Oxidative Stress Is a Mediator of Glucose Toxicity in Insulin-secreting Pancreatic Islet Cell Lines
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m307097200
ISSN1083-351X
AutoresLan Wu, Wendell E. Nicholson, Susan M. Knobel, Robert Steffner, James M. May, David W. Piston, Alvin C. Powers,
Tópico(s)Advanced Glycation End Products research
ResumoPancreatic β cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on β cells and results in β cell dysfunction and ultimately β cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to β cells, we used two model systems where glucose metabolism was increased in β cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3–20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in β cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in β cells. The level of β cell GK may also be critical in this process. Pancreatic β cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on β cells and results in β cell dysfunction and ultimately β cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to β cells, we used two model systems where glucose metabolism was increased in β cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3–20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in β cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in β cells. The level of β cell GK may also be critical in this process. Pancreatic β cells play a crucial role in maintaining glucose homeostasis through secretion of insulin in response to changes in the extracellular glucose. Glucose-stimulated insulin secretion (GSIS) 1The abbreviations used are: GSIS, glucose-stimulated insulin secretion; GK, glucokinase; HK, hexokinase; ROS, reactive oxygen species; O2·¯, superoxide; MOI, multiplicity of infection; TPEM, two-photon excitation microscopy; HE, hydroethidine; DHR, dihydrorhodamine 123; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate; MDA, malonaldehyde; 4HNE, 4-hydroxyalkenals; NIC, nicotinamide; 3AB, 3-aminobenzamide; PARP, poly(ADP-ribose) polymerase; FITC, fluorescein isothiocyanate; PI, propidium iodide; ER, endoplasmic reticulum; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. is regulated by the rate of glucose metabolism within β cells (1Meglasson M.D. Matschinsky F.M. Diabetes Metab. Rev. 1986; 2: 163-214Crossref PubMed Scopus (416) Google Scholar, 2Newgard C.B. McGarry J.D. Annu. Rev. Biochem. 1995; 64: 689-719Crossref PubMed Scopus (494) Google Scholar), and a key event in this process is the phosphorylation of glucose by glucokinase (GK). GK, also referred to as hexokinase IV, is a member of the mammalian hexokinase (HK) family that catalyzes the initial step in glucose metabolism by most metabolic pathways (e.g. glycolysis, pentose phosphate cycle and glycogen synthesis) (3Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar). In pancreatic β cells, GK assumes the rate-limiting role for glucose metabolism (4Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar). GK has a Km for glucose of ∼ 5 mm, a value that is within the physiological range of blood glucose levels and almost two orders of magnitude higher than any other HK (3Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar, 4Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar, 5Iynedjian P.B. Biochem. J. 1993; 293: 1-13Crossref PubMed Scopus (280) Google Scholar). Furthermore, the activity of GK is insensitive to feedback inhibition by physiological concentrations of its product, glucose 6-phosphate (Glc-6-P) (3Wilson J.E. Rev. Physiol. Biochem. Pharmacol. 1995; 126: 65-198Crossref PubMed Google Scholar). These characteristics of GK enable β cells to increase glucose metabolism in proportion to elevations in extracellular glucose. Subsequent to glucose phosphorylation, glucose metabolism involves both cytosolic and mitochondrial processes and generates signals leading to insulin secretion (2Newgard C.B. McGarry J.D. Annu. Rev. Biochem. 