Background: We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability. Methods. Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results: HTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions: These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia. © 2013 Alberici et al.; licensee BioMed Central Ltd.121 Martins De Lima, T., Gorjao, R., Hatanaka, E., Cury-Boaventura, M.F., Portioli Silva, E.P., Procopio, J., Curi, R., Mechanisms by which fatty acids regulate leucocyte function (2007) Clinical Science, 113 (1-2), pp. 65-77. , DOI 10.1042/CS20070006Yaqoob, P., Fatty acids as gatekeepers of immune cell regulation (2003) Trends in Immunology, 24 (12), pp. 639-645. , DOI 10.1016/j.it.2003.10.002Calder, P.C., Yaqoob, P., Thies, F., Wallace, F.A., Miles, E.A., Fatty acids and lymphocyte functions (2002) Br J Nutr, 87, pp. 1931-S48. , 10.1079/BJN2001455 11895154Ito, Y., Azrolan, N., O'Connell, A., Walsh, A., Breslow, J.L., Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice (1990) Science, 249, pp. 790-793. , 10.1126/science.2167514 2167514Aalto-Setala, K., Fisher, E.A., Chen, X., Chajek-Shaul, T., Hayek, T., Zechner, R., Walsh, A., Breslow, J.L., Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo e on the particles (1992) J Clin Invest, 90, pp. 1889-1900. , 10.1172/JCI116066 1430212Reaven, G.M., Mondon, C.E., Chen, Y.-D.I., Breslow, J.L., Hypertriglyceridemic mice transgenic for the human apolipoprotein C-III gene are neither insulin resistant nor hyperinsulinemic (1994) Journal of Lipid Research, 35 (5), pp. 820-824Amaral, M.E.C., Oliveira, H.C.F., Carneiro, E.M., Delghingaro-Augusto, V., Vieira, E.C., Berti, J.A., Boschero, A.C., Plasma glucose regulation and insulin secretion in hypertriglyceridemic mice (2002) Hormone and Metabolic Research, 34 (1), pp. 21-26. , DOI 10.1055/s-2002-19962Alberici, L.C., Oliveira, H.C.F., Patricio, P.R., Kowaltowski, A.J., Vercesi, A.E., Hyperlipidemic Mice Present Enhanced Catabolism and Higher Mitochondrial ATP-Sensitive K+ Channel Activity (2006) Gastroenterology, 131 (4), pp. 1228-1234. , DOI 10.1053/j.gastro.2006.07.021, PII S0016508506016647Alberici, L.C., Oliveira, H.C.F., Bighetti, E.J.B., De Faria, E.C., Degaspari, G.R., Souza, C.T., Vercesi, A.E., Hypertriglyceridemia Increases Mitochondrial Resting Respiration and Susceptibility to Permeability Transition (2003) Journal of Bioenergetics and Biomembranes, 35 (5), pp. 451-457. , DOI 10.1023/A:1027343915452Alberici, L.C., Oliveira, H.C., Paim, B.A., Mantello, C.C., Augusto, A.C., Zecchin, K.G., Gurgueira, S.A., Vercesi, A.E., Mitochondrial ATP-sensitive K(+) channels as redox signals to liver mitochondria in response to hypertriglyceridemia (2009) Free Radic Biol Med, 47, pp. 1432-1439. , 10.1016/j.freeradbiomed.2009.08.013 19703550Alberici, L.C., Vercesi, A.E., Oliveira, H.C., Mitochondrial energy metabolism and redox responses to hypertriglyceridemia (2011) J Bioenerg Biomembr, 43, pp. 19-23. , 10.1007/s10863-011-9326-y 21258853Facundo, H.T.F., Fornazari, M., Kowaltowski, A.J., Tissue protection mediated by mitochondrial K+ channels (2006) Biochimica et Biophysica Acta - Molecular Basis of Disease, 1762 (2), pp. 202-212. , DOI 10.1016/j.bbadis.2005.06.003, PII S0925443905001031, Mitochondria in Diseases and TherapeuticsSkulachev, V.P., Uncoupling: New approaches to an old problem of bioenergetics (1998) Biochimica et Biophysica Acta - Bioenergetics, 1363 (2), pp. 100-124. , DOI 10.1016/S0005-2728(97)00091-1, PII S0005272897000911Brookes, P.S., Mitochondrial H+ leak and ROS generation: An odd couple (2005) Free Radical Biology and Medicine, 38 (1), pp. 12-23. , DOI 10.1016/j.freeradbiomed.2004.10.016, PII S089158490400838XDahlem, Y.A., Horn, T.F.W., Buntinas, L., Gonoi, T., Wolf, G., Siemen, D., The human mitochondrial KATP channel is modulated by calcium and nitric oxide: A patch-clamp approach (2004) Biochimica et Biophysica Acta - Bioenergetics, 1656 (1), pp. 46-56. , DOI 10.1016/j.bbabio.2004.01.003, PII S0005272804000209Ellis, J.A., Mayer, S.J., Jones, O.T., The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils (1988) Biochem J, 251, pp. 887-891. , 2843166Degasperi, G.R., Velho, J.A., Zecchin, K.G., Souza, C.T., Velloso, L.A., Borecky, J., Castilho, R.F., Vercesi, A.E., Role of mitochondria in the immune response to cancer: A central role for Ca2+ (2006) Journal of Bioenergetics and Biomembranes, 38 (1), pp. 1-10. , DOI 10.1007/s10863-006-9000-yPurushothaman, D., Sarin, A., Cytokine-dependent regulation of NADPH oxidase activity and the consequences for activated T cell homeostasis (2009) J Exp Med, 206, pp. 1515-1523. , 10.1084/jem.20082851 19546249Birx, D.L., Berger, M., Fleischer, T.A., The interference of T cell activation by calcium channel blocking agents (1984) Journal of Immunology, 133 (6), pp. 2904-2909Allen, R.G., Tresini, M., Oxidative stress and gene regulation (2000) Free Radical Biology and Medicine, 28 (3), pp. 463-499. , DOI 10.1016/S0891-5849(99)00242-7, PII S0891584999002427Finkel, T., Holbrook, N.J., Oxidants, oxidative stress and the biology of ageing (2000) Nature, 408, pp. 239-247. , 10.1038/35041687 11089981Yaqoob, P., Newsholme, E.A., Calder, P.C., Influence of cell culture conditions on diet-induced changes in lymphocyte fatty acid composition (1995) Biochim Biophys Acta, 1255, pp. 333-340. , 10.1016/0005-2760(94)00251-S 7734450Mebius, R.E., Kraal, G., Structure and function of the spleen (2005) Nature Reviews Immunology, 5 (8), pp. 606-616. , DOI 10.1038/nri1669Delamaire, M., Maugendre, D., Moreno, M., Le Goff, M.-C., Allannic, H., Genetet, B., Impaired leucocyte functions in diabetic patients (1997) Diabetic Medicine, 14 (1), pp. 29-34. , DOI 10.1002/(SICI)1096-9136(199701)14:1<29::AID-DIA300>3.0.CO;2-VBouter, K.P., Meyling, F.H., Hoekstra, J.B., Masurel, N., Erkelens, D.W., Diepersloot, R.J., Influence of blood glucose levels on peripheral lymphocytes in patients with diabetes mellitus (1992) Diabetes Res, 19, pp. 77-80. , 1286542Chang, F.Y., Shaio, M.F., Decreased cell-mediated immunity in patients with non-insulin-dependent diabetes mellitus (1995) Diabetes Res Clin Pract, 28, pp. 137-146. , 10.1016/0168-8227(95)00168-8 7587921Kimura, M., Tanaka, S.-I., Isoda, F., Sekigawa, K.-I., Yamakawa, T., Sekihara, H., T lymphopenia in obese diabetic (db/db) mice is non-selective and thymus independent (1998) Life Sciences, 62 (14), pp. 1243-1250. , DOI 10.1016/S0024-3205(98)00054-X, PII S002432059800054XTanaka, I.S., Inoue, S., Isoda, F., Waseda, M., Ishihara, M., Yamakawa, T., Sugiyama, A., Okuda, K., Impaired immunity in obesity: Suppressed but reversible lymphocyte responsiveness (1993) International Journal of Obesity, 17 (11), pp. 631-636Tanaka, S.