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• W2074417648 abstract "Excessive free fatty acid (FFA) exposure represents a potentially important diabetogenic condition that can impair insulin secretion from pancreatic β-cells. Because mitochondrial oxidative phosphorylation is a main link between glucose metabolism and insulin secretion, in the present work we investigated the effects of the FFA oleate (OE) on mitochondrial function in the clonal pancreatic β-cell line, MIN6. Both the long term (72 h) and short term (immediately after application) impact of OE exposure on β-cells was investigated. After 72 h of exposure to OE (0.4 mm, 0.5% bovine serum albumin) cells were washed and permeabilized, and mitochondrial function (respiration, phosphorylation, membrane potential formation, production of reactive oxygen species) was measured in the absence or presence of OE. MIN6 cells exposed to OE for 72 h showed impaired glucose-stimulated insulin secretion and decreased cellular ATP. Mitochondria in OE-exposed cells retained normal functional characteristics in FFA-free medium; however, they were significantly more sensitive to the acute uncoupling effect of OE treatment. The mitochondria of OE-exposed cells displayed increased depolarization caused by acute OE treatment, which is attributable to the elevation in the FFA-transporting function of uncoupling protein 2 and the dicarboxylate carrier. These cells also had an increased production of reactive oxygen species in complex I of the mitochondrial respiratory chain that could be activated by FFA. A high level of reduction of respiratory complex I augmented acute FFA-induced uncoupling in a way compatible with activation of mitochondrial uncoupling protein by intramitochondrial superoxide. A stronger augmentation was observed in OE-exposed cells. Together, these events may underlie FFA-induced depression of the ATP/ADP ratio in β-cells, which accounts for the defective glucose-stimulated insulin secretion associated with lipotoxicity. Excessive free fatty acid (FFA) exposure represents a potentially important diabetogenic condition that can impair insulin secretion from pancreatic β-cells. Because mitochondrial oxidative phosphorylation is a main link between glucose metabolism and insulin secretion, in the present work we investigated the effects of the FFA oleate (OE) on mitochondrial function in the clonal pancreatic β-cell line, MIN6. Both the long term (72 h) and short term (immediately after application) impact of OE exposure on β-cells was investigated. After 72 h of exposure to OE (0.4 mm, 0.5% bovine serum albumin) cells were washed and permeabilized, and mitochondrial function (respiration, phosphorylation, membrane potential formation, production of reactive oxygen species) was measured in the absence or presence of OE. MIN6 cells exposed to OE for 72 h showed impaired glucose-stimulated insulin secretion and decreased cellular ATP. Mitochondria in OE-exposed cells retained normal functional characteristics in FFA-free medium; however, they were significantly more sensitive to the acute uncoupling effect of OE treatment. The mitochondria of OE-exposed cells displayed increased depolarization caused by acute OE treatment, which is attributable to the elevation in the FFA-transporting function of uncoupling protein 2 and the dicarboxylate carrier. These cells also had an increased production of reactive oxygen species in complex I of the mitochondrial respiratory chain that could be activated by FFA. A high level of reduction of respiratory complex I augmented acute FFA-induced uncoupling in a way compatible with activation of mitochondrial uncoupling protein by intramitochondrial superoxide. A