1995; 64: 689-719Crossref PubMed Scopus (494) Google Scholar). In contrast to the ability of glucose to acutely stimulate insulin secretion, chronic exposure of β cells to increased glucose concentrations results in β cell dysfunction and ultimately β cell death, a phenomenon termed β cell glucose toxicity (glucotoxicity) (6DeFronzo R.A. Diabetes Rev. 1997; 5: 177-269Google Scholar, 7Rossetti L. Giaccari A. DeFronzo R.A. Diabetes Care. 1990; 13: 610-630Crossref PubMed Scopus (895) Google Scholar, 8Robertson R.P. Harmon J. Tran P.O. Tanaka Y. Takahashi H. Diabetes. 2003; 52: 581-587Crossref PubMed Scopus (688) Google Scholar). During the progression of type 2 diabetes, glucose toxicity is likely an important factor that contributes to progressive β cell failure and development of overt diabetes. The molecular mechanisms of how chronic exposure to elevated glucose impairs β cell function and survival are incompletely understood. Emerging evidence suggests that oxidative stress contributes to β cell glucose toxicity (8Robertson R.P. Harmon J. Tran P.O. Tanaka Y. Takahashi H. Diabetes. 2003; 52: 581-587Crossref PubMed Scopus (688) Google Scholar). Cellular oxidative stress results from a persistent imbalance between antioxidant defenses and production of highly reactive molecular species, including reactive oxygen species (ROS) such as superoxide ( O2·¯) and hydrogen peroxide (H2O2) (9Rosen P. Nawroth P.P. King G. Moller W. Tritschler H.J. Packer L. Diabetes Metab. Res. Rev. 2001; 17: 189-212Crossref PubMed Scopus (813) Google Scholar). Since both O2·¯) and H2O2 are normal byproducts of cellular oxidative metabolism (10Yu B.P. Physiol. Rev. 1994; 74: 139-162Crossref PubMed Scopus (2223) Google Scholar), increased glucose metabolism could lead to excessive production of ROS. Pancreatic β cells express low levels of antioxidant enzymes and do not up-regulate these enzymes upon exposure to high concentrations of glucose (11Lenzen S. Drinkgern J. Tiedge M. Free Radic. Bio. Med. 1996; 20: 463-466Crossref PubMed Scopus (934) Google Scholar, 12Tiedge M. Lortz S. Drinkgern J. Lenzen S. Diabetes. 1997; 46: 1733-1742Crossref PubMed Google Scholar). Thus, increased ROS production in the face of low antioxidant defenses could result in ROS accumulation and oxidative stress in β cells. Elevated ROS affect the function and survival of β cells through direct oxidization of cellular macromolecules such as DNA and lipids (13Dypbukt J.M. Ankarcrona M. Burkitt M. Sjoholm A. Strom K. Orrenius S. Nicotera P. J. Biol. Chem. 1994; 269: 30553-30560Abstract Full Text PDF PubMed Google Scholar, 14Takasu N. Asawa T. Komiya I. Nagasawa Y. Yamada T. J. Biol. Chem. 1991; 266: 2112-2114Abstract Full Text PDF PubMed Google Scholar), and activation of cellular stress-sensitive signaling pathways (15Evans J.L. Goldfine I.D. Maddux B.A. Grodsky G.M. Diabetes. 2003; 52: 1-8Crossref PubMed Scopus (1198) Google Scholar). To test the hypothesis that accelerated glucose metabolism leads to oxidative stress and oxidative damage in β cells, we utilized β cell models in which glucose metabolism was increased by enhancing GK activity and exposing cells to increased concentrations of extracellular glucose. The rationale for this experimental approach is that both protein content and activity of GK in β cells are regulated by extracellular glucose and that elevated glucose concentrations enhance GK activity (16Liang Y. Najafi H. Smith R.M. Zimmerman E.C. Magnuson M.A. Tal M. Matschinsky F.M. Diabetes. 1992; 41: 792-806Crossref PubMed Scopus (147) Google Scholar, 17Sreenan S.K. Cockburn B.N. Baldwin A.C. Ostrega D.M. Levisetti M. Grupe A. Bell G.I. Stewart T.A. Roe M.W. Polonsky K.S. Diabetes. 1998; 47: 1881-1888Crossref PubMed Scopus (19) Google Scholar, 18Chen C. Hosokawa H. Bumbalo L.M. Leahy J.L. J. Clin. Investig. 1994; 94: 1616-1620Crossref PubMed Scopus (57) Google Scholar). Consistent with these observations, GK activity and glucose metabolism in β cells are increased in several animal models of type 2 diabetes (19Liang Y. Bonner-Weir S. Wu Y.J. Berdanier C.D. Berner D.K. Efrat S. Matschinsky F.M. J. Clin. Investig. 