Olives and olive oil are nutritionally valuable parts of a diet, since the consumption of olives and olive oil is associated with a variety of health benefits, such as lower incidences of heart disease and certain types of cancer [1] -[4] . These findings have prompted a considerable amount of research into the composition of olive fruits, and nature of those components responsible for the observed beneficial health effects [4] .

The olive oil industry produces large amounts and varieties of waste that are rich in phenol compounds [5] . HPLC analyses of olive oil extracts indicate that hydroxyl-tyrosol and p-tyrosol were the most abundant phenolic compounds; among these extracts alperujo has the highest concentration in hydroxyl-tyrosol and antioxidant activity [6] . Alperujo, potentially a natural source of antioxidant, may therefore protect an organism against damage caused by oxidative agents involved in the etiology of chronic diseases such as diabetes [7] -[12] .

A central component to the development of type-2 diabetes mellitus is the progressive failure of pancreatic islet β-cells [13] [14] . Pancreatic β-cells secrete insulin to regulate blood glucose homeostasis. In the pancreas, glucose is transported into β-cells using glucose transporter type-2 (GLUT-2), and is subsequently oxidized by glycolytic pathways, the tri-carboxylic acid cycle, and mitochondrial oxidative phosphorylation [15] -[17] . Electrons that are derived from glucose metabolism move down the electron-transport chain, producing ATP through ATP synthase. ATP facilitates the release of insulin from pancreatic β-cells. Under glucotoxic conditions, mitochondrial superoxide production is elevated, resulting from an increased donation of electrons to the electron transport chain in addition to an increase in protons pumped out of the mitochondrial matrix [17] -[19] . These events activate mitochondrial uncoupling protein 2 (UCP-2), which is not normally highly expressed in β-cells, whose physiological function is to protect the cell against reactive oxygen species (ROS). However, its protective function results in a β-cell dysfunction because it negatively regulates insulin secretion [14] [17] .

When oxidative stress induced by high metabolic loads exceeds the repair mechanism of the cell, oxidized molecules accumulate and compromise mitochondrial membrane integrity, releasing cytochrome c into the cytosol, thereby inducing apoptosis [20] .

As in other cells types, the mitochondrial respiratory chains represent a major source of ROS in β-cells [21] However, NADPH oxidases are another source of ROS associated with the plasma membrane. These enzymes catalyze the NADPH-dependent cytosolic reduction of molecular oxygen (O2) to superoxide. Superoxide is converted spontaneously, converted through manganese superoxide dismutase (Mn-SOD) activity in the mitochondria or through copper-zinc superoxide dismutase (Cu/Zn-SOD) in the cytosol, to peroxide hydrogen (H₂O₂) [22] . H₂O₂ can be removed by either catalase (CAT) or peroxidases. CAT catalyzes direct decomposition of H₂O₂ to O₂ and water. In contrast, glutathione peroxidase (GSH-Px) removes H₂O₂ using glutathione (GSH) oxidation coupling. Thioredoxin peroxidase, or peroxiredoxin, removes H₂O₂ through thioredoxin (TRDX) oxidation coupling, a widely distributed polypeptide containing 2 thiol groups. Thioredoxin peroxidase catalyzes H₂O₂ more slowly, but is more abundant than glutathione peroxidase in most tissues [22] .

Since β-cells have a low antioxidant defense capacity, they are very sensitive to ROS-induced stress associated with an increase in glucose metabolism. Therefore, protecting β-cells against oxidative stress is important in preventing diabetes, and natural antioxidants could be useful in this endeavor. Our aim was to investigate the anti-oxidative effects, in vitro, of an aqueous alperujo extract on ROS production and MIN6 β-cell function subjected to stress with glucose and H₂O₂.