stronger augmentation was observed in OE-exposed cells. Together, these events may underlie FFA-induced depression of the ATP/ADP ratio in β-cells, which accounts for the defective glucose-stimulated insulin secretion associated with lipotoxicity. Chronic elevation of circulating free fatty acids (FFAs) 1The abbreviations used are: FFA, free fatty acid; OE, oleate; UCP, uncoupling protein; DIC, dicarboxylate carrier; SOD, superoxide dismutase; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; ROS, reactive oxygen species; BSA, bovine serum albumin; KRB, Krebs-Ringer buffer; TBS, Tris-buffered saline; TBS-T, TBS-Tween.1The abbreviations used are: FFA, free fatty acid; OE, oleate; UCP, uncoupling protein; DIC, dicarboxylate carrier; SOD, superoxide dismutase; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; ROS, reactive oxygen species; BSA, bovine serum albumin; KRB, Krebs-Ringer buffer; TBS, Tris-buffered saline; TBS-T, TBS-Tween. is associated with obesity and type 2 diabetes. Long term exposure of insulin-secreting pancreatic β-cells to elevated concentrations of FFAs alters glucose-induced insulin secretion and is considered as an important factor in the pathogenesis of diabetes (1Lameloise N. Muzzin P. Prentki M. Assimacopoulos-Jeannet F. Diabetes. 2001; 50: 803-809Crossref PubMed Scopus (214) Google Scholar, 2Liang Y. Buettger C. Berner D.K. Matschinsky F.M. Diabetologia. 1997; 40: 1018-1027Crossref PubMed Scopus (73) Google Scholar, 3McGarry J.D. Diabetes. 2002; 51: 7-18Crossref PubMed Scopus (1214) Google Scholar). In pancreatic β-cells, mitochondrial oxidative phosphorylation is a crucial intermediate between glucose metabolism and insulin secretion (4Langin D. N. Engl. J. Med. 2001; 345: 1772-1774Crossref PubMed Scopus (47) Google Scholar, 5Antinozzi P.A. Ishihara H. Newgard C.B. Wollheim C.B. J. Biol. Chem. 2002; 277: 11746-11755Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and thus the effect of fatty acids on the functional state of mitochondria in this cell type is an important area of investigation. Previous work demonstrated that fatty acids applied for 48–72 h cause partial uncoupling of oxidative phosphorylation (decreased mitochondrial membrane potential and ATP content and increased respiration) (1Lameloise N. Muzzin P. Prentki M. Assimacopoulos-Jeannet F. Diabetes. 2001; 50: 803-809Crossref PubMed Scopus (214) Google Scholar, 6Carlsson C. Borg L.A. Welsh N. Endocrinology. 1999; 140: 3422-3428Crossref PubMed Google Scholar). Although fatty acids are natural weak uncouplers of oxidative phosphorylation, a direct uncoupling effect of fatty acids on β-cell mitochondria was considered as unlikely since these effects were observed after long term (48–72 h) but not short term (15 min–2 h) application (1Lameloise N. Muzzin P. Prentki M. Assimacopoulos-Jeannet F. Diabetes. 2001; 50: 803-809Crossref PubMed Scopus (214) Google Scholar, 6Carlsson C. Borg L.A. Welsh N. Endocrinology. 1999; 140: 3422-3428Crossref PubMed Google Scholar). Instead, alteration in activity and/or expression of certain mitochondrial enzymes and transporters was suggested as the most likely cause of the fatty acid effect on oxidative phosphorylation in β-cells (7Segall L. Lameloise N. Assimacopoulos-Jeannet F. Roche E. Corkey P. Thumelin S. Corkey B.E. Prentki M. Am. J. Physiol. 1999; 277: E521-E528PubMed Google Scholar). In particular, it was suggested that the lowered mitochondrial membrane potential and impaired glucose-induced rise in the ATP/ADP ratio in FFA-exposed β-cells are caused by induction of uncoupling protein 2 (UCP2) in mitochondria (1Lameloise N. Muzzin P. Prentki M. Assimacopoulos-Jeannet F. Diabetes. 