1994; 93: 2473-2481Crossref PubMed Scopus (40) Google Scholar, 20Cockburn B.N. Ostrega D.M. Sturis J. Kubstrup C. Polonsky K.S. Bell G.I. Diabetes. 1997; 46: 1434-1439Crossref PubMed Scopus (46) Google Scholar, 21Liu Y.Q. Nevin P.W. Leahy J.L. Am. J. Physiol. Endocrinol. Metab. 2000; 279: E68-E73Crossref PubMed Google Scholar, 22Chen C. Bumbalo L. Leahy J.L. Diabetes. 1994; 43: 684-689Crossref PubMed Scopus (35) Google Scholar, 23Milburn Jr., J.L. Hirose H. Lee Y.H. Nagasawa Y. Ogawa A. Ohneda M. BeltrandelRio H. Newgard C.B. Johnson J.H. Unger R.H. J. Biol. Chem. 1995; 270: 1295-1299Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Furthermore, the susceptibility to high glucose-induced β cell failure in DBA/2 mice is accompanied by enhanced GK activity and increased glucose metabolism in β cells (24Kooptiwut S. Zraika S. Thorburn A.W. Dunlop M.E. Darwiche R. Kay T.W. Proietto J. Andrikopoulos S. Endocrinology. 2002; 143: 2085-2092Crossref PubMed Scopus (74) Google Scholar). Our results show that enhanced GK activity accelerates glycolysis when the extracellular glucose is increased and that this is accompanied by accumulation of intracellular ROS, oxidative damage to mitochondria and DNA, and apoptotic cell death. These toxic effects are partially prevented by reducing ROS using a ROS scavenger. Cell Culture and GK Expression—RIN1046–38 cells were maintained in Dulbecco's modified Eagle's medium containing 25 mm glucose, 10% fetal bovine serum, penicillin, and streptomycin. The recombinant adenovirus carrying the human islet GK cDNA (AdGK) was generated by homologous recombination as previously described (25Wu L. Fritz J.D. Powers A.C. Endocrinology. 1998; 139: 4205-4212Crossref PubMed Scopus (27) Google Scholar). The recombinant adenovirus carrying the bacterial β-galactosidase gene (AdLacZ) has been described (25Wu L. Fritz J.D. Powers A.C. Endocrinology. 1998; 139: 4205-4212Crossref PubMed Scopus (27) Google Scholar). For adenoviral transduction, multiplicity of infection (MOI) was used to determine the amount of viral stock and was selected by examining GK protein expression and cell survival as a function of increasing MOI. An MOI of 20 for AdGK and AdLacZ was used throughout the study. Cells were transduced with AdGK or AdLacZ for 48 h in medium containing 3.3 mm glucose. Thereafter, cells were exposed to different concentrations of glucose for varying durations as indicated under "Results" and used for subsequent assays. The parental INS-1 cells expressing the reverse tetracycline-dependent transactivator (INS-1-r9) were kindly provided by Dr. Patrick B. Iynedjian (University of Geneva School of Medicine, Geneva, Switzerland). The plasmid expressing islet GK (PUHD10–3-GKI) was constructed by replacing rat liver GK cDNA with human islet GK cDNA in plasmid PUHD10–3-GK (26Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (117) Google Scholar) (kindly provided by Dr. Iynedjian). Stable transfection was conducted following the protocol described by Wang and Iynedjian (26Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (117) Google Scholar). Briefly, INS-1-r9 cells were co-transfected with PUHD10–3-GKI and a hygromycin resistance plasmid using LipofectAMINE reagent (Invitrogen Life Technologies). Individual hygromycin-resistant clones were screened by Southern blotting and maintained as described (26Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (117) Google Scholar). Two lines, termed INS-1-GK and INS-1-control, were used for this study. GK expression was induced by culturing cells in medium containing doxycycline and 3.3 mm glucose for 48 h. Thereafter, cells were either continuously cultured in 3.3 mm glucose or exposed to 20 mm glucose for different durations and used for subsequent assays. GK Assay—Protein levels of GK were examined by Western blot analysis and immunocytochemistry followed by flow cytometry (27Openshaw P. Murphy E.E. Hosken N.A. Maino V. Davis K. Murphy K. O'Garra A. J. Exp. Med. 1995; 182: 1357-1367Crossref PubMed Scopus (516) Google Scholar). Sheep anti-rat/human GK (a