Antioxidant Effects of Alperujo Extract (Arbequina and Frantoio Varieties) on MIN6 β-Cells Subjected to Stress with Glucose or H₂O₂

César González, Mónica Andrews, Elba Leiva, Cristina Quispe, Miguel Arredondo

Faculty of Health Sciences, University of Talca, Interdisciplinary Research Program on Healthy Aging (PIEI-ES), Talca, Chile.
Institute of Chemistry of Natural Resources, University of Talca, Talca, Chile.
Micronutrient Laboratory, Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile.

DOI: 10.4236/fns.2014.513139                

Abstract

Alperujo, an antioxidant-rich by-product of olive oil extraction, could protect β-cells against oxidative damage. Our goal was to study the antioxidant effects of an alperujo extract (AE) on MIN6 β-cells challenged with glucose or hydrogen peroxide. MIN6 β-cells were challenged with glucose (100 mM) or H₂O₂ (0.15 mM), with or without AE (20 μM phenol). Reactive oxygen species, intracellular iron (Fe), insulin, glucose uptake, and mRNA gene expression of Uncoupling Protein-2 (UCP-2), Thioredoxin (TRDX), p47phox, and the ratio Bax/Bcl-2 were measured. ROS increased when the stressors were incubated with AE (p < 0.05 and p < 0.01, respectively). Intracellular Fe increased in glucose presence (100 mM p < 0.001). Insulin secretion improved when cells were pre-incubated with AE (p < 0.001) and glucose uptake increased when cells were pre-incubated with AE for 3 days and then further treated with glucose (p < 0.001). After 3 days of AE alone, mRNA relative expression of UCP-2 and TRDX increased (p < 0.001) and after 5 days p47phox, also increased. The Bax/Bcl-2 ratio tended to decrease in the samples pre-incubated with AE. The Alperujo extract,in vitro, had a pro-oxidant behavior, however pre-incubating MIN6 β-cells with AE tended to protect them against apoptosis, thereby enhancing insulin secretion.