2001; 50: 803-809Crossref PubMed Scopus (214) Google Scholar). Indeed, FFA exposure leads to elevated levels of UCP2 mRNA and protein in β-cell mitochondria (1Lameloise N. Muzzin P. Prentki M. Assimacopoulos-Jeannet F. Diabetes. 2001; 50: 803-809Crossref PubMed Scopus (214) Google Scholar, 8Medvedev A.V. Robidoux J. Bai X. Cao W. Floering L.M. Daniel K.W. Collins S. J. Biol. Chem. 2002; 277: 42639-42644Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) due to transcriptional regulation in which sterol regulatory element-binding protein-1 plays a major role (8Medvedev A.V. Robidoux J. Bai X. Cao W. Floering L.M. Daniel K.W. Collins S. J. Biol. Chem. 2002; 277: 42639-42644Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). However, it is not clear if this can account entirely for alterations in mitochondrial function. In fact, the uncoupling (protonophoric) function of UCP2 and UCP3 is still a subject of debate (9Cadenas S. Echtay K.S. Harper J.A. Jekabsons M.B. Buckingham J.A. Grau E. Abuin A. Chapman H. Clapham J.C. Brand M.D. J. Biol. Chem. 2002; 277: 2773-2778Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 10Krauss S. Zhang C.Y. Lowell B.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 118-122Crossref PubMed Scopus (154) Google Scholar, 11Stuart J.A. Harper J.A. Brindle K.M. Jekabsons M.B. Brand M.D. J. Biol. Chem. 2001; 276: 18633-18639Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). There is considerable evidence to suggest that the two functions of UCPs are to regulate reactive oxygen species (ROS) production and to export FFA anions from mitochondria, rather than to provide basal proton conductance of the mitochondrial membrane (12Li L.X. Skorpen F. Egeberg K. Jorgensen I.H. Grill V. Biochem. Biophys. Res. Commun. 2001; 282: 273-277Crossref PubMed Scopus (119) Google Scholar, 13Himms-Hagen J. Harper M.E. Exp. Biol. Med. (Maywood.). 2001; 226: 78-84Crossref PubMed Scopus (279) Google Scholar). To gain further insight into the mechanism of the effect of fatty acid in the present study we applied permeabilized cell techniques to assess mitochondrial function in the β-cell and the effects of the long term (72 h) and short term (immediately after FFA addition to the assay medium) application of FFA. Studying intact cells provides only limited information about mitochondrial function because it is difficult to control the extramitochondrial medium and the mitochondrial functional state (14Bogucka K. Wroniszewska A. Bednarek M. Duszynski J. Wojtczak L. Biochim. Biophys. Acta. 1990; 1015: 503-509Crossref PubMed Scopus (23) Google Scholar, 15Saks V.A. Veksler V.I. Kuznetsov A.V. Kay L. Sikk P. Tiivel T. Tranqui L. Olivares J. Winkler K. Wiedemann F. Kunz W.S. Mol. Cell Biochem. 1998; 184: 81-100Crossref PubMed Google Scholar). Permeabilization of the plasma membrane for low molecular weight solutes allows the investigation of mitochondrial processes under precisely controlled conditions in situ, where mitochondrial interaction with intracellular structures is largely preserved. To obtain sufficient amounts of uniform material, which is required for this kind of study, we used the pancreatic β-cell line, MIN6. This cell line is one of the few that retains insulin-secretory responses to glucose and other secretagogues and has been used extensively in studies on the mechanisms controlling insulin secretion (16Soejima A. Inoue K. Takai D. Kaneko M. Ishihara H. Oka Y. Hayashi J.I. J. Biol. Chem. 1996; 271: 26194-26199Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Minami K. Yano H. Miki T. Nagashima K. Wang C.Z. Tanaka H. Miyazaki J.I. Seino S. Am. J. Physiol. Endocrinol. Metab. 2000; 279: E773-E781Crossref PubMed Google Scholar, 18Ishihara H. Asano T. Tsukuda K. Katagiri H. Inukai K. Anai M. Kikuchi M. Yazaki Y. Miyazaki J.I. Oka Y. Diabetologia. 