-I., Isoda, F., Yamakawa, T., Ishihara, M., Sekihara, H., T lymphopenia in genetically obese rats (1998) Clinical Immunology and Immunopathology, 86 (2), pp. 219-225. , DOI 10.1006/clin.1997.4467Jacotot, E., Costantini, P., Laboureau, E., Zamzami, N., Susin, S.A., Kroemer, G., Mitochondrial membrane permeabilization during the apoptotic process (1999) Annals of the New York Academy of Sciences, 887, pp. 18-30Kaufmann, T., Strasser, A., Jost, P.J., Fas death receptor signalling: Roles of Bid and XIAP (2012) Cell Death Differ, 19, pp. 42-50. , 10.1038/cdd.2011.121 21959933Harris, S.G., Phipps, R.P., The nuclear receptor PPAR gamma is expressed by mouse T lymphocytes and PPAR gamma agonists induce apoptosis (2001) Eur J Immunol, 31, pp. 1098-1105. , 10.1002/1521-4141(200104)31:4<1098: AID-IMMU1098>3.0.CO;2-I 11298334Muralidhar, B., Carpenter, K.L.H., Muller, K., Skepper, J.N., Arends, M.J., Potency of arachidonic acid in polyunsaturated fatty acid-induced death of human monocyte-macrophages: Implications for atherosclerosis (2004) Prostaglandins Leukotrienes and Essential Fatty Acids, 71 (4), pp. 251-262. , DOI 10.1016/j.plefa.2004.03.020, PII S0952327804000729Switzer, K.C., Fan, Y.-Y., Wang, N., McMurray, D.N., Chapkin, R.S., Dietary n-3 polyunsaturated fatty acids promote activation-induced cell death in Th1-polarized murine CD4+ T-cells (2004) Journal of Lipid Research, 45 (8), pp. 1482-1492. , DOI 10.1194/jlr.M400028-JLR200Fernanda Cury-Boaventura, M., Cristine Kanunfre, C., Gorjao, R., Martins De Lima, T., Curi, R., Mechanisms involved in Jurkat cell death induced by oleic and linoleic acids (2006) Clinical Nutrition, 25 (6), pp. 1004-1014. , DOI 10.1016/j.clnu.2006.05.008, PII S0261561406001166Aires, V., Hichami, A., Filomenko, R., Ple, A., Rebe, C., Bettaieb, A., Khan, N.A., Docosahexaenoic acid induces increases in [Ca2+]i via inositol 1,4,5-triphosphate production and activates protein kinase Cγ and -δ via phosphatidylserine binding site: Implication in apoptosis in U937 cells (2007) Molecular Pharmacology, 72 (6), pp. 1545-1556. , http://molpharm.aspetjournals.org/cgi/reprint/72/6/1545, DOI 10.1124/mol.107.039792Bakker, S.J., Ijzerman, R.G., Teerlink, T., Westerhoff, H.V., Gans, R.O., Heine, R.J., Cytosolic triglycerides and oxidative stress in central obesity: The missing link between excessive atherosclerosis, endothelial dysfunction, and beta-cell failure? (2000) Atherosclerosis, 148, pp. 17-21. , 10.1016/S0021-9150(99)00329-9 10580166Iverson, S.L., Orrenius, S., The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis (2004) Archives of Biochemistry and Biophysics, 423 (1), pp. 37-46. , DOI 10.1016/j.abb.2003.12.002Fang, K.-M., Lee, A.-S., Su, M.-J., Lin, C.-L., Chien, C.-L., Wu, M.-L., Free fatty acids act as endogenous ionophores, resulting in Na + and Ca2+ influx and myocyte apoptosis (2008) Cardiovascular Research, 78 (3), pp. 533-545. , DOI 10.1093/cvr/cvn030Otton, R., Curi, R., Toxicity of a mixture of fatty acids on human blood lymphocytes and leukaemia cell lines (2005) Toxicology in Vitro, 19 (6), pp. 749-755. , DOI 10.1016/j.tiv.2005.04.003, PII S0887233305000676Malhi, H., Bronk, S.F., Werneburg, N.W., Gores, G.J., Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis (2006) Journal of Biological Chemistry, 281 (17), pp. 12093-12101. , http://www.jbc.org/cgi/reprint/281/17/12093.pdf, DOI 10.1074/jbc.M510660200Otton, R., Da Silva, D.O., Campoio, T.R., Silveira, L.R., De Souza, M.O., Hatanaka, E., Curi, R., Non-esterified fatty acids and human lymphocyte death: A mechanism that involves calcium release and oxidative stress (2007) Journal of Endocrinology, 195 (1), pp. 133-143. , DOI 10.1677/JOE-07-0195Yuan, H., Zhang, X., Huang, X., Lu, Y., Tang, W., Man, Y., Wang, S., Li, J., NADPH oxidase 2-derived reactive oxygen species mediate FFAs-induced dysfunction and apoptosis of β-cells via JNK, p38 MAPK and p53 pathways (2010) PLoS One, 5, p. 515726. , 10.1371/journal.pone.0015726 21209957Schrauwen, P., Schrauwen-Hinderling, V., Hoeks, J., Hesselink, M.K., Mitochondrial dysfunction and lipotoxicity (1801) Biochim Biophys Acta, 2010, pp. 266-271Dikalov, S., Cross talk between mitochondria and NADPH oxidases (2011) Free Radic Biol Med, 51, pp. 1289-1301. , 10.1016/j.freeradbiomed.2011.06.033 21777669Zhang, D.X., Chen, Y.