Keywords

Alperujo, Oxidative Stress, Apoptosis, Glucose, β-Cells

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Tuck, K.L. and Hayball, P.J. (2002) Major Phenolic Compounds in Olive Oil: Metabolism and Health Effects. The Journal of Nutritional Biochemistry, 13, 636-644. http://dx.doi.org/10.1016/S0955-2863(02)00229-2
[2]	Aruoma, O.I. (2003) Methodological Considerations for Characterizing Potential Antioxidant Actions of Bioactive Components in Plant Foods. Mutation Research, 523-524, 9-20. http://dx.doi.org/10.1016/S0027-5107(02)00317-2
[3]	Perez-Jimenez, F., Alvarez de Cienfuegos, G., Badimon, L., Barja, G., et al. (2005) International Conference on the Healthy Effect of Virgin Olive Oil. European Journal of Clinical Investigation, 35, 421-424.
[4]	Fernández-Bolaños, G.J., Guillén, R., Jiménez, A., Rodríguez, R. and Rodríguez, G. (2006) Extraction of Interesting Organic Compounds from Olive Oil Waste. Grasas y Aceites, 57, 95-106.
[5]	Tripoli, E., Giammanco, M., Tabacchi, G., Di Majo, D., et al. (2005) The Phenolic Compounds of Olive Oil: Structure, Biological Activity and Beneficial Effects on Human Health. Nutrition Research Reviews, 18, 98-112. http://dx.doi.org/10.1079/NRR200495
[6]	Lesage-Meessen, L., Navarro, D., Maunier, S., Sigoillot, J.C., et al. (2001) Simple Phenolic Content in Olive Oil Residues as a Function of Extraction Systems. Food Chemistry, 75, 501-507. http://dx.doi.org/10.1016/S0308-8146(01)00227-8
[7]	Robertson, R.P., Harmon, J., Tran, P.O., Tanaka, Y. and Takahashi, H. (2003) Glucose Toxicity in β-Cells: Type 2 Diabetes, Good Radicals Gone Bad, and the Glutathione Connection. Diabetes, 52, 581-587. http://dx.doi.org/10.2337/diabetes.52.3.581
[8]	Waris, G. and Ahsan, H. (2006) Reactive Oxygen Species: Role in the Development of Cancer and Various Chronic Conditions. Journal of Carcinogenesis, 5, 14. http://dx.doi.org/10.1186/1477-3163-5-14
[9]	Robertson, P. and Harmon, J.S. (2007) Pancreatic Islet Beta-Cell and Oxidative Stress: The Importance of Glutathione Peroxidase. FEBS Letters, 581, 3743-3748. http://dx.doi.org/10.1016/j.febslet.2007.03.087
[10]	Chang, Y.C. and Chuang, L.M. (2010) The Role of Oxidative Stress in the Pathogenesis of Type 2 Diabetes: From Molecular Mechanism to Clinical Implication. American Journal of Translational Research, 2, 316-331.
[11]	Johansen, J.S., Harris, A.K., Rychly, D.J. and Ergul, A. (2005) Oxidative Stress and the Use of Antioxidants in Diabetes: Linking Basic Science to Clinical Practice. Cardiovascular Diabetology, 4, 5. http://dx.doi.org/10.1186/1475-2840-4-5
[12]	Drews, G., Krippeit-Drews, P. and Dufer, M. (2010) Oxidative Stress and Beta-Cell Dysfunction. Pflügers Archiv, 460, 703-718. http://dx.doi.org/10.1007/s00424-010-0862-9
[13]	Prentki, M. and Nolan, C.J. (2006) Islet Beta Cell Failure in Type 2 Diabetes. Journal of Clinical Investigation, 116, 1802-1812. http://dx.doi.org/10.1172/JCI29103
[14]	Ma, Z.A., Zhao, Z. and Turk, J. (2012) Mitochondrial Dysfunction and B-Cell Failure in Type 2 Diabetes Mellitus. Experimental Diabetes Research, 2012, Article ID: 703538. doi:10.1155/2012/ 70353811.
[15]	Maechler, P. and Wollheim, C.B. (2001) Mitochondrial Function in Normal and Diabetic Beta-Cells. Nature, 414, 807-812. http://dx.doi.org/10.1038/414807a
[16]	Porterfield, D.M., Corkey, R.F., Sanger, R.H., Tornheim, K., et al. (2000) Oxygen Consumption Oscillates in Single Clonal Pancreatic Beta-Cells (HIT). Diabetes, 49, 1511-1516. http://dx.doi.org/10.2337/diabetes.49.9.1511
[17]	Krauss, S., Zhang, C.Y. and Lowell, B.B. (2005) The Mitochondrial Uncoupling-Protein Homologues. Nature Reviews Molecular Cell Biology, 6, 248-261. http://dx.doi.org/10.1038/nrm1592
[18]	Patane, G., Anello, M., Piro, S., Vigneri, R., et al. (2002) Role of ATP Production and Uncoupling Protein-2 in the Insulin Secretory Defect Induced by Chronic Exposure to High Glucose or Free Fatty Acids and Effects of Peroxisome Proliferator-Activated Receptor-Gamma Inhibition. Diabetes, 51, 2749-2756. http://dx.doi.org/10.2337/diabetes.51.9.2749
[19]	Newsholme, P., Morgan, D., Rebelato, E., Oliveira-Emilio, H.C., et al. (2009) Insights into the Critical Role of NADPH Oxidase(s) in the Normal and Dysregulated Pancreatic Beta Cell. Diabetologia, 52, 2489-2498. http://dx.doi.org/10.1007/s00125-009-1536-z