1993; 36: 1139-1145Crossref PubMed Scopus (342) Google Scholar). In the present study, it was found that long term exposure of MIN6 cells to oleate (OE) sensitizes mitochondria to its direct uncoupling (acute) action. Another result of the long term OE application was stimulation of mitochondrial ROS production, mostly accounted for by the activity of complex I of the respiratory chain. In turn, the high level of reduction of complex I was accompanied by an additional uncoupling effect (mitochondrial depolarization), which was more pronounced in the OE-exposed cells. Dependence of this depolarization on the presence of FFA in the assay medium, GDP, inhibitors of complex I and a superoxide dismutase (SOD) mimetic suggests that it was caused by activation of UCP2 (the only UCP found in pancreatic β-cells) with intramitochondrial superoxide. These findings are of interest in light of continuing debate on UCPs potential functions (9Cadenas S. Echtay K.S. Harper J.A. Jekabsons M.B. Buckingham J.A. Grau E. Abuin A. Chapman H. Clapham J.C. Brand M.D. J. Biol. Chem. 2002; 277: 2773-2778Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 10Krauss S. Zhang C.Y. Lowell B.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 118-122Crossref PubMed Scopus (154) Google Scholar, 19Fink B.D. Hong Y.S. Mathahs M.M. Scholz T.D. Dillon J.S. Sivitz W.I. J. Biol. Chem. 2002; 277: 3918-3925Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 20Echtay K.S. Roussel D. St. Pierre J. Jekabsons M.B. Cadenas S. Stuart J.A. Harper J.A. Roebuck S.J. Morrison A. Pickering S. Clapham J.C. Brand M.D. Nature. 2002; 415: 96-99Crossref PubMed Scopus (1146) Google Scholar). In addition, OE-induced mitochondrial depolarization was sensitive to the dicarboxylate carrier (DIC) substrate malonate and OE-exposed cells demonstrated an elevated level of this carrier. Together these observations suggest that an increase in OE uncoupling efficiency caused by the long term incubation with OE is associated with a FFA-transporting function of the mitochondrial inner membrane proteins UCP2 and DIC. Growth, Fatty Acid Treatment, and Permeabilization of Cells—MIN6 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 0.05 mm β-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin for 3–4 days. For fatty acid exposure, growth medium was changed to Roswell Park Memorial Institute (RPMI 1640) medium containing the ingredients listed above in addition to 0.4 mm OE bound to bovine serum albumin (BSA) (0.5% BSA for control experiments). After 72 h of incubation, with daily medium change, trypsinized cells were washed in Ca2+-free Krebs-Ringer buffer (KRB buffer, 120 mm NaCl, 1.0 mm MgCl2, 24 mm NaHCO3, and 10 mm HEPES, pH 7.3) and permeabilized essentially as described by Civelek et al. (21Civelek V.N. Deeney J.T. Shalosky N.J. Tornheim K. Hansford R.G. Prentki M. Corkey B.E. Biochem. J. 1996; 318: 615-621Crossref PubMed Scopus (78) Google Scholar). Briefly, cells from two 10-cm dishes were suspended in 0.7 ml of KRB buffer containing 80 μg/ml saponin. After incubation at room temperature for 5 min, the cells were pelleted by centrifugation at 4 °C, washed in cold buffer, and suspended in cold 0.25 m sucrose containing 10 mm HEPES, pH 7.3. This produced stable cell preparations that retained permeable plasma membranes and coupled mitochondria when stored overnight at 4 °C or for at least 2 months at –80 °C. Insulin Secretion Assay—MIN6 cells cultured in 12- or 24-well plates after OE exposure as described above were washed and preincubated twice for 30 min in a modified KRB (115 mm NaCl, 5.0 mm KCl, 2.5 mMCaCl2, 1.0 mm MgCl2, 24 mm NaHCO3, 1.25 mm glucose, and 10 mm HEPES, pH 7.3) with 0.1% BSA, then incubated for 1 h in the same buffer containing 1.25–25 mm glucose similar to a previous report (22Chan C.B. De Leo D. Joseph J.W. McQuaid T.S. Ha X.F. Xu F. Tsushima R.G. Pennefather P.S. Salapatek A.M. Wheeler M.B. Diabetes. 