gift from Dr. Mark Magnuson at Vanderbilt University Medical Center) was used as primary antibody in these assays. Flow cytometry was carried out on a FACSCalibur flow cytometer using Cellquest v3.1 software (BD Biosiences). Cell debris was excluded, and the fluorescence intensity from GK staining was plotted on a logarithmic scale against cell number. The activity of GK was assayed in cell extracts fluorometrically by a Glc-6-P dehydrogenase-coupled assay (28Liang Y. Najafi H. Matschinsky F.M. J. Biol. Chem. 1990; 265: 16863-16866Abstract Full Text PDF PubMed Google Scholar). Protein content was determined by Bradford assay (Bio-Rad) and used to normalize the results. Glucose Metabolism and Metabolic Derivatives—The rate of glycolysis was assayed by measuring the production of [3H]water from d-[5-3H]glucose (PerkinElmer Life Science Products). Cells were incubated with d-[5-3H]glucose in the presence of different concentrations of glucose as described (26Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (117) Google Scholar). [3H]water in the supernatant was separated using borate affinity chromatography on AG 1-X8 anion exchange resin (Bio-Rad). Radioactivity in the water eluate was measured by liquid scintillation counting. Cellular NAD(P)H was assayed by measuring NAD(P)H autofluorescence in live cells using two-photon excitation microscopy (TPEM) (29Bennett B.D. Jetton T.L. Ying G. Magnuson M.A. Piston D.W. J. Biol. Chem. 1996; 271: 3647-3651Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Images of NAD(P)H autofluorescence from cell monolayers were acquired in the presence of different concentrations of glucose. Fluorescence intensity of NAD(P)H was analyzed using NIH Image 1.61 (Bethesda, MD). ATP was measured using the Bioluminescent Somatic Cell Assay Kit (Sigma). Cellular glutathione (GSH) was assayed as previously described (30Hissin P.J. Hilf R. Anal. Biochem. 1976; 74: 214-226Crossref PubMed Scopus (3699) Google Scholar, 31May J.M. Qu Z. Li X. Biochem. Pharmacol. 2001; 62: 873-881Crossref PubMed Scopus (66) Google Scholar). Insulin Secretion—INS-1 cells were either continuously cultured in 3.3 mm glucose or exposed to 20 mm glucose for 4 h after GK induction with doxycycline. GSIS was then assayed during a 60-min static incubation as described by Wang and Iynedjian (26Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (117) Google Scholar). Insulin was measured by radioimmunoassay and normalized for cellular protein (32Brissova M. Shiota M. Nicholson W.E. Gannon M. Knobel S.M. Piston D.W. Wright C.V. Powers A.C. J. Biol. Chem. 2002; 277: 11225-11232Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Intracellular ROS—Intracellular O2·¯ and H2O2 were measured in RIN 1046–38 cells using hydroethidine (HE) and dihydrorhodamine 123 (DHR) (Molecular Probes) as described (33Huang P. Feng L. Oldham E.A. Keating M.J. Plunkett W. Nature. 2000; 407: 390-395Crossref PubMed Scopus (749) Google Scholar, 34Such L. O'Connor J.E. Saez G.T. Gil F. Beltran J.F. Moya A. Alberola A. Cytometry. 1999; 37: 140-146Crossref PubMed Scopus (6) Google Scholar). HE (5 μm) was loaded for 1 h and DHR (5 μm) was loaded for 30 min before harvesting. The cells were then washed and analyzed by flow cytometry. Intracellular peroxides were detected in INS-1 cells using probe 5-(and-6)c-hloromethyl-2′,7′-dichlorodihydrofluoresceindiacetate(CM-H2DCFDA) as described (35Tanaka Y. Tran P.O. Harmon J. Robertson R.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12363-12368Crossref PubMed Scopus (266) Google Scholar). CM-H2DCFDA (5 μm) was loaded for 1 h before harvesting. The cells were then washed and analyzed by flow cytometry. Oxidative Damage—Lipid peroxidation was examined by measuring malonaldehyde (MDA) and 4-hydroxyalkenals (4HNE) using a colorimetric assay kit (LPO 586, OXIS International, Inc.). The integrity of the mitochondrial membrane was examined using rhodamine 123 (Molecular Probes) as described (33Huang P. Feng L. Oldham E.A. Keating M.J. Plunkett W. Nature. 