-F., Campbell, W.B., Zou, A.-P., Gross, G.J., Li, P.-L., Characteristics and superoxide-induced activation of reconstituted myocardial mitochondrial ATP-sensitive potassium channels (2001) Circulation Research, 89 (12), pp. 1177-1183Queliconi, B.B., Wojtovich, A.P., Nadtochiy, S.M., Kowaltowski, A.J., Brookes, P.S., Redox regulation of the mitochondrial K(ATP) channel in cardioprotection (1813) Biochim Biophys Acta, 2011, pp. 1309-1315Andrukhiv, A., Costa, A.D., West, I.C., Garlid, K.D., Opening mitoKATP increases superoxide generation from complex i of the electron transport chain (2006) Am J Physiol Heart Circ Physiol, 291, pp. 82067-H2074. , 10.1152/ajpheart.00272.2006 16798828Facundo, H.T.F., De Paula, J.G., Kowaltowski, A.J., Mitochondrial ATP-sensitive K+ channels are redox-sensitive pathways that control reactive oxygen species production (2007) Free Radical Biology and Medicine, 42 (7), pp. 1039-1048. , DOI 10.1016/j.freeradbiomed.2007.01.001, PII S0891584907000081Cardoso, A.R., Queliconi, B.B., Kowaltowski, A.J., Mitochondrial ion transport pathways: Role in metabolic diseases (2010) Biochim Biophys Acta, 1797, pp. 832-838. , 10.1016/j.bbabio.2009.12.017 20044972Jun, D.W., Cho, W.K., Jun, J.H., Kwon, H.J., Jang, K.S., Kim, H.J., Jeon, H.J., Lee, M.H., Prevention of free fatty acid-induced hepatic lipotoxicity by carnitine via reversal of mitochondrial dysfunction (2011) Liver Int, 31, pp. 1315-1324. , 10.1111/j.1478-3231.2011.02602.x 22093454Sato, T., Sasaki, N., O'Rourke, B., Marban, E., Nicorandil, a potent cardioprotective agent, acts by opening mitochondrial ATP-dependent potassium channels (2000) Journal of the American College of Cardiology, 35 (2), pp. 514-518. , DOI 10.1016/S0735-1097(99)00552-5, PII S0735109799005525Teshima, Y., Akao, M., Baumgartner, W.A., Marban, E., Nicorandil prevents oxidative stress-induced apoptosis in neurons by activating mitochondrial ATP-sensitive potassium channels (2003) Brain Research, 990 (1-2), pp. 45-50. , DOI 10.1016/S0006-8993(03)03383-3Hansen, L.K., Grimm Jr., R.H., Neaton, J.D., The relationship of white blood cell count to other cardiovascular risk factors (1990) Int J Epidemiol, 19, pp. 881-888. , 10.1093/ije/19.4.881 2084016Inoue, T., Iseki, K., Iseki, C., Kinjo, K., Elevated resting heart rate is associated with white blood cell count in middle-aged and elderly individuals without apparent cardiovascular disease (2012) Angiology, 63, pp. 541-546. , 10.1177/0003319711428071 22144667Walsh, A., Azrolan, N., Wang, K., Marcigliano, A., O'Connell, A., Breslow, J.L., Intestinal expression of the human apoA-I gene in transgenic mice is controlled by a DNA region 3' to the gene in the promoter of the adjacent convergently transcribed apoC-III gene (1993) Journal of Lipid Research, 34 (4), pp. 617-623Boyum, A., Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g (1968) Scand J Clin Lab Invest Suppl, 97, pp. 77-89. , 4179068Lebel, C., Ischiropoulos, H., Bondy, S., Evaluation of the probe 2 V,7 V-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress (1992) Chem Res Toxicol, 5, pp. 227-231. , 10.1021/tx00026a012 1322737Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding (1976) Anal Biochem, 72, pp. 248-254. , 10.1016/0003-2697(76)90527-3 942051