2001; 50: 1302-1310Crossref PubMed Scopus (315) Google Scholar). Insulin secretion in response to glucose was quantified using a radio-immunoassay (Linco Research, St. Charles, MO) according to manufacturer's instruction and normalized by cellular protein content. Respiration Measurements—Oxygen consumption by permeabilized MIN6 cells was measured using a Clark-type electrode connected to an Oxygraph unit (Hansatech, Pentney, England). Cells were suspended at a concentration of 0.6–0.9 mg of protein/ml in incubation medium containing 0.25 m sucrose, 10 mm HEPES, 10 mm Pi, 2 mm MgCl2, 1 mm EGTA, and 0.2% BSA. 10 mm succinate, 5 mm glutamate/5 mm malate or 7.5 mm glycerol-3-phosphate were added as respiratory substrates. Oxygen kinetic traces were treated as described by Estabrook (23Estabrook R.W. Methods Enzymol. 1967; 10: 41-47Crossref Scopus (1893) Google Scholar), and respiration rates were converted into molar oxygen units using O2 solubility in sucrose medium, as reported by Reynafarje et al. (24Reynafarje B. Costa L.E. Lehninger A.L. Anal. Biochem. 1985; 145: 406-418Crossref PubMed Scopus (195) Google Scholar). Mitochondrial Membrane Potential Monitoring—Mitchondrial membrane potential was monitored by following safranin O fluorescence in suspensions of permeabilized cells (25Vercesi A.E. Bernardes C.F. Hoffmann M.E. Gadelha F.R. Docampo R. J. Biol. Chem. 1991; 266: 14431-14434Abstract Full Text PDF PubMed Google Scholar) and rhodamine 123 fluorescence in intact cells (1Lameloise N. Muzzin P. Prentki M. Assimacopoulos-Jeannet F. Diabetes. 2001; 50: 803-809Crossref PubMed Scopus (214) Google Scholar). Different methods for measurements were due to a superior responsiveness to K+/valinomycin titration by safranin in permeabilized cells and higher loading ability of rhodamine 123 in intact cells. Measurements were performed using a FluoroCount plate reader (Packard Instrument Company, Meriden, CT) with excitation/emission wavelengths of 530/590 nm for safranin and 485/530 nm for rhodamine 123, respectively. A decrease in fluorescence corresponded to an increase in mitochondrial membrane potential. Incubation medium for permeabilized cells was identical to that used for respiratory assays but was supplemented with 2.5 μm safranin. Intact cells were incubated in KRB buffer. The magnitude of the fatty acid-induced depolarization in permeabilized cells was estimated by titration with K+/valinomycin by supplementing the assay medium with 0.7 μm valinomycin and the addition of 0.05–7.4 mm K+. Intact cells were loaded with rhodamine-123 at 10 μg/ml as previously described (22Chan C.B. De Leo D. Joseph J.W. McQuaid T.S. Ha X.F. Xu F. Tsushima R.G. Pennefather P.S. Salapatek A.M. Wheeler M.B. Diabetes. 2001; 50: 1302-1310Crossref PubMed Scopus (315) Google Scholar, 26Duchen M.R. Smith P.A. Ashcroft F.M. Biochem. J. 1993; 294: 35-42Crossref PubMed Scopus (153) Google Scholar) and the fluorescence response to the addition of glucose and the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) was monitored. ATP Assay—To monitor synthesis and hydrolysis of ATP, samples were withdrawn from the cell suspension at appropriate time points and fixed with dimethyl sulfoxide (Me2SO) (27Ouhabi R. Boue-Grabot M. Mazat J.P. Anal. Biochem. 