2000; 407: 390-395Crossref PubMed Scopus (749) Google Scholar). 5 μm Rhodamine 123 was loaded for 30 min. The cells were harvested and analyzed by flow cytometry. TUNEL assay was used to detect DNA damage. DNA strand breaks were labeled using APO-DIRECT kit (BD Pharmingen) and analyzed by flow cytometry. Poly(ADP-ribose) polymerase (PARP) activity was measured using [2,8-3H]NAD+ (PerkinElmer Life Science Products) as described (36Cardinal J.W. Margison G.P. Mynett K.J. Yates A.P. Cameron D.P. Elder R.H. Mol. Cell Biol. 2001; 21: 5605-5613Crossref PubMed Scopus (58) Google Scholar). DNA content was assayed in parallel as described (37West D.C. Sattar A. Kumar S. Anal. Biochem. 1985; 147: 289-295Crossref PubMed Scopus (261) Google Scholar) and used to normalize the results. Cell Death—Cell death was detected using FITC-labeled annexin V (annexin V-FITC) and propidium iodide (PI) (ApoAlert Annexin V-FITC Apoptosis kit, Clontech). Cells were stained with annexin V-FITC/PI and analyzed by flow cytometry. Cell debris was gated out and the results were displayed as 2-parameter dot plots with the X-axis representing annexin V-FITC and the Y-axis representing PI. This assay separates cells into three populations: viable cells, early apoptotic cells and PI positive cells. Early apoptotic cells and PI-positive cells were pooled together and counted as dead cells. Cell death was examined in the absence or presence of either a ROS scavenger or PARP inhibitors. The ROS scavenger EUK-134 was obtained from Eukarion Inc. (Bedford, MA) (38Rong Y. Doctrow S.R. Tocco G. Baudry M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9897-9902Crossref PubMed Scopus (246) Google Scholar), dissolved in culture medium. PARP inhibitors Nicotinamide (NIC) and 3-Aminobenzamide (3AB) were purchased from Sigma, and solubilized in culture medium or dimethyl sulfoxide (Me2SO) respectively. Statistical Analysis—The data are presented as the mean ± S.E. Statistical analysis was performed using analysis of variance or Student's t test where appropriate. Differences were considered significant when p < 0.05. Increased GK Protein and Activity in RIN 1046-38 Cells— Introduction of the islet GK cDNA using AdGK increased both GK protein and activity in RIN 1046-38 cells. Fig. 1a shows the results of Western blot analysis of GK protein in AdGK- and AdLacZ-treated cells. To evaluate the percentage of cells with increased GK protein, we performed immunocytochemistry followed by flow cytometry. Fig. 1b demonstrates that, compared with AdLacZ-treated cells, the majority of AdGK-treated cells have increased GK protein. To assess the activity of GK in adenovirally transduced cells, we measured the rate of glucose phosphorylation in cell extracts at various glucose concentrations. Increased GK expression enhanced GK activity whereas the activity of HK remained unchanged. The rate of glucose phosphorylation in the presence of 20 mm glucose was ∼10-fold higher in AdGK-treated cells as compared with AdLacZ-treated cells (Fig. 1c). Metabolic Response to Glucose in RIN 1046-38 Cells with Increased GK Activity—When RIN 1046-38 cells were transduced with AdGK, as opposed to AdLacZ, they began to undergo cell death in culture medium containing 25 mm glucose (see below). Transduction of cells with the adenoviruses was therefore performed in medium containing 3.3 mm glucose. To investigate the mechanisms underlying the toxic effects of increased GK activity, we examined the following parameters in RIN 1046-38 cells that were exposed to different concentrations of glucose after adenoviral transduction. We first assayed the rate of glucose metabolism by measuring glycolysis, because β cells respond to elevations in extracellular glucose by facilitating glycolysis and oxidative metabolism. Cells with increased GK activity had an accelerated rate of glycolysis when the extracellular glucose was raised above 5 mm (Fig. 2a). The rate of glycolysis in AdGK-treated cells in the presence of 20 mm glucose was ∼2-fold higher than AdLacZ-treated cells. In pancreatic