Oxidative stress is harmful to β-cell function. Johansen et al. [11] demonstrated that excessive and/or sustained ROS production affects the function and survival of β-cells, contributing to diabetes pathogenesis. Alperujo, an antioxidant-rich by-product of olive extraction [6] [27] , could protect the β-cell against oxidative stress. In our research the pancreatic β-cell line, MIN6, was challenged with two stress conditions, H2O2 and glucose. The H₂O₂ concentration used as the stressor agent decreased cell viability by about 50%, which coincides with the results of Lu et al. [28] . With respect to the glucose concentration used, some authors have reported using concentrations between 5.6 and 16.7 mM [29] as the stressor agent; however, the MIN6 β-cells used did not survive at concentrations lower than 25 mM (data not shown). For this reason, cells were exposed to high glucose concentrations to determine their survival capacity. For glucose concentrations above 150 mM, cell viability decreased by only 26%. Aqueous alperujo extract, a potential anti-oxidant containing 0.5 g/100 ml of gallic acid equivalent or phenol (20 μM) was used. This phenol concentration did not induce changes in MIN6 β-cells viability.

ROS production for each of the treatment conditions was measured over a 24 hour period, demonstrating that AE had a higher pro-oxidant effect on MIN6 β-cells than H₂O₂ or glucose, which did not differentiate ROS production with respect to the control. This finding parallels the results of Oliveira et al. [29] who measured SOD enzymatic activity in pancreatic islets exposed to glucose (5.5 to 16.7 mM). They found that when glucose concentrations increased, SOD activity also increased. Also, Lu et al. [28] observed that ROS production with 0.15 mM H₂O₂ exceeded the control more than twice in cells exposed to low glucose concentration (14mM). Likewise, Rebelato et al. [30] established that glucose concentrations above 8.3 mM suppress ROS production in a time-dependent manner, due to pentose-phosphate pathway activation and the consequent formation of NADPH, an important substrate to antioxidant cellular defense. They also confirmed that by inhibiting the enzymatic activity of Glucose-6-phosphate dehydrogenase (G6PDH) the glucose flow stopped, thus increasing ROS production. Perhaps, both increased SOD activity and activation of the pentose-phosphate pathway may explain the ROS production level found in cells challenged with glucose (100 mM) and H₂O₂ (0.15 mM) for 24 h. Contrary to previous results, challenging cells with AE alone or in combination with glucose or H₂O₂ produced an increment in ROS production, which suggests that AE contains an oxidizing component that may mask its antioxidant activity.

Alburquerque et al. [31] measured metal content in AE. They identified Fe, Cu, Mn and Zn in different concentrations, the highest of which was iron (614 mg/K). The other compound concentrations ranged from 16 to 21 mg/K. This finding suggests that intra- or extracellular H₂O₂ would react with the iron present in the AE and generate hydroxyl radicals that are highly reactive and detectable [14] . Besides, Babich et al. [32] discovered that some natural anti-oxidants such as green tea, black tea, apple extract, and granate extract, in vitro are pro-oxidants depending on the culture medium used. DMEM medium, compared to RPMI 1640 and McCoy media, generates high levels of H₂O₂ in the presence of extracts rich in polyphenols. At alkaline pH, polyphenol auto-oxidation occurs which induces an increase in ROS production. Compounds in the medium, such as inorganic salts, vitamins, and amino acids, also contribute to ROS production. To determine if iron would exercise a pro-oxidant role, we quantified Fe content in AE and MIN6 β-cells after 5 days of challenging the cells in different culture conditions. The Fe content in AE, 600 mg/K, is concordant with the levels reported by Alburquerque et al. [31] . There is a close relationship between iron metabolism and glucose levels with oxidative stress [33] . In our study we observed that cells challenged with AE, compound with elevated iron content, had an increased intracellular iron concentration, but also the cells with high glucose in the medium, demonstrating that glucose could stimulate the iron entrance in the cell. However, although both conditions had high intracellular iron the ROS production was higher in AE challenging, suggesting that AE together with iron could be more pro-oxidant that iron lonely.