1998; 263: 169-175Crossref PubMed Scopus (63) Google Scholar). ATP was measured using the luciferin-luciferase kit from Sigma according to the manufacturer's instructions. Fluorometric Determination of Hydrogen Peroxide Production—Generation of ROS was estimated as hydrogen peroxide formation by monitoring catalase-sensitive appearance of dichlorofluorescein (excitation 490 nm, emission 530 nm) from non-fluorescent dichlorofluorescein in the presence of horseradish peroxidase (28Cocco T. Di Paola M. Papa S. Lorusso M. Free Radic. Biol. Med. 1999; 27: 51-59Crossref PubMed Scopus (192) Google Scholar, 29Di Paola M. Cocco T. Lorusso M. Biochemistry. 2000; 39: 6660-6668Crossref PubMed Scopus (204) Google Scholar, 30Black M.J. Brandt R.B. Anal. Biochem. 1974; 58: 246-254Crossref PubMed Scopus (110) Google Scholar). High concentrations (1.55 units/ml) and the low Km of peroxidase helps to circumvent interference from endogenous hydrogen peroxide-metabolizing enzymes (31Kwong L.K. Sohal R.S. Methods Enzymol. 2002; 349: 341-346Crossref PubMed Scopus (6) Google Scholar). Dichlorofluorescein was obtained from the stable compound dichlorofluorescin diacetate by alkaline hydrolysis. Western Blot for DIC—Whole cell protein extracts from MIN6 cells (40 μg of protein/lane) were separated on 14% polyacrylamide gels and electroblotted to nitrocellulose membrane (Millipore Corp., Bedford, MA). Blots were blocked with 5% (w/v) nonfat dry skimmed milk in Tris-buffered saline with 0.1% (v/v) Tween (TBS-T) for 1 h and incubated with DIC antibody (polyclonal rabbit antibody against mitochondrial dicarboxylate carrier, 1:1000 dilution in TBS-T with 0.5% BSA) (32Das K. Lewis R.Y. Combatsiaris T.P. Lin Y. Shapiro L. Charron M.J. Scherer P.E. Biochem. J. 1999; 344: 313-320Crossref PubMed Scopus (25) Google Scholar) for 2 h. Blots were washed with TBS-T and exposed to horseradish peroxidase-coupled anti-rabbit IgG (Amersham Biosciences) for 1 h at room temperature. Blots were washed again with TBS-T and developed by enhanced chemiluminescence using a standard kit (ECL, Amersham Biosciences). Band intensity was measured by densitometry, analyzed using image analysis software (Scion Image v.4.02; Scion Corporation, Frederick, MD) and normalized by cytochrome c content in parallel samples. Statistics—In all cases a Student's t test was used to analyze data between groups. A p ≤ 0.05 was considered statistically significant. Effect of Oleate Exposure on Insulin Secretion—Incubation with 0.4 mm OE for 72 h caused an increase in the basal level of insulin secretion in MIN6 cells, while inhibiting glucose-stimulated insulin secretion (Fig. 1). These alterations are very similar to those observed in β-cell lines and pancreatic islets by us and others (6Carlsson C. Borg L.A. Welsh N. Endocrinology. 1999; 140: 3422-3428Crossref PubMed Google Scholar, 7Segall L. Lameloise N. Assimacopoulos-Jeannet F. Roche E. Corkey P. Thumelin S. Corkey B.E. Prentki M. Am. J. Physiol. 1999; 277: E521-E528PubMed Google Scholar, 22Chan C.B. De Leo D. Joseph J.W. McQuaid T.S. Ha X.F. Xu F. Tsushima R.G. Pennefather P.S. Salapatek A.M. Wheeler M.B. Diabetes. 2001; 50: 1302-1310Crossref PubMed Scopus (315) Google Scholar, 33Busch A.K. Cordery D. Denyer G.S. Biden T.J. Diabetes. 2002; 51: 977-987Crossref PubMed Scopus (154) Google Scholar). Effect of Oleate on Mitochondrial Function—Permeabilization of the plasma membrane with saponin allows observation of metabolic transitions accompanying oxidative phosphorylation in β-cell mitochondria in situ. Representative oxygen kinetic traces in control and OE-exposed cells (Fig. 2) demonstrate that oxygen consumption in permeabilized cells responds to respiratory substrate, ADP, oligomycin, and FCCP in a way typical for isolated mitochondria (15Saks V.A. Veksler V.I. Kuznetsov A.V. Kay L. Sikk P. Tiivel T. Tranqui L. Olivares J. Winkler K. Wiedemann F. Kunz W.S. Mol. Cell Biochem. 