactivity was visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce) and Molecular Imager ChemiDoc XRS system (Bio-Rad). Total RNA from INS-1E cells and rat islets was extracted using the RNeasy Mini Kit (Qiagen) and TRIzol reagent (Invitrogen), respectively, according to the manufacturer's instructions. First-strand cDNA synthesis was performed with 2 μg of RNA, reverse transcriptase (Super Script II, Invitrogen), and 1 μg of random primers (Promega, Madison, WI) (32.Boengler K. Gres P. Dodoni G. Konietzka I. Di Lisa F. Heusch G. Schulz R. J. Mol. Cell Cardiol. 2007; 43: 610-615Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Primers for NADH dehydrogenase 6 (ND6), cytochrome c oxidase I (COX I), mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor α coactivator 1 α (PGC-1α), copper/zinc (Cu/Zn)-dependent SOD (SOD1), manganese-dependent SOD (SOD2), catalase (CAT), glutathione peroxidase (GPx), uncoupling protein 2 (UCP2), were designed using the Primer Express Software (Applera Europe, Rotkreuz, Switzerland) and are listed in supplemental Table S1. Real-time PCR was performed using an ABI 7000 Sequence Detection System (Applera), and PCR products were quantified fluorometrically using the Power SYBR Green PCR Master Mix kit (Applied Biosystems). Values were normalized to the reference mRNA ribosomal protein subunit 29 (RPS29). Mitochondria were revealed by MitoTracker Orange (Molecular Probes, Eugene, OR) staining on fixed cells. Briefly, after the mentioned recovery period following transient oxidative stress, cells on polyornithine-coated glass coverslips were washed with PBS twice and incubated with 100 nm MitoTracker at 37 °C for 25 min. After another wash, cells were fixed with 4% paraformaldehyde and washed extensively before mounting on glass slides. Cells were viewed using a confocal laser scanning 410 microscope (Carl Zeiss). Data are presented as the means ± S.E. unless otherwise indicated. One-way analysis of variance and two-tailed unpaired t test were used for statistical analysis. Results were considered statistically significant at p < 0.05. Basal apoptotic rate in control INS-1E cells was ∼2%, in agreement with previous observations (31.Brun T. Duhamel D.L. Hu He K.H. Wollheim C.B. Gauthier B.R. Oncogene. 2007; 26: 4261-4271Crossref PubMed Scopus (35) Google Scholar). Single 10-min oxidative stress induced 20–25% TUNEL-positive cells 8-h post-stress, an apoptotic rate maintained throughout a 5-day period post-stress (Fig. 1A). INS-1E cells exhibited typical proliferation rate with 30% BrdUrd labeling (31.Brun T. Duhamel D.L. Hu He K.H. Wollheim C.B. Gauthier B.R. Oncogene. 2007; 26: 4261-4271Crossref PubMed Scopus (35) Google Scholar), while oxidative stress transiently decreased cell proliferation 24 h post-injury (Fig. 1B). Five days later, stressed cells exhibited higher proliferation rate compared with naïve cells, the latter proliferating at a slower pace along with increasing cell to cell contacts favored by confluency (Fig. 1B). Representative pictures are shown in supplemental Fig. S3. Insulin secretion from control INS-1E cells (Fig. 1C) evoked by 15 mm glucose was stimulated 4.8-fold versus control basal release (p < 0.005). Compared with controls, cells subjected to oxidative stress 3 days before experiments had a 110% increase in basal insulin release (p < 0.05) but did not respond to 15 mm glucose, corresponding to a 40% inhibition of the secretory response versus glucose-stimulated control cells (p < 0.05). Exocytosis evoked by 30 mm KCl, used to raise cytosolic Ca2+ independently of mitochondrial activation, was not different between the two groups. However, insulin release under basal conditions was elevated in stressed cells to levels comparable to KCl-induced responses of controls, therefore showing no further stimulation. Because it is known that, immediately after stress, H2O2 exposure raises Ca2+ to ∼400 nm (12.Maechler P. Jornot L. Wollheim C.B. J. Biol. Chem. 1999; 274: 27905-27913Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), we checked