[20]	Orrenius, S. and Zhivotovsky, B. (2005) Cardiolipin Oxidation Sets Cytochrome c Free. Nature Chemical Biology, 1, 188-189. http://dx.doi.org/10.1038/nchembio0905-188
[21]	Newsholme, P., Haber, E.P., Hirabara, S.M., Rebelato, E.L., et al. (2007) Diabetes Associated Cell Stress and Dysfunction: Role of Mitochondrial and Non-Mitochondrial ROS Production and Activity. The Journal of Physiology, 583, 9-24. http://dx.doi.org/10.1113/jphysiol.2007.135871
[22]	Bindoli, A., Fukuto, J.M. and Forman, H.J. (2008) Thiol Chemistry in Peroxidase Catalysis and Redox Signaling. Antioxidants Redox Signaling, 10, 1549-1564. http://dx.doi.org/10.1089/ars.2008.2063
[23]	Monari, M., Sanchez, G., Cadiz, I., Mera, S. and Castaneda, P. (1993) A Simple Sensitive and Inexpensive Micro Method for the High Performance Liquid Chromatographic Determination of Total and Free Valproic Acid in Human Plasma and Serum. Giornale Italiano Di Chimica Clinica, 17, 353-360.
[24]	Singleton, V.L. and Rossi, J.A. (1965) Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. American Journal of Enology and Viticulture, 16, 144-158.
[25]	Pfaffl, M.W. (2001) A New Mathematical Model for Relative Quantification in Real-Time RT-PCR. Nucleic Acids Research, 29, Article ID: e45. http://dx.doi.org/10.1093/nar/29.9.e45
[26]	Yamamoto, N., Ueda, M., Sato, T., Kawasaki, K., Sawada, K., Kawabata, K. and Ashida, H. (2011) UNIT 12.14 Measurement of Glucose Uptake in Cultured Cells. In: Enna, S.J., Eds., Current Protocols in Pharmacology, Chapter 12, 11-22. http://dx.doi.org/10.1002/0471141755.ph1214s55
[27]	Galli, C. and Visioli, F. (1999) Antioxidant and Other Activities of Phenolic in Olives/Olive Oil, Typical Components of the Mediterranean Diet. Lipids, 34, S23-S26. http://dx.doi.org/10.1007/BF02562224
[28]	Lu, X.Y., Guo, H. and Zhang, Y.L. (2012) Protective Effects of Sulfated Chitooligosaccharides against Hydrogen Peroxide-Induced Damage in MIN6 Cells. International Journal of Biological Macromolecules, 50, 50-58. http://dx.doi.org/10.1016/j.ijbiomac.2011.09.020
[29]	Oliveira, H.R., Curi, R. and Carpinelli, A.R. (1999) Glucose Induces an Acute Increase of Superoxide Dismutase Activity in Incubated Rat Pancreatic Islets. American Journal of Physiology, 276, C507-C510.
[30]	Rebelato, E., Abdulkader, F., Curi, R. and Carpinelli, A.R. (2011) Control of the Intracellular Redox State by Glucose Participates in the Insulin Secretion Mechanism. PLoS ONE, 6, Article ID: e24507. http://dx.doi.org/10.1371/journal.pone.0024507
[31]	Alburquerque, J.A., Gonzalvez, J., Garcia, D. and Cegarra, J. (2004) Agrochemical Characterisation of “Alperujo”, a Solid By-Product of the Two-Phase Centrifugation Method for Olive Oil Extraction. Bioresource Technology, 91, 195-200. http://dx.doi.org/10.1016/S0960-8524(03)00177-9
[32]	Babich, H., Schuck, A.G., Weisburg, J.H. and Zuckerbraun, H.L. (2011) Research Strategies in the Study of the Pro-Oxidant Nature of Polyphenol Nutraceuticals. Journal of Toxicology, 2011, Article ID: 467305. http://dx.doi.org/10.1155/2011/467305
[33]	Fernandez-Real, J.M., Lopez-Bermejo, A. and Ricart, W. (2002) Cross-Talk between Iron Metabolism and Diabetes. Diabetes, 51, 2348-2354. http://dx.doi.org/10.2337/diabetes.51.8.2348
[34]	Hisanaga, E., Nagasawa, M., Ueki, K., Kulkarni, R.N., Mori, M. and Kojima, I. (2009) Regulation of Calcium-Permeable TRPV2 Channel by Insulin in Pancreatic β-Cells. Diabetes, 58, 174-184. http://dx.doi.org/10.2337/db08-0862
[35]	Johnston, K., Sharp, P., Clifford, M. and Morgan, L. (2005) Dietary Polyphenols Decrease Glucose Uptake by Human Intestinal Caco-2 Cells. FEBS Letters, 579, 1653-1657. http://dx.doi.org/10.1016/j.febslet.2004.12.099
[36]	Kwon, O., Eck, P., Chen, S., Corpe, C.P., Lee, J.H., Kruhlak, M. and Levine, M. (2007) Inhibition of the Intestinal Glucose Transporter GLUT2 by Flavonoids. FASEB Journal, 21, 366-377. http://dx.doi.org/10.1096/fj.06-6620com
[37]	Song, J., Kwon, O., Chen, S., Daruwala, R., et al. (2002) Flavonoid Inhibition of Sodium-Dependent Vitamin C Transporter 1 (SVCT1) and Glucose Transporter Isoform 2 (GLUT2), Intestinal Transporters for Vitamin C and Glucose. Journal of Biological Chemistry, 277, 15252-15260. http://dx.doi.org/10.1074/jbc.M110496200