1998; 184: 81-100Crossref PubMed Google Scholar, 25Vercesi A.E. Bernardes C.F. Hoffmann M.E. Gadelha F.R. Docampo R. J. Biol. Chem. 1991; 266: 14431-14434Abstract Full Text PDF PubMed Google Scholar). Occurrence of the state 3-state 4 transition is of interest since in permeabilized cells it is usually prevented by cytosolic ATPases (21Civelek V.N. Deeney J.T. Shalosky N.J. Tornheim K. Hansford R.G. Prentki M. Corkey B.E. Biochem. J. 1996; 318: 615-621Crossref PubMed Scopus (78) Google Scholar, 27Ouhabi R. Boue-Grabot M. Mazat J.P. Anal. Biochem. 1998; 263: 169-175Crossref PubMed Scopus (63) Google Scholar). Therefore, we measured the rates of ATP synthesis after the addition of ADP. We also measured ATP hydrolysis after the completion of ADP phosphorylation and the inhibition of mitochondrial ATP synthase with oligomycin. For these measurements, samples were taken from cell suspensions in the course of the reaction, fixed with Me2SO, and analyzed for ATP with luciferin-luciferase. It was found that succinate-driven ATP synthesis in state 3 in permeabilized MIN6 cells is faster than cytosolic ATP hydrolysis in state 4 by more than an order of magnitude (56.5 ± 4.8 and 3.3 ± 0.6 nmol ATP/min per mg of protein, n = 3). Thus the present experimental conditions allow an estimation of mitochondrial ATP synthesis despite interference of cytosolic enzymes. Mitochondrial respiratory and coupling parameters derived from respiratory kinetic traces suggest apparently normal functional performance of mitochondria in OE-exposed cells (Table I). This is consistent with the observation that mitochondrial membrane potential kinetics in OE-treated cells closely parallels that of control cells (Fig. 3A). Fluorescent kinetics show that quenching of safranin fluorescence is entirely dependent on the addition of succinate, suggesting a mitochondrial origin of the safranin response and low levels of endogenous mitochondrial substrates in permeabilized washed cells. Succinate (or FCCP)-dependent safranin fluorescence quenching ΔFquench/ΔFtotal of 0.647 ± 0.024 and 0.648 ± 0.032 was found for the control and OE-exposed cells, respectively (n = 4).Table IRespiratory rates and coupling parameters in control and oleate-treated permeabilized MIN6 cells (n = 4)State 3 respiration V3,ADPADP/ORespiratory control rationmol O2/min mg of proteinControl cells20.1 ± 2.01.33 ± 0.073.06 ± 0.49Oleate-treated cells18.8 ± 1.41.38 ± 0.043.33 ± 0.39 Open table in a new tab Fig. 3Mitochondrial membrane potential formation and breakdown in permeabilized and intact MIN6 cells.A, permeabilized cells (0.7 mg of protein/ml) were placed into respiratory incubation medium (see “Experimental Procedures”) supplemented with 2.5 μm safranin. Other additions are indicated by arrows: 10 mm succinate and 4.7 μm FCCP. B, intact cells were loaded with 10 μg/ml rhodamine 123 for 10 min, preincubated in glucose-free medium for 20 min, and washed and placed into KRB buffer supplemented with 0.1% (w/v) BSA. Additions are indicated by arrows: 15 mm glucose and 4.7 μm FCCP. Kinetic traces are representative of seven (A) and three (B) independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The apparent normal oxidative phosphorylation in the fatty acid-exposed cells is in contradiction with the earlier reported uncoupling effect of fatty acids on β-cell mitochondria (1Lameloise N. Muzzin P. Prentki M. Assimacopoulos-Jeannet F. Diabetes. 2001; 50: 803-809Crossref PubMed Scopus (214) Google Scholar, 6Carlsson C. Borg L.A. Welsh N. Endocrinology. 