KCl responses in cells 3 days post-stress. Supplemental Fig. S4 shows efficient KCl-induced Ca2+ rise in stressed cells, suggesting impairment in the exocytotic machinery downstream of Ca2+ elevation. Five days post-stress, we observed similar inhibition of the glucose response (2.1-fold versus 5.4-fold in stressed versus control conditions, respectively) associated with elevated basal insulin release (+127% versus control, p < 0.005; Fig. 1D). These data show that the single 10-min oxidative stress induced secretory defects lasting over days. To determine whether mitochondrial morphology was conserved after transient oxidant exposure, INS-1E mitochondrial shape was visualized (Fig. 1, E and F). Control cells exhibited normal filament-like mitochondria (Fig. 1, E and F, top), whereas exposure to 200 μm H2O2 3 days before observation resulted in discontinuous mitochondrial network, eventually presenting globular patterns (Fig. 1E, bottom). Mitochondrial morphology was restored 5 days after transient oxidative stress (Fig. 1F, bottom). Mitochondrial membrane was hyperpolarized when glucose was raised from 2.5 mm to 15 mm and depolarized by the subsequent addition of 1 μm of the protonophore FCCP (Fig. 2A). In INS-1E cells stressed 3 days before by 200 μm H2O2, glucose-induced mitochondrial hyperpolarization was inhibited by 55%, and total ΔΨm revealed by FCCP was reduced by 42% compared with non-stressed controls (Fig. 2A). Exposure to 500 μm H2O2 resulted in near complete abrogation of glucose-induced hyperpolarization measured 3 days post-stress (supplemental Fig. S1). Elevation of glucose from 2.5 mm to 15 mm in control cells resulted in a sustained elevation of cytosolic ATP levels (Fig. 2B), which were disrupted by adding the mitochondrial poison NaN3. Stressed cells exhibited a 59% inhibition in glucose-induced ATP generation. Measured as absolute levels, total cellular ATP concentrations in control INS-1E cells stimulated with 15 mm glucose were increased by 31% (p < 0.01) compared with basal levels (Fig. 2C). Stressed cells exhibited lower basal (−42%, p < 0.002) and stimulated (−46%, p < 0.0002) ATP levels versus control cells. Efficient electron transfer from NADH to complex I ensures activation of mitochondrial electron transport chain resulting in hyperpolarization of ΔΨm and ATP generation. Electron transport chain complex I activity was measured using permeabilized isolated mitochondria as the rate of NADH-induced electron flux. INS-1E cells subjected to oxidative stress 3 days before analysis exhibited reduced mitochondrial complex I activity (−14%, p < 0.001, n = 7) compared with naïve cells (data not shown). Measured in cell suspension, glucose-induced O2 consumption rate was reduced in stressed cells compared with normal respiration in naïve cells (Fig. 2D). Measured in isolated mitochondria, basal respiratory rates were similar between control mitochondria and mitochondria isolated from cells transiently stressed with 200 μm H2O2 3 days before measurements. Complex I was activated by the addition of its substrate NADH (5 mm) to permeabilized mitochondria and resulted in a 5.1-fold (p < 0.01) increase in O2 consumption in control mitochondria (Fig. 2E), which was totally abolished by the application of complex I inhibitor rotenone (data not shown). Compared with controls, mitochondria isolated from stressed cells showed a 43% (p < 0.05) reduction of the respiratory rate. Next, succinate was used as complex II substrate. In control mitochondria, state 4 respiration induced by 5 mm succinate and state 3 induced by further addition of 150 μm ADP resulted in 14.3- and 31.2-fold (p < 0.01 and p < 0.001, respectively) elevations of O2 consumption, respectively (Fig. 2F). Transient exposure to oxidative stress 3 days prior to respiration measurements caused a decrease in both state 4 (−36%, p < 0.01) and state 3 (−59%, p < 0.005) respiration compared with corresponding controls. Concentrations of H2O2 lower than 200 μm (50 and 100 μm
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