In MIN6 β-cells cultured with glucose, AE tends to increase the Bax/Bcl-2 ratio, indicating some degree of cellular apoptosis. This process leads to a decrease in cell mass and therefore a decrease in insulin releasing capacity; however, insulin secretion was not affected in these conditions. A compensatory mechanism could be elucidated by the findings of Hisanaga et al. [34] , who revealed that an insulin-induced, calcium-permeable channel—Transient Receptor Potential cation channel, subfamily V, member 2 (TRPV2)—exists in certain tissues (neurons, neuroendocrine cells in the gastrointestinal tract, macrophages, and β-cells). Insulin secretion, from MIN6 β-cells cultured with glucose (25 mM), stimulates the synthesis, translocation, and insertion of TRPV2 into the cellular membrane. This facilitates the entry of calcium into the cells, stimulating more insulin secretion into the extracellular space; this mechanism described in MIN6 could explain these finding in insulin secretion. The Bax/Bcl-2 ratio tended to decrease in MIN6 β-cells pre-incubated with AE, thereby preserving the cell mass. This observation, together with the compensatory mechanism previously described, could explain the increased insulin secretion in these cells.

mRNA relative abundance of UCP-2 and TRDX increased in cells incubated with AE for 3 days. Due to increased antioxidant activity, the Bax/Bcl-2 ratio did not change. As mentioned above, a compensatory mechanism would help sustain insulin secretion despite increasing UCP-2 under these conditions. Furthermore, the challenge lasting more than 3 days with AE tended to decrease MIN6 β-cell glucose uptake. Johnston et al. [35] demonstrated that certain dietary polyphenols decrease Caco-2 cell glucose uptake, through non-competitive inhibition of GLUT-2 transporters. This finding was confirmed by Kwon et al. [36] and Song et al. [37] . In this study, despite the decreased glucose uptake, there was a rise in p47phox mRNA expression that reflects the pro-oxidant nature of the AE. Despite this rise, the Bax/Bcl-2 ratio was not altered.

The 3-day glucose plus AE condition had a synergistic effect on the intracellular Fe concentration. This increment may have generated oxidative stress, which then triggered a slight increase in Bax/Bcl-2 ratio. The decreased cell mass, due to mitochondrial compromise during apoptosis, could explain the decreased mRNA relative abundance of UCP-2 [20] .

The H₂O₂ challenge decreased glucose uptake, had no effects on mRNA relative abundance of UCP-2, TRDX, or p47phox, and had a slightly increased the Bax/Bcl-2 ratio. These results, contradictory to what was expected, have also been reported by Lu et al. [28] , who showed that H₂O₂ (0.15 mM) decreases SOD, CAT and GSH-Px enzymatic activity, indicating antioxidant defense deregulation in MIN6 β-cells. Therefore, as expected under the 5-day H₂O₂ challenge, there was a tendency towards apoptosis, which the substantial increase in the Bax/Bcl-2 ratio reflected. When MIN6 β-cells were pre-incubated with AE and subsequently challenged with glucose or H₂O₂, an increase in the intracellular iron concentration was observed.

Increased intracellular Fe levels in cells challenged with the AE/H₂O₂ mix constitutes an unexpected finding. Also observed, the AE/glucose mix challenge increased glucose uptake a considerable amount. This may be due to an acute effect of glucose load in the culture, since the cells were exposed to AE plus 25 mM glucose for 4 days, and as stated above, AE does not affect glucose uptake. However, the treatment with glucose (100 mM) 24 hours before the assay probably increased its uptake. mRNA relative abundance of the studied genes and the Bax/Bcl-2 ratio had a tendency to decrease.

In summary, alperujo extract behaves as a pro-oxidant in the culture conditions. However, despite its nature, AE tends to protect pancreatic β-cells against apoptosis, when the cells are pre-incubated with the alperujo, thereby improving insulin secretion.

This research was conducted with support from team of Micronutrient Laboratory, Institute of Nutrition and Food Technology (INTA), in charge of Dr. Miguel Arredondo, highlighting the unconditional support of Dr. Monica Andrews during throughout the development of this research funded by Fondecyt to M. Arredondo (No. 1110080). César González was a master student from the Biomedical Sciences Program, University of Talca.