β cells, increased glucose metabolism in response to high glucose leads to increases in several metabolic derivatives, such as NAD(P)H and ATP, that are crucial for GSIS. We therefore measured cellular NAD(P)H and ATP. AdGK-treated cells cultured in 3.3 mm glucose had increases in NAD(P)H in response to 10 mm and 20 mm glucose that were comparable to similarly treated AdLacZ-treated cells (data not shown). In sharp contrast and different from the results of glycolysis, exposure of cells with increased GK activity to 20 mm glucose for 2 h significantly decreased the NAD(P)H response to 10 mm and 20 mm glucose (Fig. 2b). Similarly, exposure of AdGK-treated cells to 10 and 20 mm glucose significantly reduced cellular ATP (Fig. 2c). Since NAD(P)H and ATP are generated during both glycolysis in the cytosol and oxidative metabolism in mitochondria and are utilized during cellular metabolism as an energy source or electron donor, these results suggest increased usage and/or decreased mitochondrial generation of NAD(P)H and ATP. Accumulation of ROS in RIN 1046-38 Cells with Increased GK Activity after Exposure to High Concentrations of Glucose— One use of NAD(P)H is to provide electrons for the regeneration of different cellular antioxidants. Therefore, decreased cellular NAD(P)H in the face of increased glucose metabolism could result from cellular oxidative stress. To test this, we monitored the levels of intracellular O2·¯ and H2O2 in RIN 1046-38 cells using the intracellular probes HE and DHR, respectively. HE is oxidized specifically by O2·¯, and this process generates a fluorescent product. DHR reacts with H2O2 and generates fluorescent rhodamine 123. Cells were either continuously cultured in 3.3 mm glucose, or exposed to 20 mm glucose for different durations. These treatments did not change the levels of intracellular O2·¯ and H2O2 in AdLacZ-treated cells (Fig. 3, a and b, shaded curves). In contrast, both O2·¯ and H2O2 accumulated time-dependently in AdGK-treated cells after exposure to 20 mm glucose (Fig. 3, a and b). The increase in intracellular H2O2 became detectable after exposure to 20 mm glucose for 15 min. A further increase was only detected in a subpopulation of AdGK-treated cells at the 4-hour time point (Fig. 3b), possibly as a result of diffusion of H2O2 out of the cells. Furthermore, a subpopulation of AdGK-treated cells had decreased intracellular H2O2 at the 4-hour time point. To ascertain whether this subpopulation represented dead cells, we double labeled the cells with DHR and PI and analyzed by flow cytometry. The results showed that the PI-positive cells (dead cells) had reduced fluorescence intensity of rhodamine 123, reflecting a decreased intracellular H2O2 (data not shown). Oxidative Damage in RIN 1046-38 Cells with Increased GK Activity after Exposure to High Concentrations of Glucose— Elevated ROS can damage β cells by directly oxidizing cellular macromolecules such as lipids and DNA. To investigate if such damage was present in RIN 1046-38 cells with increased GK activity, we exposed cells to 20 mm glucose for 4 h and examined the following parameters. Lipid peroxidation was assayed by measuring MDA and 4HNE, two markers that are commonly used to reflect lipid peroxidation and lipid peroxidation-induced membrane damage (39Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5902) Google Scholar, 40van Ginkel G. Sevanian A. Methods Enzymol. 1994; 233: 273-288Crossref PubMed Scopus (119) Google Scholar). Compared with AdLacZ-treated cells, AdGK-treated cells had significantly higher levels of MDA/4HNE after exposure to 20 mm glucose for 4 h, demonstrating the existence of oxidative damage to cellular lipids (Fig. 4a). Since ROS are produced primarily in the electron transport chain associated with the mitochondrial membrane, mitochondria may be a major target for oxidative injury. Using rhodamine 123 to assess the mitochondrial membrane, a significantly higher proportion of AdGK-treated cells lost their mitochondrial membrane integrity after exposure to 