1999; 140: 3422-3428Crossref PubMed Google Scholar). For further verification of our findings, mitochondrial membrane potential was tested in the same cells before permeabilization, as shown in Fig. 3B. Typical kinetic curves demonstrate that FCCP causes a significantly smaller fluorescent response in OE-exposed cells (48 ± 8% of the fluorescent response in control cells, p < 0.05) indicating that mitochondria in OE-treated cells, in the presence of endogenous substrates or external glucose, are partially depolarized. This confirms results of previous work and indicates that mitochondria in β-cells exposed to fatty acids are partly uncoupled when functioning in their native intracellular environment but show normal functional capacity when placed into standard incubation medium (see “Experimental Procedures”). This suggests that mitochondrial uncoupling in β-cells exposed to OE is caused primarily by cytosolic mediators that appear in the cells treated with fatty acid. A natural candidate for such a role would be OE itself, which is well known as a weak mitochondrial uncoupler (34Skulachev V.P. Biochim. Biophys. Acta. 1998; 1363: 100-124Crossref PubMed Scopus (817) Google Scholar). To test this hypothesis, we titrated mitochondrial membrane potential in the control and OE-exposed permeabilized MIN6 cells with OE (35Hofmann W.E. Liu X. Bearden C.M. Harper M.E. Kozak L.P. J. Biol. Chem. 2001; 276: 12460-12465Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 36Simonyan R.A. Jimenez M. Ceddia R.B. Giacobino J.P. Muzzin P. Skulachev V.P. Biochim. Biophys. Acta. 2001; 1505: 271-279Crossref PubMed Scopus (49) Google Scholar). Typical titration kinetics (Fig. 4A) and analysis of the data (Fig. 4B) indicate that the mitochondrial potential in OE-exposed cells is more sensitive to the uncoupling effect of OE. Qualitatively similar results were observed when the cells were exposed to an oleate/palmitate mixture (2:1) of the same sum molarity (Fig. 4B). It is known that oxidative phosphorylation is characterized by a steep dependence on the mitochondrial membrane potential, which is confined within a 20–30 mV potential range (37Zoratti M. Pietrobon D. Azzone G.F. Eur. J. Biochem. 1982; 126: 443-451Crossref PubMed Scopus (73) Google Scholar, 38Woelders H. van der Velden T. van Dam K. Biochim. Biophys. Acta. 1988; 934: 123-134Crossref PubMed Scopus (14) Google Scholar). Calibration of mitochondrial depolarization shown in Fig. 4A demonstrates that fatty acid-induced alterations in membrane depolarization are located in this physiologically important interval. Formation of ROS in Mitochondria—Oxidative stress is considered an important component of disorders related to high levels of FFA (39Cuzzocrea S. Riley D.P. Caputi A.P. Salvemini D. Pharmacol. Rev. 2001; 53: 135-159PubMed Google Scholar). In particular, ROS are thought to contribute significantly to defective β-cell function associated with type 2 diabetes (40Gorogawa S. Kajimoto Y. Umayahara Y. Kaneto H. Watada H. Kuroda A. Kawamori D. Yasuda T. Matsuhisa M. Yamasaki Y. Hori M. Diabetes Res. Clin. Pract. 2002; 57: 1-10Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar); however, this notion is based on the effects of exogenous effectors such as alloxan (41Sakurai K. Katoh M. Someno K. Fujimoto Y. Biol. Pharm. Bull. 2001; 24: 876-882Crossref PubMed Scopus (65) Google Scholar) and oxygen radical scavengers (42Ho E. Chen G. Bray T.M. FASEB J. 1999; 13: 1845-1854Crossref PubMed Scopus (126) Google Scholar), while endogenous ROS production in β-cells remains poorly understood. Recently, it was shown that palmitate and cytokin" @default.
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• W2074417648 title "Mitochondrial Functional State in Clonal Pancreatic β-Cells Exposed to Free Fatty Acids" @default.
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