20 mm glucose for 4 h (reflected by a loss of the ability to retain rhodamine 123 in mitochondria, Fig. 4b). We next examined whether DNA damage was present by detecting DNA strand breaks using TUNEL assay. Fig. 4c shows that exposure of AdGK-treated cells to 20 mm glucose for 4 h increases the number of DNA strand breaks. Because excessive DNA strand breaks causes overactivation of the DNA-repairing enzyme PARP and depletion of ATP in β cells (41Heller B. Wang Z.Q. Wagner E.F. Radons J. Burkle A. Fehsel K. Burkart V. Kolb H. J. Biol. Chem. 1995; 270: 11176-11180Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 42Burkart V. Wang Z.Q. Radons J. Heller B. Herceg Z. Stingl L. Wagner E.F. Kolb H. Nat. Med. 1999; 5: 314-319Crossref PubMed Scopus (323) Google Scholar), we then measured PARP activity. AdGK-treated cells had significantly higher levels of PARP activity compared with AdLacZ-treated cells after exposure to 20 mm glucose for 4 h (Fig. 4d). Together, these results demonstrate that oxidative stress causes damage to both mitochondria and DNA. When combined with the data on cellular NAD(P)H and ATP, these results suggest that mitochondrial damage contributes to reduced cellular NAD(P)H by decreasing the generation of these nucleotides. Likewise, mitochondrial damage may impair ATP generation, in addition to depletion of cellular ATP by PARP overactivation. Oxidative Stress Causes Apoptosis in RIN 1046-38 Cells with Increased GK Activity—The initial observation during this project was that AdGK-treated RIN 1046-38 cells began to detach from culture plates and eventually died when they were cultured in medium containing 25 mm glucose. To characterize the nature of the cell death, we first examined the glucose concentration dependence. Adenovirally transduced RIN 1046-38 cells were exposed to different concentrations of glucose for 4 h, and then analyzed for cell death. High concentrations of glucose did not induce cell death in AdLacZ-treated cells, whereas cells with increased GK activity underwent cell death when they were exposed to 10 and 20 mm glucose (Fig. 5a). To determine if cell death requires the presence of metabolizable glucose and to exclude the hyperosmotic effect of high concentrations of glucose, AdGK-treated cells were exposed to 20 mm 3-O-methylglucose (3OMG) for up to 16 h. This treatment did not induce cell death (data not shown). Combined with the data on glycolysis, these results indicate that the metabolic consequences of increased glucose metabolism lead to cell death. To characterize the mode of cell death, we stained the cells with annexin V-FITC and PI, followed by flow cytometric analysis. AdGK-treated cells were separated into 3 populations. Viable cells are both annexin V-FITC and PI-negative (Fig. 5b; lower left quadrant of each panel). Early apoptotic cells are annexin V-FITC-positive but PI-negative (Fig. 5b; lower right quadrant of each panel). The cells located in the upper right quadrant of each panel in Fig. 5b are both annexin V-FITC and PI-positive and represent dead cells with disrupted cytoplasmic membrane (referred to as PI-positive cells in this study). After exposure to 20 mm glucose for 4 h, AdGK-treated cells had significantly higher numbers of both early apoptotic cells and PI-positive cells, compared with either AdGK-treated cells cultured only in 3.3 mm glucose or AdLacZ-treated cells (Fig. 5b). Together, these results demonstrate that increased glucose metabolism leads to apoptosis. To investigate if oxidative damage activated apoptosis signaling pathways, we included a ROS scavenger or PARP inhibitors in the cell death assay. EUK-134 is a synthetic superoxide dismutase (SOD)/catalase mimetic that catalytically eliminates both O2·¯ and H2O2 (38Rong Y. Doctrow S.R. Tocco G. Baudry M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9897-9902Crossref PubMed Scopus (246) Google Scholar). NIC is a PARP inhibitor and serves as the precursor of NAD+ (43Sestili P. Spadoni G. Ba
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